Abstract: Menstrual toxic shock syndrome (mTSS) is an acute febrile disease accompanied by hypotension and multiple organ involvement. Infection with Staphylococcus aureus producing the superantigen toxic shock syndrome toxin-1 (TSST-1) vaginally is necessary; however, only a small fraction of those infected with TSST-1 producing bacteria actually develop mTSS, suggesting that host factors modulate disease susceptibility. Serum antibodies to the toxin protect against development of the syndrome, but not all antibodies can neutralize the toxin. We set out to determine whether risk of developing the syndrome is related to the absence of neutralizing antibody and if antibody isotypes influence the neutralization capacity. In healthy subjects, TSST-1-binding serum antibodies were exclusively of the IgG and IgM classes; however, toxin-neutralizing capacity was correlated to the TSST-1-specific IgG1 and IgG4 antibodies (r (2)=0.88, p<0.0001 and 0.33, p<0.0086, respectively) but not with IgM antibodies. Specific IgA was not detectable. Compared to healthy matched controls who were colonized vaginally with S. aureus, IgG1 anti-TSST-1 antibodies and toxin neutralizing activity was lacking in all of the acute phases and in the majority of convalescent sera, suggesting that these patients may be incapable of generating TSST-1 neutralizing antibodies. These new findings support the hypothesis that host factors are important in the development of mTSS and that the anti-toxin isotype impacts antibody functionality.

Abstract: Our epidemiologic studies on invasive Group A Streptococci (GAS) infections identified specific HLA class II haplotypes/alleles conferring high-risk or protection from streptococcal toxic shock syndrome with a strong protection conferred by the DRB1*15/DQB1*06 haplotype. We used HLA-transgenic mice to provide an in vitro and in vivo validation for the direct role of HLA class II allelic variation in streptococcal toxic shock syndrome. When splenocytes from mice expressing the protective HLA-DQB1*06 (DQ6) allele were stimulated with a mixture of streptococcal superantigens (SAgs), secreted by the prevalent M1T1 strain, both proliferative and cytokine responses were significantly lower than those of splenocytes from mice expressing the neutral DRB1*0402/DQB1*0302 (DR4/DQ8) alleles (p < 0.001). In crisscross experiments, the presentation of SAgs to pure T cells from either the DQ6 or the DR4/DQ8 mice resulted in significantly different levels of response depending on the HLA type expressed on the APCs. Presentation by HLA-DQ6 APCs elicited significantly lower responses than the presentation by HLA-DR4/DQ8 APCs. Our in vitro data were supported by in vivo findings, as the DQ6 mice showed significantly longer survival post-i.v. infection with live M1T1 GAS (p < 0.001) and lower inflammatory cytokine responses as compared with the DR4/DQ8 mice (p < 0.01). The data presented here provide evidence for a direct role of HLA class II molecules in modulating responses to GAS SAgs and underscore the dominant role of HLA class II allelic variation in potentiating the severity of GAS systemic infections.

Abstract: Group A Streptococcus (GAS) is a leading human pathogen associated with a wide spectrum of mucosal and invasive infections. GAS expresses a large number of virulence determinants whose expression is under the control of several transcriptional regulatory networks. Here we performed the first mutational analysis of a genetic locus immediately upstream of the streptolysin S biosynthetic operon in several GAS genome sequences, including that of the M1T1 serotype, the leading isolates associated with serious invasive disease. The locus consists of a predicted RofA-like stand-alone transcriptional regulator (RALP3) and the largest open reading frame in the GAS genome, encoding a predicted LPXSG motif cell wall-anchored protein we have named LSA (for "large surface-anchored" protein). Comparative reverse transcription-PCR analysis of wild-type M1T1 GAS and an isogenic RALP3-deficient mutant identifies RALP3 as a global transcriptional regulator affecting expression of numerous virulence factor genes, including those for strong repression of the hyaluronic acid capsule and cysteine protease production. RALP3 contributed to GAS epithelial cell invasion and bloodstream survival. LSA was found to be under negative regulation by RALP3 and to influence GAS-epithelial cell interactions and GAS antimicrobial peptide sensitivity. Isogenic M1T1 GAS mutants lacking either RALP3 or LSA were attenuated in a murine model of systemic infection, indicating that this locus plays a role in the virulence potential of the organism.

Abstract: Transforming growth factor-beta1 (TGF-beta1) is a pleotropic cytokine that promotes angiogenesis and extracellular matrix protein synthesis in addition to its immunosuppressive effects. The purpose of this study is to identify optimal conditions for in vivo expression of TGF-beta1 by human islets to exploit the possible beneficial effects and minimize undesirable side effects. We transduced human islets with adenoviral vectors encoding the active form of Ad-TGF-beta1 or Ad-LacZ to test the effects of TGF-beta1 gene expression on islet in vivo function following their transplantation into a NOD-SCID mouse model. Islets were transduced with multiplicity of infection (MOI) of 20, 10, 5, and 2.5 per islet cell. At a MOI ranging from 2.5 to 20, expression of TGF-beta1 in islet supernatant persisted for 1-2 months and ranged from 153 +/- 5 to 2574 +/- 1299 pg/ml, respectively. Transduction with the lowest MOI (2.5) did not compromise the in vivo production of human C-peptide. We conclude that TGF-beta1 expression in transplanted islets does not compromise viability and that adenoviral transduction with the TGF-beta1 gene has a dose-dependent effect, with larger MOIs being deleterious. The data also indicate that in vitro culture system and the in vivo NOD-SCID model could be used successfully to evaluate the nonimmune effects of gene transduction.

Abstract: Most invasive bacterial infections are caused by species that more commonly colonize the human host with minimal symptoms. Although phenotypic or genetic correlates underlying a bacterium's shift to enhanced virulence have been studied, the in vivo selection pressures governing such shifts are poorly understood. The globally disseminated M1T1 clone of group A Streptococcus (GAS) is linked with the rare but life-threatening syndromes of necrotizing fasciitis and toxic shock syndrome. Mutations in the GAS control of virulence regulatory sensor kinase (covRS) operon are associated with severe invasive disease, abolishing expression of a broad-spectrum cysteine protease (SpeB) and allowing the recruitment and activation of host plasminogen on the bacterial surface. Here we describe how bacteriophage-encoded GAS DNase (Sda1), which facilitates the pathogen's escape from neutrophil extracellular traps, serves as a selective force for covRS mutation. The results provide a paradigm whereby natural selection exerted by the innate immune system generates hypervirulent bacterial variants with increased risk of systemic dissemination.

Abstract: Variation in responses to pathogens is influenced by exposure history, environment and the host's genetic status. We recently demonstrated that human leukocyte antigen class II allelic differences are a major determinant of the severity of invasive group A streptococcal (GAS) sepsis in humans. While in-depth controlled molecular studies on populations of genetically well-characterized humans are not feasible, it is now possible to exploit genetically diverse panels of recombinant inbred BXD mice to define genetic and environmental risk factors. Our goal in this study was to standardize the model and identify genetic and nongenetic covariates influencing invasive infection outcomes. Despite having common ancestors, the various BXD strains (n strains=33, n individuals=445) showed marked differences in survival. Mice from all strains developed bacteremia but exhibited considerable differences in disease severity, bacterial dissemination and mortality rates. Bacteremia and survival showed the expected negative correlation. Among nongenetic factors, age -- but not sex or weight -- was a significant predictor of survival (P=0.0005). To minimize nongenetic variability, we limited further analyses to mice aged 40-120 days and calculated a corrected relative survival index that reflects the number of days an animal survived post-infection normalized to all significant covariates. Genetic background (strain) was the most significant factor determining susceptibility (P< or =0.0001), thus underscoring the strong effect of host genetic variation in determining susceptibility to severe GAS sepsis. This model offers powerful unbiased forward genetics to map specific quantitative trait loci and networks of pathways modulating the severity of GAS sepsis.

Abstract: The serine protease high-temperature requirement A (HtrA) (DegP) of the human pathogen Streptococcus pyogenes (group A Streptococcus; GAS) is localized to the ExPortal secretory microdomain and is reportedly essential for the maturation of cysteine protease streptococcal pyrogenic exotoxin B (SpeB). Here, we utilize HSC5 (M5 serotype) and the in-frame isogenic mutant HSC5DeltahtrA to determine whether HtrA contributes to the maturation of other GAS virulence determinants. Mutanolysin cell wall extracts and secreted proteins were arrayed by 2-DE and identified by MALDI-TOF PMF analysis. HSC5DeltahtrA had elevated levels of cell wall-associated M protein, whilst the supernatant had higher concentrations of M protein fragments and a reduced amount of mature SpeB protease, compared to wild-type (WT). Western blot analysis and protease assays revealed a delay in the maturation of SpeB in the HSC5DeltahtrA supernatant. HtrA was unable to directly process SpeB zymogen (proSpeB) to the active form in vitro. We therefore conclude that HtrA plays an indirect role in the maturation of cysteine protease SpeB.

Abstract: Monarch-1/NLRP12 is expressed in myeloid cells and functions as a negative regulator of inflammation by inducing proteasome-mediated degradation of NF-kappaB-inducing kinase. Monarch-1 is a member of the CATERPILLER gene family, also known as the nucleotide-binding domain leucine-rich repeat gene family. This family shares strong structural homology to major immune regulators expressed in lower organisms, including plants. In plants, these disease-resistance proteins (R proteins) sense pathogenic insult and initiate a protective response to limit pathogen growth. To perform this role, many R proteins require the highly conserved chaperone molecule, heat shock protein (Hsp) 90. Using a two-dimensional gel/mass spectrometry system, we detected the association of the nucleotide-binding domain leucine-rich repeat protein Monarch-1 with heat shock proteins. Further analysis indicates that analogous to plant R proteins, Hsp90 is required for Monarch-1 activity. In human monocytes, Monarch-1 associates with Hsp90, and these complexes are sensitive to treatment with specific Hsp90 inhibitors. Disruption of these complexes results in rapid degradation of Monarch-1 via the proteasome and prevents Monarch-1-induced proteolysis of NF-kappaB-inducing kinase. This demonstrates that Hsp90 is a critical regulator of Monarch-1 anti-inflammatory activity.

Abstract: The immunomodulatory properties of peptidoglycan (PGN), a constituent of the bacterial cell wall, have been studied extensively but with contrasting results. Recent studies have demonstrated that the TLR2-mediated inflammatory responses elicited by Gram-positive PGN preparations are in fact a result of contaminating lipoproteins and lipoteichoic acid that can be removed only through sophisticated extraction procedures. Here, we report that commercial preparations of Staphylococcus aureus or Streptococcus pyogenes PGN are contaminated with bacterial superantigens (SAg). The T cell-derived cytokines IL-17A and IL-17F were induced by PGN preparations but not by TLR agonists or nucleotide-binding and oligomerization domain-like receptor agonists in human PBMC. IL-17 induction by PGN preparations was sensitive to protease digestion and required TCR signaling. Bacterial SAg could be detected by immunoblot in the PGN preparations, and purified recombinant SAg were powerful inducers of IL-17. Finally, the PGN preparations stimulated proliferation and expansion of T cells bearing specific TCR Vbeta elements. Our results suggest that a large body of literature that relied on commercial PGN preparations to study inflammatory diseases, such as arthritis, where IL-17 also plays an important role, should be interpreted with caution and possibly revisited. Future studies aimed at characterizing the activities of PGN should use PGN preparations of proven purity.

Abstract: Streptococcus pyogenes of the M1 serotype is commonly associated with large outbreaks of invasive streptococcal infections and development of streptococcal toxic shock syndrome (STSS). The pathogenesis behind these infections is believed to involve bacterial superantigens that induce potent inflammatory responses, but the reason why strains of the M1 serotype are over-represented in STSS is still not understood. In the present investigation, we show that a highly purified soluble form of the M1 protein from S. pyogenes, which lacks the membrane-spanning region, is a potent inducer of T cell proliferation and release of Th1 type cytokines. M1 protein-evoked T cell proliferation was HLA class II-dependent but not MHC-restricted, did not require intracellular processing and was Vbeta-restricted. Extensive mass spectrometry studies indicated that there were no other detectable proteins in the preparation. Taken together, our data demonstrate that soluble M1 protein is a novel streptococcal superantigen, which likely contributes to the excessive T cell activation and hyperinflammatory response seen in severe invasive streptococcal infections.

Abstract: The innate immune response plays a crucial role in satisfactory host resolution of bacterial infection. In response to chemotactic signals, neutrophils are early responding cells that migrate in large numbers to sites of infection. The recent discovery of secreted neutrophil extracellular traps (NETs) composed of DNA and histones opened a novel dimension in our understanding of the microbial killing capacity of these specialized leukocytes. M1 serotype strains of the pathogen Group A Streptococcus (GAS) are associated with invasive infections including necrotizing fasciitis (NF) and express a potent DNase (Sda1). Here we apply a molecular genetic approach of allelic replacement mutagenesis, single gene complementation, and heterologous expression to demonstrate that DNase Sda1 is both necessary and sufficient to promote GAS neutrophil resistance and virulence in a murine model of NF. Live fluorescent microscopic cell imaging and histopathological analysis are used to establish for the first time a direct linkage between NET degradation and bacterial pathogenicity. Inhibition of GAS DNase activity with G-actin enhanced neutrophil clearance of the pathogen in vitro and reduced virulence in vivo. The results demonstrate a significant role for NETs in neutrophil-mediated innate immunity, and at the same time identify a novel therapeutic target against invasive GAS infection.

Abstract: BACKGROUND: Group A streptococcal severe soft tissue infections, such as necrotizing fasciitis, are rapidly progressive infections associated with high mortality. Group A streptococcus is typically considered an extracellular pathogen, but has been shown to reside intracellularly in host cells. METHODS AND FINDINGS: We characterized in vivo interactions between group A streptococci (GAS) and cells involved in innate immune responses, using human biopsies (n = 70) collected from 17 patients with soft tissue infections. Immunostaining and in situ image analysis revealed high amounts of bacteria in the biopsies, even in those collected after prolonged antibiotic therapy. Viability of the streptococci was assessed by use of a bacterial viability stain, which demonstrated viable bacteria in 74% of the biopsies. GAS were present both extracellularly and intracellularly within phagocytic cells, primarily within macrophages. Intracellular GAS were predominantly noted in biopsies from newly involved tissue characterized by lower inflammation and bacterial load, whereas purely extracellular GAS or a combination of intra- and extracellular GAS dominated in severely inflamed tissue. The latter tissue was also associated with a significantly increased amount of the cysteine protease streptococcal pyrogenic exotoxin SpeB. In vitro studies confirmed that macrophages serve as reservoirs for viable GAS, and infection with a speB-deletion mutant produced significantly lower frequencies of cells with viable GAS following infection as compared to the wild-type bacteria. CONCLUSIONS: This is the first study to demonstrate that GAS survive intracellularly in macrophages during acute invasive infections. This intracellular presence may have evolved as a mechanism to avoid antibiotic eradication, which may explain our finding that high bacterial load is present even in tissue collected after prolonged intravenous antibiotic therapy. This new insight into the pathogenesis of streptococcal soft tissue infections highlights a need for alternative therapeutic strategies.

Abstract: Acute rejection after pancreas transplantation remains a significant problem and contributes to immunologic graft loss. No clinical markers of pancreas rejection have been universally accepted. Recent studies have identified several cytotoxic genes as possible markers of acute rejection in renal and islet cell transplant recipients. However, these markers of rejection have not been evaluated in pancreas transplant recipients. This study evaluated the differential expression of granzyme B, perforin, and human leukocyte antigen (HLA)-DRA in peripheral blood between patients with and without biopsy-proven pancreas rejection (n = 7 per group). Gene expression levels were analyzed using real time reverse transcriptase polymerase chain reaction assays. Expression of these genes in controls (n = 17) with and without type 1 diabetes was also analyzed. Patients with biopsy-proven pancreas rejection had higher levels of granzyme B, perforin, and HLA-DRA than patients who did not have rejection, although the difference was not statistically significant. Moreover, patients with biopsy-proven pancreas rejection had a significantly higher level of granzyme B than control subjects with type 1 diabetes (p <or= 0.03). Our findings suggest that, with additional evaluation of a larger number of patients as well as a longitudinal approach, analyses of granzyme B expression levels in peripheral blood may be shown to be a non-invasive method of monitoring pancreas allograft rejection.

Abstract: S-Adenosylmethionine (SAM, AdoMet) is the most important methyl donor used for synthesis of nucleic acids, phospholipids, creatine, and polyamines and for methylation of many bioactive molecules. The metabolic response of the lung to oxidative stress of hyperoxia requires increased RNA and protein synthesis for energy metabolism, growth arrest, and antioxidant defense. We studied the production of SAM and other aspects of methionine metabolism in lung epithelial cells exposed to hyperoxia. Human lung epithelial-like (A549) and primary small airway epithelial (SAE) cells were exposed to normoxia (21% O(2)) or hyperoxia (95% O(2)). Cell methionine and S-adenosylmethionine content increased in response to hyperoxia in SAE and A549 cells. Because methionine adenosyl transferase (MAT) is the rate-limiting enzyme of the pathway, we examined the expression of a lung epithelial isoform of MAT 2A in hyperoxia. Western blots revealed a novel MAT 2A isoform expressed in both cell types, with a lower molecular mass than that described in Jurkat cells. Cloning and sequencing of the MAT 2A cDNA revealed one silent nucleotide substitution compared to that expressed in Jurkat. The lower mass of MAT 2A in both lung epithelial cells indicated that the absence of the major posttranslational modification of MAT 2A found in Jurkat. MAT 2A protein progressively increased during hyperoxic exposure in both transformed and primary lung epithelium. Increased flux of (13)C-labeled methionine to S-adenosylhomocysteine (SAH) in A549 demonstrated that SAM's methyl group was utilized, and increased formation of cystathionine indicated that at least part of SAM generated was directed toward cysteine/GSH in the transsulfuration pathway. These results indicate activation of MAT 2A and the transmethylation pathway in the metabolic response to hyperoxia in lung epithelium.

Abstract: The globally disseminated Streptococcus pyogenes M1T1 clone causes a number of highly invasive human diseases. The transition from local to systemic infection occurs by an unknown mechanism; however invasive M1T1 clinical isolates are known to express significantly less cysteine protease SpeB than M1T1 isolates from local infections. Here, we show that in comparison to the M1T1 strain 5448, the isogenic mutant delta speB accumulated 75-fold more human plasmin activity on the bacterial surface following incubation in human plasma. Human plasminogen was an absolute requirement for M1T1 strain 5448 virulence following subcutaneous (s.c.) infection of humanized plasminogen transgenic mice. S. pyogenes M1T1 isolates from the blood of infected humanized plasminogen transgenic mice expressed reduced levels of SpeB in comparison with the parental 5448 used as inoculum. We propose that the human plasminogen system plays a critical role in group A streptococcal M1T1 systemic disease initiation. SpeB is required for S. pyogenes M1T1 survival at the site of local infection, however, SpeB also disrupts the interaction of S. pyogenes M1T1 with the human plasminogen activation system. Loss of SpeB activity in a subpopulation of S. pyogenes M1T1 at the site of infection results in accumulation of surface plasmin activity thus triggering systemic spread.

Abstract: Superantigens (SAgs) play an important role in the pathogenesis of severe invasive infections caused by Group A Streptococcus (GAS). We had shown earlier that the expression of streptococcal cysteine protease SpeB results in partial loss of the immune-stimulating activity of the native secreted GAS SAgs, namely the streptococcal pyrogenic exotoxins produced by the globally disseminated M1T1 GAS strain, associated with invasive infections worldwide. In this study, we examined the susceptibility of each of the M1T1 recombinant SAgs to degradation by rSpeB. Whereas SmeZ was degraded completely within 30 min of incubation with rSpeB, SpeG, and SpeA were more resistant and SpeJ was completely unaffected by the proteolytic effects of this protease. Proteomic analyses demonstrated that the order of susceptibility of the M1T1 SAgs to SpeB proteolysis is unaltered when they are present in a mixture that reflects their native physiological status. As expected, the degradation of SmeZ abolished its immune stimulatory activity. In silico sequence disorder and structural analyses revealed that SmeZ, unlike the three other structurally related SAgs, possesses a putative SpeB cleavage site within an area of the protein likely to be exposed to the surface. The study provides evidence for the effect of subtle structural differences between highly similar SAgs on their biological activity.

Abstract: Group A Streptococcus pyogenes causes a distinctive clinical disorder, streptococcal toxic shock syndrome, mediated by superantigenic bacterial exotoxins. Oncology patients with viridans group streptococcal sepsis frequently present with a streptococcal toxic shocklike syndrome of unclear pathogenesis. Viridans group streptococci isolated from pediatric oncology patients with streptococcal toxic shocklike illnesses do not possess homologs of known superantigen genes. Supernatants from cultures of these bacteria also fail to stimulate T-cell proliferation, suggesting these bacteria do not commonly elaborate superantigens. Adjunctive treatment with intravenous immunoglobulin, which is advantageous in streptococcal toxic shock syndrome, may not benefit these patients.

Abstract: The pathogen group A Streptococcus (GAS) produces a wide spectrum of infections including necrotizing fasciitis (NF). Streptolysin S (SLS) produces the hallmark beta-haemolytic phenotype produced by GAS. The nine-gene GAS locus (sagA-sagI) resembling a bacteriocin biosynthetic operon is necessary and sufficient for SLS production. Using precise, in-frame allelic exchange mutagenesis and single-gene complementation, we show sagA, sagB, sagC, sagD, sagE, sagF and sagG are each individually required for SLS production, and that sagE may further serve an immunity function. Limited site-directed mutagenesis of specific amino acids in the SagA prepropeptide supports the designation of SLS as a bacteriocin-like toxin. No significant pleotrophic effects of sagA deletion were observed on M protein, capsule or cysteine protease production. In a murine model of NF, the SLS-negative M1T1 GAS mutant was markedly diminished in its ability to produce necrotic skin ulcers and spread to the systemic circulation. The SLS toxin impaired phagocytic clearance and promoted epithelial cell cytotoxicity, the latter phenotype being enhanced by the effects of M protein and streptolysin O. We conclude that all genetic components of the sag operon are required for expression of functional SLS, an important virulence factor in the pathogenesis of invasive M1T1 GAS infection.

Abstract:

Abstract: The aim of this study was to identify the molecular signatures that are predictive of nonfunctional islet preparations. We examined functional outcomes of six islet preparations accepted for research purposes from human donors. Islet were maintained on culture in M-SFM media for 7 to 14 days then transplanted into NOD-SCID mice. At the time of transplant, RNA was extracted from a second aliquot of cultured islets for expression analysis. We also performed gene expression analysis using high-density Affymetrix U133A GeneChips on these preparations. Among 1833 genes selected, hierarchical clustering was performed using the GeneSpring software package (Silicon Genetics, Inc.), where 754 genes (higher in nonfunctional) and 177 genes (lower in nonfunctional) were differentially expressed with tight pattern of expression. Islets with low functionality showed high relative levels of expression of hypoxia-induced genes and increased frequency of expression of proinflammatory and proangiogenic genes, such as vascular endothelial growth factor. Conversely, nonfunctional islets had low levels of insulin-processing message. The general profile of these low-functionality islets shows attempted recovery from hypoxic assault and little effort directed toward insulin production and secretion. Further identification of the molecular signature of nonfunctional islets could allow the development of a potency assay for human transplantation.

Abstract: The recrudescence of severe invasive group A streptococcal (GAS) diseases has been associated with relatively few strains, including the M1T1 subclone that has shown an unprecedented global spread and prevalence and high virulence in susceptible hosts. To understand its unusual epidemiology, we aimed to identify unique genomic features that differentiate it from the fully sequenced M1 SF370 strain. We constructed DNA microarrays from an M1T1 shotgun library and, using differential hybridization, we found that both M1 strains are 95% identical and that the 5% unique M1T1 clone sequences more closely resemble sequences found in the M3 strain, which is also associated with severe disease. Careful analysis of these unique sequences revealed three unique prophages that we named M1T1.X, M1T1.Y, and M1T1.Z. While M1T1.Y is similar to phage 370.3 of the M1-SF370 strain, M1T1.X and M1T1.Z are novel and encode the toxins SpeA2 and Sda1, respectively. The genomes of these prophages are highly mosaic, with different segments being related to distinct streptococcal phages, suggesting that GAS phages continue to exchange genetic material. Bioinformatic and phylogenetic analyses revealed a highly conserved open reading frame (ORF) adjacent to the toxins in 18 of the 21 toxin-carrying GAS prophages. We named this ORF paratox, determined its allelic distribution among different phages, and found linkage disequilibrium between particular paratox alleles and specific toxin genes, suggesting that they may move as a single cassette. Based on the conservation of paratox and other genes flanking the toxins, we propose a recombination-based model for toxin dissemination among prophages. We also provide evidence that a minor population of the M1T1 clonal isolates have exchanged their virulence module on phage M1T1.Y, replacing it with a different module identical to that found on a related M3 phage. Taken together, the data demonstrate that mosaicism of the GAS prophages has contributed to the emergence and diversification of the M1T1 subclone.

Abstract: Ethical considerations constrain the in vivo study of human hemopoietic stem cells (HSC). To overcome this limitation, small animal models of human HSC engraftment have been used. We report the development and characterization of a new genetic stock of IL-2R common gamma-chain deficient NOD/LtSz-scid (NOD-scid IL2Rgamma(null)) mice and document their ability to support human mobilized blood HSC engraftment and multilineage differentiation. NOD-scid IL2Rgamma(null) mice are deficient in mature lymphocytes and NK cells, survive beyond 16 mo of age, and even after sublethal irradiation resist lymphoma development. Engraftment of NOD-scid IL2Rgamma(null) mice with human HSC generate 6-fold higher percentages of human CD45(+) cells in host bone marrow than with similarly treated NOD-scid mice. These human cells include B cells, NK cells, myeloid cells, plasmacytoid dendritic cells, and HSC. Spleens from engrafted NOD-scid IL2Rgamma(null) mice contain human Ig(+) B cells and lower numbers of human CD3(+) T cells. Coadministration of human Fc-IL7 fusion protein results in high percentages of human CD4(+)CD8(+) thymocytes as well human CD4(+)CD8(-) and CD4(-)CD8(+) peripheral blood and splenic T cells. De novo human T cell development in NOD-scid IL2Rgamma(null) mice was validated by 1) high levels of TCR excision circles, 2) complex TCRbeta repertoire diversity, and 3) proliferative responses to PHA and streptococcal superantigen, streptococcal pyrogenic exotoxin. Thus, NOD-scid IL2Rgamma(null) mice engrafted with human mobilized blood stem cells provide a new in vivo long-lived model of robust multilineage human HSC engraftment.

Abstract: The role played by host-pathogen interactions in regulation of expression of streptococcal virulence factors in vivo is beginning to become clear. We have reported that the expression of 2 streptococcal virulence factors, the streptococcal pyrogenic exotoxin (Spe) A and the cysteine protease SpeB, was reciprocally modulated during infection with Streptococcus pyogenes. To identify host signals mediating this reciprocal regulation, we cocultured clonal M1T1 isolates with human peripheral blood mononuclear cells (PBMCs). In accordance with our in vivo findings, when bacteria were in direct contact with human PBMCs or were separated in transwells, expression of speA was induced, whereas expression of speB was down-regulated. This phenomenon was mediated by transferrin and lactoferrin and was influenced by the iron-saturation status of these proteins. Iron chelation from media induced expression of speA, but to a much lesser degree than did that with apotransferrin and lactoferrin, suggesting additional effects of these ferrins on modulation of expression of speA and speB. Thus, ferrins may play an important role in host-pathogen interactions in skin and mucosal tissues.

Abstract: PURPOSE: Islet transplantation is limited by islet graft failure because of poor revascularization, host immune rejection, and nonspecific inflammatory response. Human vascular endothelial growth factor (hVEGF) gene delivery is likely to promote islet revascularization and survival. METHODS: We evaluated gene expression from a bicistronic plasmid encoding hVEGF and enhanced green fluorescent protein (EGFP) (pCMS-EGFP-hVEGF). Glucose responsiveness of islets was evaluated both in vitro and in vivo, and revascularization in islet graft was evaluated by immunohistochemistry. RESULTS: After transfection, hVEGF and EGFP expression levels were comparable with original monocistronic plasmids in Jurkat cells but higher and prolonged hVEGF expression in islets transfected with the bicistronic plasmid was observed, possibly as the result of differences in promoter strength and hypoxia response. The 3:1 w/w complexes showed little toxicity to islets at a dose of 5 microg DNA per 2000 islets. On glucose challenge, insulin release from transfected islets as well as secretion from islets after transplantation under the mouse kidney capsules in response to glucose stimulation, increased with time. Immunohistochemical staining of transplanted islets using mouse anti-human insulin, mouse anti-human von Willebrand factor, and rat anti-mouse CD31 antibodies suggests that islets are functional and there is new blood vessel formation. CONCLUSIONS: These findings suggest that transient hVEGF gene expression by the islets may promote islet revascularization and prolong islet survival after transplantation.

Abstract: A globally disseminated strain of M1T1 group A Streptococcus (GAS) has been associated with severe infections in humans including necrotizing fasciitis and toxic shock syndrome. Recent clinicoepidemiologic data showed a striking inverse relationship between disease severity and the degree to which M1T1 GAS express the streptococcal cysteine protease, SpeB. Electrophoretic 2-D gel analysis of the secreted M1T1 proteome, coupled with MALDI-TOF mass spectroscopy, revealed that expression of active SpeB caused the degradation of the vast majority of secreted GAS proteins, including several known virulence factors. Injection of a SpeB+/SpeA- M1T1 GAS strain into a murine subcutanous chamber model of infection selected for a stable phase-shift to a SpeB-/SpeA+ phenotype that expressed a full repertoire of secreted proteins and possessed enhanced lymphocyte-stimulating capacity. The proteome of the SpeB-in vivo phase-shift form closely matched the proteome of an isogenic speB gene deletion mutant of the original M1T1 isolate. The absence or the inactivation of SpeB allowed proteomic identification of proteins in this M1T1 clone that are not present in the previously sequenced M1 genome including SpeA and another bacteriophage-encoded novel streptodornase allele. Further proteomic analysis of the M1T1 SpeB+ and SpeB- phase-shift forms in the presence of a cysteine protease inhibitor demonstrated differences in the expression of several proteins, including the in vivo upregulation of SpeA, which occurred independently of SpeB inactivation.

Abstract: BACKGROUND: Culturing human islets in Memphis serum-free media (M-SFM) is associated with excellent postculture recovery, in vitro function, and in vivo survival. The authors investigate the possibility of preserving islet function for extended periods (6 months) in culture and describe the in vitro and in vivo functional outcomes associated with these extended culture times. METHODS: Human islets isolated from three cadaveric donor organs were cultured in M-SFM for 1, 3, or 6 months before transplantation under the kidney capsule of nonobese diabetic (NOD)-severe combined immunodeficiency (SCID) mice. In vitro function was measured by static incubation at the time of transplantation. In vivo function was assessed by measuring human insulin and C-peptide production, and by the ability of 6-month cultured islets to cure streptozotocin-induced diabetes in this mouse model. RESULTS: Islet recovery ratios after 1 month in culture ranged from 85% to 88% and declined to 28% to 53% after 6 months of culture (P <0.01). Insulin stimulation indices did not differ among the fresh or the 6-month cultured preparations. All preparations cultured for 1 to 3 months functioned in the NOD-SCID mice. After 6 months of culture, two of the three preparations demonstrated in vivo function and were able to cure streptozotocin-induced diabetes. CONCLUSIONS: These data demonstrate that human islets can be cultured in M-SFM for extended periods and still retain in vitro and in vivo function and the ability to cure experimental diabetes. The ability to maintain islets in culture for prolonged periods is an important step toward the development of islet tissue repositories and distribution centers.

Abstract: The goal of the Complex Trait Consortium is to promote the development of resources that can be used to understand, treat and ultimately prevent pervasive human diseases. Existing and proposed mouse resources that are optimized to study the actions of isolated genetic loci on a fixed background are less effective for studying intact polygenic networks and interactions among genes, environments, pathogens and other factors. The Collaborative Cross will provide a common reference panel specifically designed for the integrative analysis of complex systems and will change the way we approach human health and disease.

Abstract: The purpose of this study was to evaluate the utility of nondiabetic immune-deficient NOD-SCID mouse model in assessing the functional capacity of isolated human islets. We transplanted 2000 islet equivalents obtained from six preparations used for human islet transplantation in three patients under the kidney capsule of groups of 10 mice. Human (Hu) C-peptide and insulin levels were determined following intraperitoneal (i.p.) glucose challenge at days 0, 7, 14, 21, 30, 60, 90, and 120. The Hu C-peptide level >1.5 ng/mL was the threshold for islet function in this model. The first patient did not achieve insulin independence and had minimal (0.5 ng/mL) fasting C-peptide levels that mirrored the low C-peptide levels observed in the mice. After the first infusion, the insulin requirements were reduced by 50% in the second patient. She became insulin free 10 days after her second infusion with a C-peptide level of 3.0 ng/mL, which corresponded to the peak C-peptide level (3.9 ng/mL) observed in the mice. By 150 days' posttransplant, the decline in C-peptide level paralleled the decline observed in mice. Within 2 weeks after the first transplant, insulin dose was reduced by 75% in the third patient, which corresponded to the robust C-peptide production in mice (7.3 ng/mL). Both patient and mice had a delay in islet function following the second infusion. She remained with a C-peptide level of 1.8 ng/mL and insulin free until suffering a rejection episode 3 months later. We observed that human islet graft function in NOD-SCID mice correlated with clinical response in islet transplant recipients.

Abstract: The M1T1 strain remains the most frequently isolated strain from group A streptococcal (GAS) infection cases worldwide. We previously reported that M1T1 differs from the fully sequenced M1 SF370 strain. To better understand the reason for the persistence and increased virulence of M1T1, we analysed its secreted proteome and identified two virulence proteins that are not present in the sequenced M1 SF370 strain: streptococcal pyrogenic exotoxin A (SpeA) and a streptodornase D (SdaD) homologue. In the present study, we determined the nucleotide sequence of the M1T1 streptodornase and found that its deduced amino acid sequence is highly similar to other streptococcal streptodornases, and is most closely related to the SdaD of GAS strain M49. M1T1 Sda shares two highly conserved domains with several DNases and putative DNases in streptococci; however, it possesses a unique C-terminal amino acid sequence. Thus, we named the protein Sda1, and we detected the presence of the sda1 gene in 16 M1T1 clinical isolates. The cloned and expressed Sda1 degrades both streptococcal and mammalian DNA at physiological pH. Amino acid similarity analyses of known GAS deoxyribonucleases suggest that Sda1 may be a chimeric protein created through recombination events. Moreover, a natural mutation that resulted in longer Sda1 and SdaD as compared to other GAS DNases was found to confer increased activity on the protein. Analysis of the sequences flanking sda1 determined that it is carried by a prophage or a prophage-like element inserted in the tRNA-Ser gene of M1T1 GAS. Ongoing studies in our laboratory aim to determine the contribution of Sda1 to the virulence of this globally disseminated M1T1 strain.

Abstract: Islet transplantation is limited by islet graft failure due to poor revascularization, host immune rejection and nonspecific inflammatory response. Delivery of human vascular endothelial growth factor (hVEGF) gene to the islets is likely to promote islet revascularization and survival. We used a bicistronic adenoviral vector encoding hVEGF and CpG-free allele of green fluorescent protein (Adv-GFP-hVEGF) and introduced into human pancreatic islets by transfection. We found that transfection efficiency and apoptosis were dependent on the multiplicity of infection (MOI). Compared to Adv-GFP transfected and nontransfected islets, the levels of hVEGF secreted from Adv-GFP-hVEGF transfected islets were higher and exhibit a linear relationship between hVEGF expression and MOI (10-5000). Persistent, but low level expression of hVEGF from nontransfected islets was also observed. This may be due to expression of the endogenous hVEGF gene under hypoxic conditions. The levels of DNA fragmentation determined by ELISA of islet lysates were dependent on the MOI of Adv-GFP-hVEGF. On glucose challenge, insulin release from transfected islets was comparable to nontransfected islets. Immunohistochemical staining for hVEGF was very high in Adv-GFP-hVEGF transfected islets. Weak staining was also observed for hCD31 in both transfected and nontransfected islets. These findings suggest that Adv-GFP-hVEGF is a potential candidate for promoting islet revascularization.

Abstract: Streptococcal pyrogenic exotoxin (Spe) B, a streptococcal cysteine protease, is believed to be important in group A streptococcal (GAS) pathogenesis. The present study examined the effect of SpeB on the activity of superantigenic exotoxins secreted by M1T1 GAS isolates. The proliferative response of human lymphocytes to culture supernatant (SUP) from an SpeB(+) isolate increased significantly (P<.05) when the isolate was grown with N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]-agmatine, a cysteine protease inhibitor. The lymphocyte-stimulating activity of SUP from a spontaneous SpeB(-) variant or SpeB(-) knockout (DeltaSpeB) mutant was also significantly higher than that of SUP from the SpeB(+) parent isolate (P<.001). The addition of recombinant SpeB to the DeltaSpeB mutant reduced the lymphocyte response to a level comparable to that with the SpeB(+) isolate. SpeB affected superantigens that stimulate cells expressing T cell receptor Vbeta (TCRBV)-4, TCRBV7, and TCRBV8 but not those that stimulate TCRBV2. SpeB has a selective proteolytic effect on GAS superantigens.

Abstract: BACKGROUND: Lung injury in severe acute pancreatitis is mediated by infiltrating leukocytes. Our laboratory has previously demonstrated that acute lung injury in acute pancreatitis results in an up-regulation of vascular adhesion molecule-1 (VCAM-1) cell surface receptor expression on pulmonary vascular endothelium and neutrophil sequestration. The objective of this study was to determine whether blocking expression of VCAM-1 in acute pancreatitis would modify acute pulmonary injury. METHODS: Young female mice were fed a choline-deficient ethionine (CDE) supplemented diet to induce acute pancreatitis. After initiation of the diet, one group (acute pancreatitis treated [n = 18]) was treated with blocking doses (2.35 mg/kg) of monoclonal anti-VCAM-1 receptor antibody (Ab) at 48, 96, and 120 hours. A second group (acute pancreatitis treated control [n = 5]) was treated with a similar dose of an isotypic control for VCAM-1 (nonbinding Ab) at the same time points. A third group (acute pancreatitis untreated [n = 12]) received a CDE diet, and a fourth group (control [n = 11]) received standard food with no Ab treatment. All animals were killed at 144 hours. The dual radiolabeled monoclonal Ab method was used to quantitate VCAM-1 cell surface expression in lung tissue. Lung injury was assessed histologically, and apoptosis was detected by transferase-mediated deoxyuridine triphosphate nick end labeling assay. Pulmonary leukocyte sequestration was determined by myeloperoxidase (MPO) assay and CD18 staining. RESULTS: Pulmonary VCAM-1 cell surface expression was significantly increased in animals with acute pancreatitis when compared to controls (P <.001) and was reduced to near control levels in acute pancreatitis treated animals. On histologic examination, treated animals with acute pancreatitis exhibited significantly less lung injury and apoptosis than did untreated animals with acute pancreatitis. Leukocyte sequestration and MPO activity were significantly reduced in the treated animals with pancreatitis compared to untreated animals with pancreatitis (P <.0001) or acute pancreatitis treated controls (P <.03). CONCLUSIONS: Blocking VCAM-1 on pulmonary vascular endothelium decreases leukocyte adherence and recruitment into the lung, hence reducing lung injury in severe acute pancreatitis. Clinically, VCAM-1 antagonism may be an important adjunct to evolving therapy for distant organ injury in severe acute pancreatitis.

Abstract: Transplantation of pancreatic islets has great potential for treating Type I diabetes. Ex vivo gene therapy may promote re-vascularization or inhibit apoptosis of the islets and promote graft. In this study, we investigated the feasibility of non-viral gene delivery using Enhanced Green Fluorescent Protein (EGFP) and human Vascular Endothelial Growth Factor (hVEGF(165)) expression plasmids as model reporter and therapeutic genes. LipofectAMINE/pDNA and Superfect/pDNA complexes showed high transfection efficiency in rapidly dividing Jurkat cells, but low transfection in non-dividing human islets. LipofectAMINE/pCAGGS-hVEGF transfected islets showed relatively higher levels of hVEGF than in those transfected with LipofectAMINE/pCMS-EGFP complexes or 5% glucose. To exclude endogenously secreted hVEGF, real time RT-PCR experiment was repeated using pCAGGS vector-specific forward primer and hVEGF gene-specific reverse primer. In this case, both non-transfected islets and the islets transfected with LipofectAMINE/pCMS-EGFP complexes showed negligible amplification of hVEGF. On glucose challenge, insulin release from LipofectAMINE/pCAGGS-hVEGF transfected human islets increased from 10.78 +/- 4.56 to 65 +/- 5 ng/ml, suggesting little adverse effect on islet beta cell response to glucose challenge. The low transfection efficiency is due to the islets being a cluster of approximately 1000 non-dividing cells. This underscores the importance of experimentation with the actual human islets.

Abstract: Variability in CYP3A (CYP3A4/5) and P-glycoprotein (human MDR1 gene product) activity underlies interindividual differences in oral cyclosporine (CsA) bioavailability. Racial differences in polymorphic expression of CYP3A5 and MDR1 may explain observed interracial variability in oral bioavailability. Our objective was to evaluate the effect of CYP3A5 and MDR1 polymorphic expression on CsA oral disposition. Steady-state plasma concentration profiles (n = 19) were sampled in renal transplant recipients receiving concentration-adjusted CsA maintenance therapy. CsA plasma concentrations were measured by fluorescence polarization immunoassay. CYP3A5 and MDR1 genotypes were determined by real-time polymerase chain reaction. Noncompartmental pharmacokinetic analysis and nonlinear mixed-effects modeling (NONMEM) were performed to assess the effect of genotype on CsA pharmacokinetics. MDR1 C3435T genotype was identified as the best predictor of CsA systemic exposure. CsA oral clearance was significantly higher in subjects who carried at least one 3435T allele compared to homozygous wild-type individuals (40.0 +/- 2.2 vs. 26.4 +/- 3.1 L/h, p = 0.007). MDR1 C3435T genotype accounted for 43% of the interindividual variability of CsA oral clearance in the study population after accounting for interoccasion variability. The authors were unable to independently assess whether CYP3A5 correlated with any CsA pharmacokinetic parameter since all CYP3A5 nonexpressors were also 3435T allele carriers. MDR1 3435T allele carriers have enhanced oral clearance compared to individuals with the CC genotype. The frequency of the 3435T allele is lower in African Americans compared to Caucasians. Thus, the MDR1 C3435T genotype offers a potential mechanistic basis to explain interracial differences in CsA oral bioavailability. Further studies are needed to explore the relationship between CYP3A5 and MDR1 genotype and phenotype.

Abstract: The role of host genetic factors in determining susceptibility to infections has become more evident. Certain individuals appear to be predisposed to certain infections, whereas others are protected. By studying the immune response and the genetic makeup of susceptible and resistant individuals a better understanding of the disease process can be achieved. Infections caused by group A streptococci offer an excellent model to study host-pathogen interactions and how the host genetic variation can influence the infection outcome. These studies showed that the same clone of these bacteria can cause severe or non-severe invasive disease. This difference was largely related to the human leukocyte antigen class 11 type of the patient. Certain class II haplotypes present the streptococcal superantigens in a way that results in responses, whereas others present the same superantigens in a way that elicits very potent inflammatory responses that can lead to organ failure and shock. These findings underscore the role of host genetic factors in determining the outcome of serious infections and warrants further investigations into how the same or different genetic factors affect susceptibility to other emerging and re-emerging pathogens.

Abstract: The objective of this study was to develop a real-time polymerase chain reaction (PCR) method to detect MDR1 (human multidrug resistance gene) single nucleotide polymorphisms (SNPs) C3435T and G2677T. C3435T and G2677T are linked to MDR1*2, which is associated with enhanced efflux activity in vitro. Using the Smart Cycler, an allele-specific real-time PCR-based genotyping method was developed to detect C3435T and G2677T. The MDR1 genotype of human genomic DNA templates was determined by direct DNA sequencing. PCR reactions for genotyping C3435T and G2677T by using allele-specific primers were conducted in separate tubes. An additional nucleotide mismatch at the third position from the 3' end of each allele-specific primer was used to abrogate nonspecific PCR amplification. The fluorescence emitted by SYBR Green I was monitored to detect formation of specific PCR products. PCR growth curves exceeding the threshold cycle were considered positive. Fluorescence melt-curve analysis was used to corroborate results from PCR growth curves. Using PCR growth curves, our assay accurately determined hetero- and homozygosity for C3435T and G2677T. Genotype assignments based on PCR growth curve, melt-curve analysis, agarose gel electrophoresis, and direct DNA sequencing results of PCR products were in perfect agreement. We have developed a rapid MDR1 genotyping method that can be used to assess the contribution of MDR1*2 to pharmacokinetic and pharmacodynamic variability of P-glycoprotein substrates.

Abstract: Superantigens have been implicated as pivotal mediators of severe invasive group A streptococcal (GAS) infections, by virtue of their potent immunostimulatory activity. HLA polymorphism has been suggested to influence the susceptibility to severe invasive GAS infection. Here we studied the influence of allelic and isotypic variation of HLA class II molecules on GAS superantigen-induced immune responses using cells derived from patients with bare lymphocyte syndrome, untransfected or transfected with various HLA class II alleles. Significantly higher proliferative responses were detected when streptococcal pyrogenic exotoxin (Spe) A was presented by cells expressing DQA1*0101/DQB1*0302 (DQ3.2), as compared to cells expressing DR1, DR4, or DR5 alleles (p=0.0002-0.01). In contrast to SpeA, SpeC was preferentially presented by DR4 as compared to DQB1*03 (p=0.04). In agreement with the proliferation results, a significantly higher frequency of IL-2-, TNF-alpha-, TNF-beta-, and IFN-gamma-producing cells was detected when SpeA was presented by HLA class II DQB1*03 alleles as compared to DR4 (p=0.0002-0.04). Binding experiments showed a high affinitybinding of SpeA to both class II DR4 and DQB1*0302, and there was no significant difference in SpeA binding affinity between the two alleles. The data confirm the effect of allelic polymorphism in superantigen responses and show that different superantigens are preferentially presented by distinct class II alleles.

Abstract: BACKGROUND: The quest for noninvasive methods to diagnose rejection in solid-organ transplants has been rejuvenated by recent observations that specific cytotoxic T-cell markers are up-regulated during rejection. METHODS: We developed a one-step real-time polymerase chain reaction (PCR) method allowing reliable detection of the expression of several T-cell genes within a relatively short period of time. The assay is highly sensitive and reproducible with a wide dynamic range allowing accurate quantification of target mRNA in as little as 3 pg total RNA. The utility of this assay in detecting renal allograft rejection was evaluated. Peripheral blood mononuclear cells were collected from 27 patients undergoing kidney allograft biopsies for renal dysfunction after transplantation. Expression of the T-cell activation markers, granzyme B, perforin, and HLA-DRA, was quantified and correlated to the histopathologic changes in the renal biopsies. RESULTS: In cases with allograft rejection (n=8), peripheral lymphocyte expression was increased for granzyme B (P <0.001) and perforin (P <0.08) compared with cases without rejection (n=19). Granzyme B mRNA up-regulation showed the highest specificity for detecting rejection (95%). Moreover, HLA-DRA mRNA was significantly up-regulated (P <0.0016) and had the highest sensitivity (88%) detecting rejection. The up-regulation of both granzyme B and HLA-DRA was most specific in detecting rejection, P<0.001. CONCLUSIONS: These data demonstrate that a rapid test of target gene up-regulation using real-time PCR can be used as an aid in the diagnosis of kidney allograft rejection. This is also the first report on the possible utility of HLA-DRA mRNA up-regulation as a marker for kidney transplant rejection.

Abstract: A novel water-soluble lipopolymer was synthesized by linking cholesteryl chloroformate to the secondary amino groups of branched poly(ethylenimine) (PEI) of 1,800 and 10,000 Da. Conjugation through PEI secondary amines gives this newly synthesized lipopolymer (abbreviated as PEI-Chol) special advantage over our previously synthesized lipopolymers, which utilized the primary amino groups for conjugation, as the primary amino groups have a significant role in DNA condensation. Also, significantly, only one cholesterol molecule was grafted onto each PEI molecule (confirmed by (1)H NMR and MALDI-TOF mass spectrometry), leaving enough space for the steric interactions of the PEI's primary amines with the DNA. The PEI-Chol lipopolymer was characterized for the critical micellar concentration (cmc), buffer capacity, DNA condensation (by band retardation and circular dichroism), in vitro transfection efficiency, and cell viability. The cmcs of PEI-Chol 1,800 and PEI-Chol 10,000 were 496.6 and 1,330.5 microg/mL, respectively. The acid-base titration indicated high buffering capacity of the polymers around the pH range of 5-7, which indicated their potential for buffering in the acidic pH environment of the endosomes. The band retardation studies indicated that efficient condensation of the plasmid DNA could be achieved using these lipopolymers. The circular dichroism spectra indicated a change in DNA conformation and adoption of lower energy state upon condensation with these lipopolymers when an N/P ratio of 2.5/1 or above was formulated. The mean particle size of these complexes was in the range 110-205 nm, except for the complexes prepared using PEI of 1,800 Da, which had a mean particle size of 384 +/- 300 nm. The zeta potential of DNA complexes prepared using PEI-Chol 1,800, PEI-Chol 10,000 and PEI of 1,800, 10,000, and 25,000 Da at an N/P ratio of 15/1 was in the range 23-30 mV and was dependent on the N/P ratios. The in vitro transfection of PEI-Chol/pCMS-EGFP complexes in Jurkat cells showed high levels of expressed Green Fluorescent Protein (GFP) with little toxicity as determined by flow cytometry. These novel water-soluble lipopolymers provided good transfection efficiency with other desirable characteristics such as water solubility, free primary amino groups for efficient DNA condensation and high buffer capacity that indicated the possibility of efficient endosomal release.

Abstract: The role of host genetic factors in conferring predisposition or protection in infectious diseases has become evident. Infection with group A streptococci causes a wide spectrum of disease ranging from pharyngitis to streptococcal toxic shock syndrome. The release of inflammatory cytokines triggered by streptococcal superantigens has a pivotal role in invasive streptococcal disease. However, individuals infected with the same strain can develop very different manifestations. We report here that the immunogenetics of the host influence the outcome of invasive streptococcal infection, and demonstrate the underlying mechanism for these genetic associations. Specific human leukocyte antigen class II haplotypes conferred strong protection from severe systemic disease, whereas others increased the risk of severe disease. Patients with the DRB1*1501/DQB1*0602 haplotype mounted significantly reduced responses and were less likely to develop severe systemic disease (P < 0.0001). We propose that human leukocyte antigen class II allelic variation contributes to differences in severity of invasive streptococcal infections through their ability to regulate cytokine responses triggered by streptococcal superantigens.

Abstract: Infections with Group C Streptococci can lead to severe disease, particularly in individuals with underlying illnesses such as cardiovascular disease, malignancy or immunosuppression. We report the first case of rhabdomyolysis and disseminated intravascular coagulation secondary to Group C Streptococcus in a previous healthy male. A toxic shock-like syndrome associated with Group C and Group G Streptococci has been reported. However, unlike with Group A Streptococci, production of endotoxins by these organisms is less well defined. We tested the patient's isolate for its ability to produce superantigenic toxins and to induce a mitogenic response. Although it is not known whether Group C Streptococci require special growth conditions for the production of superantigens, we could not demonstrate either the production of exotoxins or the induction of a mitogenic response.

Abstract: OBJECTIVE: To determine whether blocking the cell surface expression of intracellular adhesion molecules (ICAM-1) in established severe acute pancreatitis (AP) would ameliorate pulmonary injury. SUMMARY BACKGROUND DATA: Lung injury in AP is in part mediated by infiltrating leukocytes, which are directed to lung tissue by ICAM-l. The authors' laboratory has previously demonstrated that AP results in overproduction of inflammatory cytokines, upregulation of pulmonary ICAM-1 expression, and a concomitant infiltration of neutrophils, which results in lung injury. METHODS: Young female mice were fed a choline-deficient/ethionine-supplemented diet to induce AP and were treated with a blocking dose of monoclonal antibody specific to the ICAM-1 receptor. Antibody treatment was administered at 72, 96, and 120 hours after beginning the diet, and all animals were killed at 144 hours. The degree of pancreatitis was evaluated by serum biochemical and tumor necrosis factor alpha levels as well as histology. The dual radiolabeled monoclonal antibody method was used to quantitate ICAM-1 cell surface expression in pulmonary tissue. Lung injury was assessed histologically and by determining lung microvascular permeability by measuring accumulated 125I-radiolabeled albumin. Pulmonary neutrophil sequestration was determined by the myeloperoxidase assay. RESULTS: All mice developed severe AP, and pancreatic injury was equally severe in both treated and untreated groups. Pulmonary ICAM-1 expression was significantly upregulated in animals with AP compared with controls. Treatment with a blocking dose of anti-ICAM-1 antibody after the induction of AP resulted in inhibited ICAM-1 cell surface expression to near control levels. Compared to untreated animals with AP, mice treated with anti-ICAM-1 mice had significantly reduced histologic lung injury and neutrophil sequestration, and a decreased microvascular permeability by more than twofold. CONCLUSIONS: These results demonstrate for the first time that treatment targeting the cell surface expression of ICAM-1 after the induction of AP ameliorates pulmonary injury, even in the face of severe pancreatic disease.

Abstract: Mammalian methionine adenosyltransferase II (MAT II) consists of a catalytic alpha2/alpha2' and a regulatory beta subunit. Up-regulation of alpha2 subunit expression is associated with increased intracellular levels of S-adenosylmethionine, the major methyl group donor and a key compound in cell metabolism and polyamine synthesis. Previous studies have shown that expression of the alpha2 subunit is differentially regulated in normal and malignant cells. To delineate the molecular basis for the differential regulation of alpha2 subunit expression, we cloned and characterized the human MAT2A gene and its promoter and defined regions that contain enhancer and repressor elements. Detailed functional characterization of the proximal promoter of the MAT2A gene revealed the formation of three major protein-DNA complexes with probes containing three Sp1 sites (Sp1-1 at -14, Sp1-2 at -47, and Sp1-3 at -69). Competition with a probe copying sequence between -76 and -54, which contains the Sp1-3 site only, or mutation of this site, abolished complex formation. Furthermore, mutation of the Sp1-3 site, but not the Sp1-1 or Sp1-2 sites, inhibited the in vivo promoter activity by approximately 85%. Supershift assays showed that the transcription factors Sp2 and Sp3 are part of the complexes formed at the Sp1-3 site, and that Sp1 does not appear to be directly involved. The data indicate that complex formation is initiated at site Sp1-3, which appears to be essential for promoter activity. However, other regions of the proximal promoter may also contribute to the regulation of MAT2A gene expression. These studies may lead to the delineation of the molecular basis for the differential regulation of MAT2A expression in normal and leukemic T cells.

Abstract: Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine (AdoMet), a key molecule in transmethylation reactions and polyamine biosynthesis. The MAT II isozyme consists of a catalytic alpha2 and a regulatory beta subunit. Down-regulation of the MAT II beta subunit expression causes a 6-10-fold increase in intracellular AdoMet levels. To understand the mechanism by which the beta subunit expression is regulated, we cloned the MAT2B gene, determined its organization, characterized its 5'-flanking sequences, and elucidated the in vitro and in vivo regulation of its promoter. Transcription of the MAT2B gene initiates at position -203 relative to the translation start site. Promoter deletion analysis defined a minimal promoter between positions +52 and +93 base pairs, a GC-rich region. Inclusion of the sequences between -4 and +52 enhanced promoter activity; this was primarily because of an Sp1 recognition site at +9/+15. The inclusion of sequences up to position -115 provided full activity; this was attributed to a TATA at -32. The Sp1 site at position +9 was key for the formation of protein.DNA complexes. Mutation of both the Sp1 site at +9 and the TATA at -32 reduced promoter activity to its minimal level. Supershift assays showed no effect of the anti-Sp1 antibody on complex formation, whereas the anti-Sp3 antibody had a strong effect on protein.DNA complex formation, suggesting that Sp3 is one of the main factors binding to this Sp1 site. Chromatin immunoprecipitation assays supported the involvement of both Sp1 and Sp3 in complexes formed on the MAT2B promoter. The data show that the 5'-untranslated sequences play an important role in regulating the MAT2B gene and identifies the Sp1 site at +9 as a potential target for modulating MAT2B expression, a process that can have a major effect on intracellular AdoMet levels.

Abstract: The metabolism of S-adenosylmethionine (AdoMet), a key molecule in regulating T cell differentiation and proliferation, is different in normal and leukemic T cells. To delineate the basis for these differences we studied the transcriptional regulation of human methionine adenosyltransferase II (MAT II), which catalyzes AdoMet synthesis in these cells. Recently, we identified an Sp1 site in the proximal promoter of the MAT2A gene, which encodes the alpha2 catalytic subunit of MAT II, that is essential for the in vitro and in vivo promoter activity in Jurkat leukemic T cells, and that involves binding of the nuclear factors Sp2 and Sp3, but not Sp1. Here, the in vitro and in vivo activity of the proximal MAT2A promoter in normal resting, PHA-stimulated, and leukemic human T cells was compared. Significantly different patterns of protein factor interaction in the proximal region of the MAT2A promoter were found. Normal resting and activated T cells produced complexes of significantly lower molecular weight than those formed in leukemic T cells. Supershift studies coupled with analysis of proteins bound to the proximal promoter suggest that low levels of expression of Sp2 and Sp3 in normal T cells may be responsible for the difference in the in vitro promoter activity between normal and leukemic cells. Mutation of the key Sp1 site equally reduced the in vivo promoter activity in normal and malignant T cells; by contrast, it had significantly different effects on protein-DNA interactions in normal and leukemic T cells. Together, the data support the idea that differences in protein-DNA interactions may contribute to significant differences in MAT2A regulation in normal and malignant cells.

Abstract: The streptococcal pyrogenic exotoxins (Spes) play a central role in the pathogenesis of invasive group A streptococcal (GAS) infections. The majority of recent invasive GAS infections have been caused by an M1T1 strain that harbors the genes for several streptococcal superantigens, including speA, speB, speF, speG, and smeZ. However, considerable variation in the expression of Spe proteins among clonal M1 isolates has been found, and many of the speA-positive M1 strains do not produce detectable amounts of SpeA in vitro. This study was designed to test the hypothesis that speA gene expression can be induced in vivo. A mouse infection chamber model that allows sequential sampling of GAS isolates at various time points postinfection was developed and used to monitor the kinetics of Spe production in vivo. Micropore Teflon diffusion chambers were implanted subcutaneously in BALB/c mice, and after 3 weeks the pores became sealed with connective tissue and sterile fluid containing a white blood cell infiltrate accumulated inside the infection chambers. Representative clonal M1T1 isolates expressing no detectable SpeA were inoculated into the implanted chambers, and the expression of SpeA in the aspirated aliquots of the chamber fluid was analyzed on successive days postinfection. Expression of SpeA was detected in the chamber fluid as early as days 3 to 5 postinfection in most animals, with a significant increase in expression by day 7 in all infected mice. Isolates recovered from the chamber and grown in vitro continued to produce SpeA even after 21 passages in vitro, suggesting stable switch on of the speA gene. A temporal relation between the upregulation of SpeA expression and the downregulation of SpeB expression was observed in vivo. These data suggest that in vivo host and/or environmental signals induced speA gene expression and suppressed speB gene expression. This underscores the role of the host-pathogen interaction in regulating the expression of streptococcal virulence factors in vivo. The model described here should facilitate such studies.

Abstract: BACKGROUND: Difficulties with recovering and preserving pancreatic islets have hampered progress in islet transplantation. In previous in vitro studies, our laboratory successfully demonstrated that using serum-free medium for prolonged pancreatic islet culture allows postculture recovery ratios greater than those obtained with standard media with sustained in vitro islet function. The goal of this study was to determine whether culturing of islets in a modified serum-free medium (M-SFM) would sustain function in vivo. METHODS: Islets were isolated from pancreata procured from 12 cadaveric organ donors and cultured in the M-SFM for up to 2 months, cryopreserved at -70 degrees C within 1-3 days of isolation for 2 months, or placed in short-term culture (3-5 days) before their transplantation under the kidney capsule of nonobese diabetic-severe combined immunodeficient mice (n=4-7 per group/time point). In vivo islet function was assessed by measuring the production of human insulin and C-peptide over a period of 3-15 months. RESULTS: After extended culture of islets in M-SFM for 1 or 2 months, transplanted islets maintained their viability, and in some instances in vivo function improved when compared with short-term cultured islets transplanted from the same preparation (P<0.01). Improvement was particularly evident for islets cultured for 1 month. Furthermore, when compared with cryopreserved preparations, early function (postoperative day 7) of islets from 1-month culture preparations was statistically better (P<0.05). Prolonged culture in M-SFM had no significant impact on long-term function, inasmuch as cultured islets functioned for more than 120 days. CONCLUSION: These data demonstrate that prolonged islet culture in M-SFM sustained viability and function, and in some instances had a positive effect on in vivo islet function, particularly in the 1-month cultures. No negative effect on long-term in vivo function was demonstrated in this study. Confirmation in clinical models utilizing extended (1-2 months) islet culture in M-SFM could significantly enhance islet transplantation by allowing the identification of best-matched recipients, pretransplantation recipient conditioning, and possible pretransplantation islet modifications to promote engraftment and prolonged graft function.

Abstract: This paper reports clinical and metabolic studies of two Italian siblings with a novel form of persistent isolated hypermethioninaemia, i.e. abnormally elevated plasma methionine that lasted beyond the first months of life and is not due to cystathionine beta-synthase deficiency, tyrosinaemia I or liver disease. Abnormal elevations of their plasma S-adenosylmethionine (AdoMet) concentrations proved they do not have deficient activity of methionine adenosyltransferase I/III. A variety of studies provided evidence that the elevations of methionine and AdoMet are not caused by defects in the methionine transamination pathway, deficient activity of methionine adenosyltransferase II, a mutation in methylenetetrahydrofolate reductase rendering this activity resistant to inhibition by AdoMet, or deficient activity of guanidinoacetate methyltransferase. Plasma sarcosine (N-methylglycine) is elevated, together with elevated plasma AdoMet in normal subjects following oral methionine loads and in association with increased plasma levels of both methionine and AdoMet in cystathionine beta-synthase-deficient individuals. However, plasma sarcosine is not elevated in these siblings. The latter result provides evidence they are deficient in activity of glycine N-methyltransferase (GNMT). The only clinical abnormalities in these siblings are mild hepatomegaly and chronic elevation of serum transaminases not attributable to conventional causes of liver disease. A possible causative connection between GNMT deficiency and these hepatitis-like manifestations is discussed. Further studies are required to evaluate whether dietary methionine restriction will be useful in this situation.

Abstract: Host-pathogen interactions were studied in tissue biopsy samples from patients with severe invasive group A streptococcus (GAS) infections. Skin, subcutaneous tissue, and fascia biopsy samples were divided into clinical grade 1 (no evidence of inflammation [n=7]) or clinical grade 2 (inflamed tissue--erythema and edema including cellulitis, fasciitis, and necrotizing fasciitis [n=24]). In situ imaging demonstrated significantly higher bacterial load in biopsy samples of higher clinical grade (P<.05), and the bacterial load correlated with the in vivo expression of the superantigen streptococcal pyrogenic exotoxin F (P<.02). Increased expression of the interleukin-1 cytokines and significantly higher expression of tumor necrosis factor-beta, interferon-gamma, and the homing receptors CC chemokine receptor 5, CD44, and cutaneous lymphocyte-associated antigen (P<.002-.05) were observed in biopsy samples of higher clinical grade. Thus, the cytokine profile at the local site of infection mimics that of a typical superantigen cytokine response. The findings of this study demonstrate a critical role for superantigens and Th1 cytokines in GAS tissue infections.

Abstract: Lung injury is a major cause of patient morbidity in acute pancreatitis. The purpose of this study was to examine the mechanism of pulmonary infiltration and lung injury in acute pancreatitis. Mice were fed a choline-deficient/ethionine-supplemented (CDE) diet for 144 hours to induce severe acute pancreatitis. Serum samples were collected for measurement of biochemical markers of disease and for the detection of tumor necrosis factor-alpha (TNF-alpha). Cell surface adhesion molecule expression was quantified by the sensitive radiolabeled dual monoclonal antibody technique. Neutrophil sequestration in lung tissue was measured by the myeloperoxidase assay. Lung injury was determined histologically and lung edema was assessed by wet/dry ratios. Pancreatic injury was demonstrated to occur in all CDE-fed mice, which developed significant hyperamylasemia and hypoglycemia by 48 hours (P <0.0001). Serum TNF-alpha levels increased significantly by 48 hours over baseline values (P <0.02). Expression of intracellular adhesion molecule (ICAM-1) in pulmonary endothelia was significantly increased above baseline by 30% at 48 hours (P <0.02) and peaked at 120 hours by 100% (P <0.0001). Vascular cellular adhesion molecule (VCAM-1) was constitutively expressed at baseline and was upregulated threefold by 48 hours (P <0.0001). Neutrophil infiltration increased gradually 24 hours after ICAM-1 and VCAM-1 were upregulated with significant elevation of myeloperoxidase activity over baseline at 72 hours (7.2 +/- 1.2 vs. 18.1 +/- 2.2 activity units/gram tissue; P <0.05). Neutrophil infiltration peaked at 144 hours (26.24 +/- 10.49 activity units/gram tissue P <0.0001), and its kinetics correlated with the onset and progression of morphologic injury as well as increased lung edema. These results show that acute pancreatitis is associated with a systemic release of inflammatory cytokines, followed by increased expression of pulmonary ICAM-1 and VCAM-1, neutrophil infiltration, and histologic lung injury. The adhesion molecule axis may be a potential target for practical intervention to ameliorate lung injury and morbidity in acute pancreatitis.

Abstract: Immunostimulatory oligodeoxynucleotides (ODN) containing CpG dinucleotides have been shown to stimulate murine and human lymphocytes. We investigated the presence of stimulatory DNA motifs in specific group A streptococcal (GAS) genes to elucidate the potential role of DNA in the immunopathogenesis of GAS infections. Despite low GC content in GAS DNA, the emm1 gene encoding the streptococcal M1 protein contained a relatively high frequency of TTCG(T/C), TCGTCG and (G/A)TCGT motifs that preferentially stimulated human lymphocytes. Interestingly, these motifs were also found in genes encoding M-like proteins of group C and G streptococci. ODN copying the emm1 gene sequences harboring these motifs induced the proliferation of human B cells and up-regulated the expression of CD25 on their surface. T cells were not required for the response to the ODN, indicating a direct effect of these motifs on B lymphocytes. Inter-individual variations in responsiveness to ODN were observed, suggesting that host factors potentiate these responses. The finding that GAS DNA contains stimulatory motifs that induce activation of human B cells in a T cell-independent manner suggests that this may be an important mechanism by which the bacteria can target the innate arm of the immune system in the early stages of invasive infections.

Abstract: MAT II, the extrahepatic form of methionine adenosyltransferase (MAT), consists of catalytic alpha(2)/alpha(2') subunits and a noncatalytic beta subunit, believed to have a regulatory function. The full-length cDNA that encodes the beta subunit of human MAT II was cloned and found to encode for a 334-amino acid protein with a calculated molecular weight of 37,552. Analysis of sequence homology showed similarity with bacterial enzymes that catalyze the reduction of TDP-linked sugars. The beta subunit cDNA was cloned into the pQE-30 expression vector, and the recombinant His tagged protein, which was expressed in Escherichia coli, was recognized by antibodies to the human MAT II, to synthetic peptides copying the sequence of native beta subunit protein, and to the rbeta protein. There is no cross-reactivity between the MAT II alpha(2) or beta subunits. None of the anti-beta subunit antibodies reacted with protein extracts of E. coli host cells, suggesting that these bacteria have no beta subunit protein. Interestingly, the rbeta subunit associated with E. coli as well as human MAT alpha subunits. This association changed the kinetic properties of both enzymes and lowered the K(m) of MAT for L-methionine. Together, the data show that we have cloned and expressed the human MAT II beta subunit and confirmed its long suspected regulatory function. This knowledge affords a molecular means by which MAT activity and consequently the levels of AdoMet may be modulated in mammalian cells.

Abstract: The relatedness of group A streptococcal (GAS) strains isolated from 35 Canadian patients with invasive disease of different severity was investigated by a variety of molecular methods. All patients were infected with M1T1 strains and, based on clinical criteria, were classified as severe (n = 21) and nonsevere (n = 14) invasive GAS infection cases. All the M1 strains studied had the emm1.0 allele and the same streptococcal pyrogenic exotoxin (Spe) genotype, speA(+) speB(+) speC speF(+) speG(+) speH smeZ(+) ssa. All isolates had the same speA allotype, speA2. The randomly amplified polymorphic DNA banding pattern with two different primers was identical for all strains, and pulsed field gel electrophoresis analysis showed that 33 and 30 isolates had identical banding patterns after DNA digestion with SfiI or SmaI, respectively; the nonidentical isolates differed from the main pattern by only one band. A relatively high degree of polymorphism in specific regions of the sic gene was observed among isolates; however, this polymorphism was not associated with disease severity. Likewise, although the phenotypic expression of SpeA, SpeB, and SpeF proteins varied among the M1T1 isolates, there was no correlation between the amount of Spe expressed and disease severity. Importantly, mitogenic and cytokine responses induced by partially purified bacterial culture supernatants containing a mixture of expressed superantigens were very similar for isolates from severe and nonsevere cases (P > 0.1). Together, the data indicate that highly related invasive M1T1 isolates, some indistinguishable, can cause disease of varying severity in different individuals. These findings underscore the contribution of host factors to the outcome of invasive GAS infections.

Abstract: OBJECTIVE: To determine whether expression of P- and E-selectin molecules is associated with the development of systemic organ manifestations in acute pancreatitis (AP). SUMMARY BACKGROUND DATA: Overproduction of inflammatory cytokines in AP induces expression of adhesion molecules, which may lead to increased leukocytic infiltration and tissue damage. Understanding the temporal expression of these molecules could afford better measures for therapeutic intervention. METHODS: Acute pancreatitis was induced in 30-day-old female C57/ bI/6J mice by feeding a choline-deficient/ethionine-supplemented diet (n = 95). Mice were divided into three groups. Group I (n = 35) was used to study the biochemical and histologic manifestations of AP and to evaluate the neutrophilic infiltration by myeloperoxidase activity and immunofluorescence. Groups II (n = 35) and III (n = 25) were used to evaluate expression of P- and E-selectin by the dual radiolabeled monoclonal antibody technique. RESULTS: Biochemical and histologic evidence of AP developed in all mice. The inflammatory cytokine tumor necrosis factor-alpha gradually increased in serum as early as 18 hours, reaching more than 800-fold background levels by 72 hours. Biphasic P-selectin expression in the lung was seen with peaks at 24 and 48 hours; E-selectin expression peaked at 48 hours. CD18-positive leukocytes and increased myeloperoxidase activity in the lung were demonstrated at 24 hours, correlating with the onset of selectin upregulation. Histologic scoring of lung tissue demonstrated mild damage at 24 hours, with progressive injury occurring from 48 to 72 hours. CONCLUSIONS: In AP, the production of inflammatory cytokines precedes up-regulation of P- and E-selectin, whose expression coincided with the increased infiltration of CD18-positive cells and neutrophil sequestration in lung tissue. Temporally, these events correlate with evidence of histologic pulmonary injury and underscore the role of adhesion molecules as mediators of pathophysiologic events. This mechanistic pathway may afford novel therapeutic interventions in clinical disease by using blocking agents to ameliorate the systemic manifestations of AP.

Abstract: In this study we compared the ability of different immunoglobulin (Ig) preparations containing IgG, IgM, and/or IgA to neutralize the activity of streptococcal pyrogenic exotoxin A (SpeA) or culture supernatant from a clinical group A streptococcal isolate. All Ig preparations markedly inhibited the mitogenic and cytokine-inducing activity of SpeA and culture supernatant at concentrations of 0.05-0.5 mg/mL, and at 0.5 mg/mL, most caused 95-100% inhibition of both stimuli. A significantly higher (P< or =.05) inhibition of SpeA was achieved by Pentaglobin (IgG, IgM, and IgA) and IgAbulin (IgA and IgG), as compared with pure IgG preparations. IgM- and IgA-enriched preparations had significantly higher inhibitory activity against SpeA than against culture supernatant, whereas the reverse was true for the IgG preparations (P< or =.05). The data show that IgM and IgA are potent inhibitors of specific streptococcal superantigens. These findings may have implications for the optimization of immunotherapy in invasive streptococcal infections.

Abstract: Cytokines elicited by superantigens have been suggested to play a central role in severe systemic clinical manifestations of gram-positive sepsis. Here we provide evidence for a potent inflammatory cytokine response in acute invasive group A streptococcal infections, and show a direct correlation between the magnitude of this response and the severity of systemic manifestations of the disease. Severe invasive cases suffering from toxic shock and/or necrotizing fasciitis had significantly higher frequencies of IL-2-, IL-6-, and TNF-alpha-producing cells in their circulation as compared to non-severe invasive cases (p=0.05-0.01). This difference was even more accentuated when severe and non-severe cases infected with a clonal M1T1 strain were compared (p=0.03-0. 004). To determine whether host factors were responsible for this difference in magnitude of cytokine responses, paired age- and gender-matched severe and non-severe M1T1 cases (n=8) were tested in vitro during their convalescent phase for immune response to superantigens produced by their infecting isolate. The results showed persistent and inherent differences in the magnitude of proliferative and cytokine responses of severe and non-severe patients to the streptococcal superantigens to which they had been exposed during infection. Thus, the study provides evidence that patients with a propensity to produce higher levels of inflammatory cytokines in response to streptococcal superantigens develop significantly more severe systemic manifestations than patients who have a propensity to produce lower levels of inflammatory cytokines to the same superantigens. We therefore conclude that host factors influence the magnitude of cytokine responses to superantigens and consequently the clinical outcome of the infection.

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Abstract: The streptococcal cysteine protease (SpeB) is one of the major virulence factors produced by group A streptococci (GAS). In this study we investigated if differences exist in SpeB production by clonally related M1T1 clinical isolates derived from patients with invasive infections. Twenty-nine of these isolates were from nonsevere cases and 48 were from severe cases, including streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) cases. The expression and amount of the 28-kDa SpeB protein produced were determined by quantitative Western blotting, and protease activity was measured by a fluorescent enzymatic assay. A high degree of variation in SpeB expression was seen among the isolates, and this variation seemed to correlate with the severity and/or clinical manifestation of the invasive infection. The mean amount of 28-kDa SpeB protein and cysteine protease activity produced by isolates from nonsevere cases was significantly higher than that from STSS cases (P = 0.001). This difference was partly due to the fact that 41% of STSS isolates produced little or no SpeB compared to only 14% of isolates recovered in nonsevere cases. Moreover, the cysteine protease activity among those isolates that expressed SpeB was significantly lower for STSS isolates than for isolates from nonsevere cases (P = 0.001). Increased SpeB production was also inversely correlated with intact M protein expression, and inhibition of cysteine protease activity blocked the cleavage of the surface M protein. Together, the data support the existence of both an "on-off" and a posttranslational regulatory mechanism(s) controlling SpeB production, and they suggest that isolates with the speB gene in the "off" state are more likely to spare the surface M protein and to be isolated from cases of severe rather than nonsevere invasive infection. These findings may have important implications for the role of SpeB in host-pathogen interactions via regulation of the expression of GAS virulence genes and the severity of invasive disease.

Abstract: Acute pancreatitis (AP) is characterized by release of proteolytic enzymes from the pancreas and a powerful inflammatory cytokine cascade that mediates the systemic manifestations and contributes to the mortality of the disease. The purpose of this study was to examine a potential link between pancreatic proteolytic enzymes, which are increased in AP, and cytokine production. To evaluate this, we incubated rat peritoneal macrophages (PMO) with increasing concentrations of trypsin and measured cytokine production. Supernatants from the cell cultures were assayed for TNF-alpha and IL-1beta, and the PMO were collected for the evaluation of cytokine mRNA by polymerase chain reaction (PCR). Further to evaluate the role of pancreatic proteases in triggering the cytokine cascade in AP, trypsin was injected into the peritoneal cavity of Sprague-Dawley rats, and the production of cytokines was measured in the peritoneal fluid. Controls included injection of inactivated trypsin. Incubation of PMO with trypsin in vitro resulted in a dose-dependent increase in TNF-alpha production with maximal response (2,660.5+/-748.8 pg/mL) at 10 microg/mL protease. Peak TNF-alpha and IL-1beta release was noted 16 h after stimulation of the PMO (2,759.5+/-698.0 pg/mL and 160,596+/-4,065 cpm, respectively). Trypsin-induced TNF-alpha production was not due to release of cell-associated cytokine, inasmuch as activation of PMO with this protease causing an increase in TNF-alpha mRNA by 30 minutes, reaching a 14-fold increase at 4 h. Trypsin-injected animals produced TNF-alpha-containing ascitic fluid in a dose-dependent manner with peak TNF-alpha at 2 h (371.3+/-180 pg/mL) versus control (53.8+/-11.2 pg/mL; p < 0.022). No TNF-alpha was found in ascites of rats injected with heat-inactivated trypsin. Histologic examination of trypsin-injected animals revealed evidence of pulmonary inflammation at 2 and 4 hours. We conclude that the proteolytic enzyme trypsin stimulates cytokine production from macrophages in vitro and in vivo. This model demonstrates for the first time that trypsin is a potential mediator of the cytokine response seen during AP.

Abstract: An impressive change in the epidemiology and severity of invasive group A streptococcal infections occurred in the 1980s, and the incidence of streptococcal toxic shock syndrome cases continues to rise. The reason for the resurgence of severe invasive cases remains a mystery-has there been a change in the pathogen or in host protective immunity? To address these questions, we have studied 33 patients with invasive infection caused by genotypically indistinguishable M1T1 strains of Streptococcus pyogenes who had different disease outcomes. Patients were classified as having severe (n = 21) and nonsevere (n = 12) invasive infections based on the presence or absence of shock and organ failure. Levels of anti-M1 bactericidal antibodies and of anti-streptococcal superantigen neutralizing antibodies in plasma were significantly lower in both groups than in age- and geographically matched healthy controls (P < 0.01). Importantly, the levels of these protective antibodies in plasma samples from severe and nonsevere invasive cases were not different. Together the data suggest that low levels of protective antibodies may contribute to host susceptibility to invasive streptococcal infection but do not modulate disease outcome. Other immunogenetic factors that regulate superantigen responses may influence the severity of systemic manifestations associated with invasive streptococcal infection.

Abstract: We performed retrospective flow cytometry crossmatch (FCXM) on 106 renal graft recipients who were transplanted based on current T cell negative serologic crossmatch. T and B cell FCXMs were performed on current and historical peak reactive post-transplant sera using 1024-channel flow cytometer and the shift in median channel fluorescence (SMCF) over the negative control was calculated. Cut-off values for a positive T and B crossmatch, > 40 and > 80 SMCF, respectively, were determined based on previous retrospective analysis of the data in the context of clinical outcome in our center, and were 1.5 times the standard deviation (SD) above the mean median channel fluorescence (MCF) of normal sera controls. The 1-yr graft survival was 95% for the total group of patients studied, and 87% for the recipients who had a positive T cell FCXM. To focus on the influence of a positive B cell FCXM on the incidence of rejection, primary transplant recipients who had a negative T cell FCXM (n = 81) were studied. Fifteen of 30 (50%) recipients with a positive B cell FCXM experienced at least one rejection episode within the first year. By contrast, only 15 of 51 (29.4%) of patients with a negative B cell FCXM experienced rejection (p = 0.05). The mean B cell SMCF in the group of patients who had no rejections was 45 +/- 59, while that of the group of patients who experienced at least one rejection was 97 +/- 97 (p = 0.012). By comparison, the rejection rate among the retransplant patients was 44.4%, and the mean B cell SMCF in the group with rejection was 94 +/- 75 while it was 5 +/- 7 among retransplant patients who did not have rejection (p = 0.031). Eighty-six percent of sensitized (panel reactivity antibodies (PRA) > 10%) patients who had a B positive/T negative FCXM experienced rejection, compared to 33% (n = 6 out of 16) of the B negative/T negative sensitized patients (p = 0.03). Furthermore, 62% (n = 13 out of 21) of donor-recipient mismatched patients with a B positive/T negative FCXM experienced rejection, compared to 38% (n = 13 out of 35) of patients with T negative/B negative FCXM who were similarly mismatched (p = 0.064). These data demonstrate the value of a positive B cell FCXM for predicting post-transplant rejections particularly when evaluated in the context of prior sensitization and/or DR mismatching. Our results suggest that B cell FCXM may have significant clinical implications, justifying its use in post-transplant management of recipients who have other risk factors of rejection.

Abstract: BACKGROUND: Discrepancies in reported HLA class II associations with rheumatic heart disease (RHD) may have been due to inaccuracies of serological typing reagents and/or lack of defined clinical classification of patients analyzed. The molecular association between HLA and RHD was investigated in patients with defined clinical outcome. METHODS AND RESULTS: Class II allele/haplotype distribution was determined in 2 groups of RHD patients (n=88) and a control group (n=59). Patients were divided into the mitral valve disease (MVD) category (ie, those with mitral regurgitation with or without mitral stenosis) and the multivalvular lesions (MVL) category, with impairment of aortic and/or tricuspid valves in addition to mitral valve damage. The MVD category (n=65) accounted for 74% of patients and included significantly fewer recurrent RF episodes compared with MVL patients (P=0.002). CONCLUSIONS: Significant increases in DRB1*0701 and DQA1*0201 alleles and DRB1*0701-DQA1*0201 haplotypes were found in patients. Removal of the MVL patients from analysis increased the strength of HLA associations among the MVD sample. The frequency of DQA1*0103 allele was decreased and the DQB1*0603 allele was absent from the patient group, suggesting that these alleles may confer protective effects against RHD. DQ alleles in linkage disequilibrium with DR alleles appear to influence risk/protection effect: whereas the DRB1*13-DQA1*0501-3-DQB1*0301 haplotype showed a trend toward risk, the DRB1*13-DQA1*0103-DQB1*0603 haplotype was absent in the RHD sample. Our data indicate that certain class II alleles/haplotypes are associated with risk or protection from RHD and that these associations appear to be stronger and more consistent when analyzed in patients with relatively more homogeneous clinical manifestations.

Abstract: Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet). The mammalian MAT II isozyme consists of catalytic alpha(2) and regulatory beta subunits. The aim of this study was to investigate the interaction and kinetic behavior of the human MAT II subunit proteins in mammalian cells. COS-1 cells were transiently transfected with pTargeT vector harboring full-length cDNA that encodes for the MAT II alpha(2) or beta subunits. Expression of the His-tagged recombinant alpha(2) (ralpha(2)) subunit in COS-1 cells markedly increased MAT II activity and resulted in a shift in the K(m) for L-methionine (L-Met) from 15 microM (endogenous MAT II) to 75 microM (ralpha(2)), and with the apparent existence of two kinetic forms of MAT in the transfected COS-1 cell extracts. By contrast, expression of the recombinant beta (rbeta) subunit had no effect on the K(m) for L-Met of the endogenous MAT II, while it did cause an increase in both the V(max) and the specific activity of endogenous MAT. Co-expression of both ralpha(2) and rbeta subunits resulted in a significant increase of MAT specific activity with the appearance of a single kinetic form of MAT (K(m) = 20 microM). The recombinant MAT II alpha(2) and rbeta subunit associated spontaneously either in cell-free system or in COS-1 cells co-expressing both subunits. Analysis of nickel-agarose-purified His-tagged ralpha(2) subunit from COS-1 cell extracts showed that the beta subunit co-purified with the alpha(2) subunit. Furthermore, the alpha(2) and beta subunits co-migrated in native polyacrylamide gels. Together, the data provide evidence for alpha(2) and beta MAT subunit association. In addition, the beta subunit regulated MAT II activity by reducing its K(m) for L-Met and by rendering the enzyme more susceptible to feedback inhibition by AdoMet. We believe that the previously described differential expression of MAT II beta subunit may be an important mechanism by which MAT activity can be modulated to provide different levels of AdoMet that may be required at different stages of cell growth and differentiation.

Abstract: Previous studies have shown D2-like dopamine receptor involvement in the regulation of phospholipid methylation (PLM), while others have documented impaired methionine and folate metabolism in schizophrenia. Utilizing [14C]formate labeling in cultured neuroblastoma cell lines, we now show that D4 dopamine receptors (D4R) mediate the stimulatory effect of dopamine (DA) on PLM. The effect of DA was potently blocked by highly D4R-selective antagonists and stimulated by the D4R-selective agonist CP-226269. DA-stimulated PLM was dependent upon the activity of methionine cycle enzymes, but DA failed to increase PLM in [3H]methionine labeling studies, indicating that a methionine residue in the D4R might be involved in mediating PLM. A direct role for MET313, located on transmembrane helix No. 6 immediately adjacent to phospholipid headgroups, was further suggested from adenosylation, site-directed mutagenesis and GTP-binding results. A comparison of PLM in lymphocytes from schizophrenia patients vs control samples showed a four-fold lower activity in the schizophrenia group. These findings reveal a novel mechanism by which the D4R can regulate membrane composition. Abnormalities in D4R-mediated PLM may be important in psychiatric illnesses such as schizophrenia.

Abstract: Here we show that in extrahepatic methionine adenosyltransferase replacement of a single amino acid (glycine 120) by cysteine is sufficient to create a functional nitric oxide binding site without affecting the kinetic properties of the enzyme. When wild-type and mutant methionine adenosyltransferase were incubated with S-nitrosoglutathione the activity of the wild-type remained unchanged whereas the activity of the mutant enzyme decreased markedly. The mutant enzyme was found to be S-nitrosylated upon incubation with the nitric oxide donor. Treatment of the S-nitrosylated mutant enzyme with glutathione removed most of the S-nitrosothiol groups and restored the activity to control values. In conclusion, our results suggest that functional S-nitrosylation sites can develop from existing structures without drastic or large-scale amino acid replacements.

Abstract: Twenty-one consecutive patients with streptococcal toxic shock syndrome (TSS) between December 1994 and April 1995 were treated with a median dose of 2 g of intravenous immunoglobulin (IVIG)/kg (cases) and were compared with 32 patients with streptococcal TSS between 1992 and 1995 who did not receive IVIG therapy (controls). The outcome measure was 30-day survival. Patient plasma was tested for its ability to inhibit T cell activation induced by the infecting strain. The proportion of cases with 30-day survival was higher than that of the controls with 30-day survival (67% vs. 34%, respectively; P = .02). Multivariate analysis revealed that IVIG administration and a lower Acute Physiology and Chronic Health Evaluation II score were associated with survival; the odds ratio for survival associated with IVIG therapy was 8.1 (95% confidence interval, 1.6-45; P = .009). IVIG therapy enhanced the ability of patient plasma to neutralize bacterial mitogenicity and reduced T cell production of interleukin-6 and tumor necrosis factor alpha. IVIG may be an effective adjunctive therapy for streptococcal TSS, possibly because of its ability to neutralize bacterial exotoxins.

Abstract: The surface M protein of group A streptococci (GAS) is one of the major virulence factors for this pathogen. Antibodies to the M protein can facilitate opsonophagocytosis by phagocytic cells present in human blood. We investigated whether pooled normal immunoglobulin G (IVIG) contains antibodies that can opsonize and enhance the phagocytosis of type M1 strains of GAS and whether the levels of these antibodies vary for different IVIG preparations. We focused on the presence of anti-M1 antibodies because the M1T1 serotype accounts for the majority of recent invasive GAS clinical isolates in our surveillance studies. The level of anti-M1 antibodies in three commercial IVIG preparations was determined by enzyme-linked immunosorbent assay (ELISA), and the opsonic activity of these antibodies was determined by neutrophil-mediated opsonophagocytosis of a representative M1T1 isolate. High levels of opsonic anti-M1 antibodies were found in all IVIG preparations tested, and there was a good correlation between ELISA titers and opsonophagocytic activity. However, there was no significant difference in the levels of opsonic anti-M1 antibodies among the various IVIG preparations or lots tested. Adsorption of IVIG with M1T1 bacteria removed the anti-M1 opsonic activity, while the level of anti-M3 opsonophagocytosis was unchanged. Plasma was obtained from seven patients with streptococcal toxic shock syndrome who received IVIG therapy, and the level of anti-M1 antibodies was assessed before and after IVIG administration. A significant increase in the level of type M1-specific antibodies was found in the plasma of all patients who received IVIG therapy (P < 0.006). The results reveal another potential mechanism by which IVIG can ameliorate severe invasive group A streptococcal infections.

Abstract: Inasmuch as normal intravenous polyspecific immunoglobulin G (IVIG) neutralizes the activity of a wide spectrum of superantigens, it may be an efficient adjunctive therapy for diseases associated with superantigen-producing organisms, including severe group A streptococcal diseases. The neutralizing activity against purified superantigens, streptococcal pyrogenic exotoxins (Spe), and a mixture of superantigens present in culture supernatant of clinical group A streptococcal isolates was determined for five IVIG preparations. A significant variation among different IVIG preparations (P < .05) and different lots of the same IVIG brand (P < .044) was found. Neutralization of SpeA activity was significantly lower than that of other streptococcal superantigens (P < .05); however, there was no correlation between SpeA binding and SpeA neutralizing activity in different IVIGs. Plasma samples obtained from patients after IVIG infusion varied in their titers of neutralizing antibodies to culture supernatants prepared from their respective isolates, and this variation paralleled differences in the neutralizing titer of the IVIG lot administered to each patient studied. The study suggests that complete neutralizing activity may be achieved by optimizing the type and/or dose of IVIG used in treatment.

Abstract: OBJECTIVE: The purpose of this study was to determine whether pathologic progression and cytokine responses in acute pancreatitis (AP) are altered in the absence of endotoxemia. SUMMARY BACKGROUND DATA: Previous studies have demonstrated that AP is characterized by rapid production and release of inflammatory cytokines, which play a major role in the local pancreatic and systemic complications of this disease. Infection and endotoxemia have been implicated as a major source of morbidity and death in AP and as possible stimuli for the overwhelming cytokine response seen in this disease. METHODS: AP was induced by a choline-deficient and ethionine-supplemented diet for 4 days in normal C57BL/6J mice (controls, n = 23) and in CD14 knockout mice (CD14KO, n = 23), which cannot produce circulating cytokines in response to endotoxin. Control and endotoxin-resistant mice were killed at time 0, then at 24, 48, 72, and 96 hours after the start of the diet. At each time point serum was collected for amylase, glucose, and cytokine measurements (tumor necrosis factor-alpha [TNFalpha] and interleukin-1beta [IL1beta]), and the pancreas was removed for histologic examination. TNFalpha was measured with a bioassay using WEHI-2F cells and IL1beta with a bioassay using D10.G4.1 cells. RESULTS: CD14KO mice developed biochemical manifestations of AP with alterations in amylase levels, hypoglycemia, weight loss, and histologic changes of pancreatitis similar to the pattern seen in control mice. TNFalpha and IL1beta production had similar kinetics in both groups, with significant peak TNFalpha serum levels at 72 hours and a progressive rise of IL1beta levels throughout the study period. Histologic changes appeared earlier and were more pronounced in the control versus the CD14KO mice. However, the mortality rate was identical (20% at 96 hours) for both groups. CONCLUSIONS: These results demonstrate that the progression of AP, the cytokine response associated with the disease, and early death are independent of endotoxin action. These findings, which suggest that an uncharacterized stimulus is responsible for triggering the cytokine cascade in this disease, may have significant implications for the management of patients with AP.

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Abstract: Just as we thought that we know everything about superantigens, new molecular and structural studies indicate that we have only just begun to unravel the secrets of these fascinating molecules. Recent structure-function analysis of superantigens from Gram-positive bacteria, with emphasis on their interaction with major histocompatibility complex molecules, could help us decipher the role of superantigens in disease, identify host factors that potentiate their effects and design drugs that specifically block their activity.

Abstract: Polyamines are important for regulation of lymphocyte differentiation and proliferation. Mitogens induce synthesis of ornithine decarboxylase (ODC), the rate limiting enzyme in polyamine biosynthesis. Since mitogens stimulate T-cells by non-physiological routes, the role of polyamine metabolism in T-cell receptor (TCR)-mediated T-cell activation has not been adequately evaluated. The effect of phytohemagglutinin (PHA) and staphylococcal enterotoxin B (SEB) on T-cell ODC and polyamine synthesis was compared. ODC activity was 6-11-fold higher in PHA compared to SEB stimulated T-cells. These differences were not attributed to differences in the magnitude of T-cell proliferation. Kinetics of ODC and polyamine synthesis were also different in PHA- and SEB-stimulated T-cells. In PHA-stimulated cells ODC levels and the induction of putrescine and spermidine synthesis peaked 6 h prior to peak IL-2 production, while in SEB-stimulated cells, ODC levels and polyamine synthesis peaked 6-12 h after IL-2 production. Differences in the temporal relationship between IL-2 production and polyamine induction in mitogen- versus superantigen-stimulated cells may account for the significant inhibition of the proliferative response by alpha-difluoromethylornithine following PHA but not SEB stimulation. Polyamine metabolism is regulated differently in T-cells stimulated via TCR engagement than with polyclonal mitogens.

Abstract: Genetically engineered fusion proteins of the super-antigen staphylococcal enterotoxin A (SEA) and tumor-reactive monoclonal antibodies, C215Fab-SEA and C242Fab-SEA, have been generated and shown to be effective in mediating superantigen-antibody directed cellular cytotoxicity against human carcinoma cells expressing the CA215 or CA242 antigens in an MHC class II-independent manner. In an attempt to reduce the in vivo toxicity of superantigen administration, alanine substitution mutations in SEA at residues F47 and D227 that affect SEA binding to class II molecules have been created and genetically linked to C215Fab or C242Fab. The purpose of this study was to determine whether these Fab-SEA mutant fusion proteins, that have low MHC class II binding affinities, were still able to stimulate human T cells in a Vbeta-specific manner in the presence or absence of MHC class II molecules. The SEA wt- and SEA-D227A-based fusion proteins shared the ability to activate V beta5. 2-, Vbeta6-, Vbeta7-, Vbeta9- and Vbeta18-bearing T cells, whereas Fab-SEA-F47A protein activated only Vbeta6- and Vbeta7-bearing T cells. The fusion of Fab fragments onto SEA wt, SEA-F47A or SEA-D227A had no effect on the Vbeta specificity of these superantigens. Fab fusion proteins containing either SEA wt or SEA mutants were presented, in the absence of class II molecules, by CHO cells transfected with CA215 and CD80 and all induced the expansion of only Vbeta6-, Vbeta7- and Vbeta 18-bearing T cells. Fab-SEA mutant fusion proteins may provide attenuated therapeutic agents that, while still able to specifically target high affinity T cells for MHC class II-independent local tumor killing, will not induce excessive systemic toxicity.

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Abstract: The majority of group A streptococcal (GAS) isolates from patients with streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) express numerous virulence factors, including several superantigens (SAgs). Purified SAgs are potent inducers of inflammatory (Th1) cytokines that contribute to the pathogenesis of severe infections. However, GAS-infected individuals are likely to be exposed to a mixture of GAS SAgs as well as other virulence factors produced by the bacteria, and therefore, our goal was to characterize the mitogenic and cytokine induction profiles of this mixture. All GAS isolates tested had brisk mitogenic activity and induced potent cytokine responses, with higher frequencies of Th1 than Th2 cytokine-producing cells. The mitogenic activity produced in culture supernatants of three selected clinical GAS isolates was significantly different, but no marked difference was found in their overall cytokine induction profiles. However, significant differences (P < 0.0062) were noted in the induction of Th2 cytokines between GAS supernatants and recombinant streptococcal pyrogenic exotoxin A (rSpeA), suggesting that the presence of other SAgs and/or the production of additional virulence factors may alter the overall cytokine induction profile of SAgs. A significant individual variation in the level of proliferative and cytokine responses to the same GAS culture supernatants or to rSpeA was noted. Individuals with higher frequencies of cells producing Th2 cytokines mounted lower levels of Th1 cytokine responses, and vice versa. Furthermore, quantification of the intensity and cell area of interleukin-1beta (IL-1beta)-producing cells by image analysis revealed that individuals with higher Th2 responses had significantly lower IL-1beta production (P < 0.0001) than the individual with a strong Th1 response. Differences in the ability to induce Th1 versus Th2 cytokines, as well as the individual variations in cytokine responses to streptococcal SAgs, may play a central role in determining the severity of invasive GAS infections.

Abstract: Superantigens interact with the T cell receptor for antigen (TCR) and are, therefore, more physiological stimulators of T lymphocytes than nonspecific polyclonal T cell mitogens. The effects of these two classes of T cell stimulators on methionine adenosyltransferase (MAT) and S-adenosylmethionine (AdoMet) levels were investigated. Activation of resting human peripheral blood T lymphocytes by the mitogen phytohemagglutinin (PHA) or the superantigen staphylococcal enterotoxin B (SEB) caused a 3- to 6-fold increase in MAT II specific activity. Although the proliferative response was higher in cultures stimulated with PHA compared with SEB, MAT II activity was comparable in both cultures. Both stimuli caused down-regulation of the MAT 68-kDa lambda subunit expression and induced a comparable increase in the expression of the catalytic alpha2/alpha2' subunit mRNA and protein. However, in superantigen-stimulated cells, the expression of the noncatalytic beta subunit was down-regulated and virtually disappeared by 72 h post-stimulation; whereas, no change in the expression of this subunit was noted in PHA-stimulated cells. Thus, at 72 h following stimulation, PHA-stimulated cells expressed MAT II alpha2/alpha2' and beta subunits while SEB-stimulated cells expressed the alpha2/alpha2' subunits only; the beta subunit was no longer expressed in superantigen-stimulated cells. Kinetic analysis of MAT II in extracts of PHA- and SEB-stimulated cells using reciprocal kinetic plots revealed that in the absence of the beta subunit the Km of the enzyme for L-methionine (L-Met) was 3-fold higher than in the presence of the beta subunit. Furthermore, AdoMet levels were 5-fold higher in cell extracts lacking the beta subunit (SEB-stimulated cell extracts) compared with extracts containing MAT II alpha2/alpha2' and beta subunits. We propose that the increased levels of AdoMet in superantigen-stimulated cells may be attributed to the absence of the beta subunit, which seems to have rendered MAT II less sensitive to product feedback inhibition by (-)AdoMet. The data suggest that the beta subunit of MAT II, which has no catalytic activity, may be a regulatory subunit that imparts a lower Km for L-Met but increases the sensitivity to feedback inhibition by AdoMet. The down-regulation of the beta subunit, which occurred when T cells were stimulated via the TCR, may be an important mechanism to regulate AdoMet levels at different stages of T cell differentiation under physiological conditions.

Abstract: The effect of the polyamines, putrescine, spermine, and spermidine, on the activity of extrahepatic methionine adenosyltransferase (MAT II) was studied. The polyamines inhibited MAT II activity at concentrations equal to or greater than 5 mm. Combinations of polyamines were more effective than individual polyamines in inhibiting MAT activity; maximum inhibition approached 80% with combinations of all three polyamines. S-Adenosylmethionine (AdoMet), Pi, and PPi, the products of the MAT reaction, are known to be synergistic inhibitors of the nonhepatic form of the enzyme. Combinations of polyamines plus Pi and/or PPi induced an additive inhibition of the enzyme. AdoMet plus polyamines also resulted in significant inhibition, but inhibition plateaued at about 80%, indicating the presence of a protective mechanism to maintain AdoMet synthesis. Extrahepatic MAT from human and rat tissues was inhibited by the polyamines, indicating that this phenomenon is not species specific. In addition, we examined the effect of polyamines on MAT activity in resting and activated human lymphocytes that were shown to differ in the relative expression of MAT II subunits. Although MAT from mitogen (phytohemagglutinin, PHA)- and superantigen (Staphylococcal enterotoxin B, SEB)-stimulated lymphocytes were similarly inhibited by 10 mM polyamines, at lower concentrations of polyamines (1-5 mM), MAT from SEB-stimulated cells appeared to be more susceptible to inhibition by the polyamines. Inasmuch as SEB is a more physiological stimulator of T cells than PHA, the data suggest a possible role of polyamines in regulating MAT activity.

Abstract: Acute rheumatic fever (ARF) and rheumatic heart disease (RHD) are autoimmune sequelae of upper respiratory infections with group A streptococci (GAS). To gain a better understanding of the pathogenesis of these diseases, we examined the in vitro proliferative responses of peripheral blood mononuclear cells (PBMC) from RHD patients to human myocardial proteins in a T-cell Western assay. A number of myocardial proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were recognized by PBMC from both patients and controls. However, PBMC from a significant percentage of RHD patients (40%) responded to a discrete band of myocardial proteins migrating with an apparent molecular mass of 50 to 54 kDa while none of the control subject PBMC responded to this protein band (P < or = 0.0001). To further investigate the link between infections with GAS and autoimmune carditis, we studied the proliferative responses of PBMC from patients and controls to myocardial proteins before and after in vitro stimulation of the cells with opsonized GAS isolated from ARF patients. Priming of PBMC with rheumatogenic GAS caused the percentage of RHD patients responding to the 50- to 54-kDa myocardial proteins to increase from 43 to 90% (P < or = 0.0284). By contrast, PBMC from control subjects failed to recognize the 50- to 54-kDa myocardial proteins even after stimulation with the opsonized streptococci (P < or = 0.0001). The assay sensitivity was increased from 40 to 90% after priming of a patient's cells with opsonized GAS, but the positive predictive value was 100% in both unprimed and primed cultures. Antibodies generated to partially purified 50- to 54-kDa myocardial proteins did not cross-react with either streptococcal homogenates, purified M protein, myosin, laminin, or vimentin, suggesting a lack of cross-reactivity at the humoral level. This study suggests that the 50- to 54-kDa myocardial proteins contain a putative antigen that is preferentially recognized by T cells from RHD patients and demonstrates that exposure to streptococcal antigens enhances the ability of patients to recognize these proteins.

Abstract: PURPOSE: To establish the maximum-tolerated dose (MTD) and define the toxicities of a single-dose infusion of PNU-214565, a recombinant Escherichia coli-derived fusion protein of Staphylococcal enterotoxin A (SEA) and the Fab-fragment of the C242 monoclonal antibody in patients with advanced colorectal and pancreatic carcinomas. To investigate the capability of PNU-214565 to induce a superantigen (SAg) response resulting in cytokine production and tumor regression. PATIENTS AND METHODS: Twenty-one patients (age range, 39 to 76 years; median, 64; 12 men, nine women; 18 colorectal, three pancreatic cancers) were treated with a single 3-hour infusion of PNU-214565, with doses ranging from 0.01 to 1.5 ng/kg. All patients had prior chemotherapy and a good performance status Eastern Cooperative Oncology Group [ECOG] performance status [PS] = 0 [n = 10]; PS = 1 [n = 11]), 10 had prior radiation, and 18 had prior surgery. RESULTS: Fever and hypotension were the most common toxicities. Fever of any grade occurred in 16 of 21 patients (76%): four of 21 (19%) with grade 2 and two of 21 (9.5%) with grade 3. Hypotension of any grade occurred in 13 of 21 (62%): four of 21 with grade 2 and one of 21 (5%) with grade 3. Interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF alpha) induction correlated with toxicity. In the two patients with grade 3 fever, peak IL-2 and TNF alpha levels were 2.9 IU/mL and 165 pg/mL, and 8.3 IU/mL and 245 pg/mL, respectively. Transient, > or = 50% decreases in circulating monocytes were observed in 17 of 21 patients as early as 0.5 hours (median time, 2 hours) from the start of infusion. Decreases (mean 33%) in circulating lymphocytes were observed in seven of 21 patients. All three patients with grade 3 toxicity were treated at the 0.5-ng/kg dose. The significance of baseline anti-SEA, human antimouse antibody (HAMA), CA242-soluble antigen levels, and T-cell receptor variable beta region (TCR V beta) subsets and histocompatibility leukocyte antigen-DR (HLA-DR) genotypes was assessed as possible predictors of toxicity. All toxicities were transient and easily managed. No grade 3 toxicity occurred at the higher dose levels. CONCLUSION: PNU-214565, a SAg-based tumor targeted therapy, is safe when given as a single 3-hour infusion at doses up to 1.5 ng/kg. The MTD for a single dose was not determined. The safety of a repeated dose schedule is currently under investigation, beginning with doses determined to be safe in this trial.

Abstract: Previous studies have suggested a central role for superantigen-induced immune responses in the pathogenesis of streptococcal toxic shock syndrome. The production of streptococcal superantigens by clinical group A streptococcal (GAS) isolates was studied, and the ability of plasma collected from patients with severe invasive GAS infections to neutralize the proliferative- and cytokine-inducing activities of these superantigens was investigated. Overnight culture supernatants from all GAS isolates obtained from patients with invasive disease were found to contain superantigenic activity, as evident from their ability to drive potent T cell proliferation, induce high production of cytokines, and stimulate T cells in a V beta-specific manner. Twelve patients with severe invasive GAS infections, including 11 streptococcal toxic shock syndrome cases and one necrotizing fasciitis without shock, were treated with i.v. infusions of normal polyspecific Ig (IVIG). Plasma samples collected from each patient before and after IVIG administration were analyzed for their ability to neutralize the activity of streptococcal superantigens produced by the GAS isolate that caused their disease. In all IVIG-treated patients, the capacity to neutralize the superantigenic activity, produced by their respective GAS isolate or by purified streptococcal pyrogenic exotoxins, increased in plasma following IVIG administration. Of particular clinical relevance, post-IVIG plasma from each patient completely blocked cytokine production elicited by their respective GAS culture supernatants or by purified streptococcal pyrogenic exotoxins. This study shows that IVIG treatment confers in vivo inhibitory activity against a large array of streptococcal superantigens and suggests that IVIG may be useful in the treatment of severe invasive streptococcal infections.

Abstract: BACKGROUND: Elevated levels of tumor necrosis factor-alpha (TNFalpha) have been measured in a lethal model acute pancreatitis (AP) and may contribute to the pathophysiologic sequelae of the disease. METHODS: To determine the significance of anti-TNFalpha therapy on survival and disease manifestations in a clinically relevant model of AP, a rat model was developed using a retrograde pancreatic ductal infusion of bile. Animals were randomized to no treatment (n = 30) or treatment with anti-TNFalpha antibody 15 minutes prior to induction of AP (n = 30). Five treated and 5 untreated rats were killed at various time periods up to 72 hours to provide temporal characterization of TNFalpha activity in AP. RESULTS: A burst Of TNFalpha activity in the serum of untreated pancreatitis animals between 1 and 3 hours after induction of the disease is prevented by pretreatment with anti-TNFalpha antibody. CONCLUSIONS: These findings provide a plausible mechanism for the improvement in biochemical and histologic parameters as well as in overall survival in an experimental model of acute pancreatitis in the rat.

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Abstract: Tumor necrosis factor-alpha (TNF alpha) has been implicated as one of the numerous likely mediators of the systemic complications of acute pancreatitis. Recent suggestions that calcium (Ca2+) acts as a signal not only for TNF alpha release but also for TNF alpha action at distant sites led us to hypothesize that the calcium channel blocker diltiazem could inhibit TNF alpha release in acute pancreatitis, ameliorating the severity of the disease and improving overall survival. A rat model of acute pancreatitis induced by retrograde ductal infusion of bile was used for two experiments (n = 120). Experiment 1 was designed to determine the effects of calcium channel blockade using diltiazem on the severity of pancreatitis as measured by changes in biochemistry, pathology, and serum TNF alpha levels. In experiment 2, effects of calcium channel blockade on animal survival were measured over 72 h. Calcium channel blockade was associated with a significant reduction in serum TNF alpha levels as well as amelioration of pancreatitis by biochemical and pathological criteria. Overall survival from bile-induced pancreatitis was dramatically improved in rats pretreated with diltiazem (80%) compared to untreated animals (40%). Our data suggest that calcium channel blockade is associated with TNF alpha inhibition and improved outcome in a rat model of acute pancreatitis.

Abstract: The development of systemic complications in acute pancreatitis is largely responsible for the mortality associated with this disease. The systemic sequelae encountered in acute pancreatitis are similar to those occurring in patients with septic shock, a syndrome of multiple organ failure thought to be related to overproduction of inflammatory cytokines. As with sepsis, data is mounting that cytokines, particularly TNF alpha, may play a central role in acute pancreatitis and mediate the systemic sequelae of the disease. We have previously shown elevated levels of TNF alpha in the serum of animals with experimental acute pancreatitis. In this study, we use a bile-infusion model of pancreatitis in the rat to show amelioration of disease severity as well as a distinct survival advantage by TNF alpha blockade using anti-TNF alpha polyclonal antibody. These data provide strong evidence that TNF alpha is a major contributor to the morbidity and mortality from acute pancreatitis.

Abstract: OBJECTIVE: The effects of the superantigens (SAgs) Staphylococcal Enterotoxin B (SEB), Toxic Shock Syndrome Toxin-1 (TSST-1) and Mycoplasma Arthritidis Mitogen (MAM) were examined on the induction and on the course of experimental SLE-like disease. METHODS: Immunization of BALB/c mice with human anti-DNA mAb (MIV-7) carrying the pathogenic idiotype 16/6 emulsified in complete Freund's adjuvant (CFA), followed by a boost of MIV-7/PBS 3 weeks later, generated an experimental SLE via an idiotypic dysregulation. RESULTS: After immunization with MIV-7/SAg, replacing the MIV-7 boost by SAg, and then injecting SAg 7 weeks after the regular induction of the SLE-like disease, the mice failed to produce anti-hIgM and dsDNA Ab up to 6 months after the induction. The mice immunized with MIV-7/CFA and boosted with the SAg had high titers of anti-hIgM but no detectable anti-dsDNA Ab. In both experimental groups low titers of anti-CL Abs developed in 25/40 (62%) and 30/38 (79%) of the mice respectively, including the control mice immunized with non-pathogenic human IgM/SAg or PBS/SAg. The mice immunized according to the "classical" protocol showed increased titers of anti-dsDNA Ab (22%) and anti-CL Ab (28%) during 10 weeks of observation. In contrast SEB, TSST-1 and MAM induced a 29%, 1% and 17% reduction in the anti-DNA titers and a 32%, 15% and 12% reduction in the anti-CL titers, respectively. CONCLUSIONS: These data suggest that the SAg tested here cannot replace the effect of CFA in the induction of the primary humoral response. The SAgs TSST-1, SEB and MAM did not induce the SLE-like disease following idiotypic modulation. Moreover, they may have had a suppressive effect on the idiotypic network in our model. The appearance of anti-CL Abs in almost all the experimental groups including the naive mice supports the possibility that microbial SAgs can induce the production of autoantibodies by different mechanisms. The SAgs TSST-1, SEB and MAM reduced autoantibody production in the serologically established idiotypic-induced experimental SLE-like murine model. This beneficial effect may indicate new directions for research on the management of SLE.

Abstract: Previous studies have provided strong evidence for restricted Vbeta gene usage in response to DR synthetic peptides presented in context by self MHC molecules, i.e., via the indirect pathway of allorecognition. Although numerous studies have suggested a role for the T cell receptor (TCR) beta chain in the direct pathway of allorecognition, it is not clear whether a particular HLA allele elicits a consistent pattern of restricted Vbeta gene usage by resting T cells from different individuals. To address this problem, HLA class II homozygous cell lines that do not express class I antigens were used to study the role of the TCR Vbeta elements in direct recognition of specific DR alleles. Analysis of TCR V gene usage revealed that each DR allele tested elicited the same pattern of preferential Vbeta gene usage in all individuals studied. For example, the DRB1*0101 allele was preferentially recognized by T cells expressing Vbeta2, Vbeta13.1, Vbeta18, and Vbeta20, and the DRB1*1301 caused expansion of Vbeta4-, Vbeta6-, Vbeta8-, and Vbeta18-bearing T cells. Similarly, analysis of TCR V gene usage in response to defined DR alleles expressed on homozygous cell lines that express class I was also made possible by using anti-class I antibodies to block class I recognition and focus the response on the DR molecule. The results showed that the DRB1*1501 allele expressed on two distinct homozygous lines elicited the preferential expansion of Vbeta2, Vbeta8, and Vbeta13.2 T cells. Similarly, the DRB1*0301 allele expressed on normal, Epstein-Barr virus-transformed, or transfected fibroblasts was found to elicit the same pattern of Vbeta expansion and to selectively stimulate T cells bearing Vbeta2, Vbeta16, and Vbeta17 elements. In contrast to this highly reproducible pattern of Vbeta gene usage elicited by specific DR alleles, extensive heterogeneity in the CDR3 region was found and no preferential Jbeta or Valpha gene usage was observed in response to any of the DR alleles tested. The data suggest that, similar to the situation with the indirect pathway, TCR Vbeta elements are involved in the direct pathway of allorecognition. Although some overlap may exist, different sets of Vbeta elements may be preferentially used for each of these pathways. Inasmuch as HLA-identical siblings, but not HLA-identical unrelated individuals, express a very similar TCR repertoire, our data suggest that additional factors are involved in shaping the repertoire. Preferential activation of T-cell subsets by specific DR alleles may play an important role in primary alloresponses, e.g., in mixed lymphocyte reactions, and organ transplantation. Elucidation of the Vbeta specificity of each DR allele may have an impact on therapeutic strategies aimed at blocking specific alloresponse and prolonging graft survival in transplant recipients while avoiding the hazardous sequelae of nonspecific immunosuppression.

Abstract: C-terminal residues of staphylococcal enterotoxin A (SEA), including H187, D225, and D227, are involved in moderate affinity binding to MHC class II beta-chain, whereas N-terminal residues, including F47, are involved in low affinity binding to MHC class II alpha-chain. The effect of alanine substitutions at residues D227 or F47 on induction of T cell proliferation and the expansion of specific TCR Vbeta families was determined. SEA wild type specifically activated T cells expressing Vbeta1, Vbeta5.2, Vbeta6, Vbeta7, Vbeta9, Vbeta18, and Vbeta22. Although SEA-D227A exhibited substantially reduced mitogenicity compared with SEA wild type, it expanded the same Vbeta-bearing T cells, except those expressing Vbeta1. By contrast, SEA-F47A, which was slightly less mitogenic than SEA wild type, induced expansion only of T cells expressing Vbeta6, Vbeta7, and to a lesser extent Vbeta22. Therefore, specific mutations affecting either MHC class II alpha or beta binding sites differentially affect the Vbeta specificity of this superantigen. The lack of expansion in four of seven Vbeta families by SEA-F47A suggests that the class II alpha binding site may position SEA on the MHC class II molecules in an appropriate conformation for interaction with certain Vbeta elements.

Abstract: Recently we demonstrated that normal polyspecific immunoglobulin given intravenously (IVIG) and plasma samples from patients treated with IVIG neutralize the mitogenic and cytokine-inducing activities of group A streptococcal (GAS) superantigens. Here we investigated whether this neutralizing activity is mediated by antibodies to these superantigens. IVIG and plasma samples collected from a patient with GAS necrotizing fasciitis post-IVIG infusions markedly inhibited the mitogenic activity elicited by the streptococcal pyrogenic exotoxins SpeB and SpeC, as well as by GAS culture supernatant. Immunoblot analysis showed marked increases in the levels of antibodies to SpeC and proteins in the GAS culture supernatant in post-IVIG over those of pre-IVIG plasma samples. Removal of antisuperantigen antibodies in IVIG by adsorption to SpeC- and GAS culture supernatant-coupled Sepharose markedly reduced the neutralizing ability of IVIG against respective stimuli. The neutralizing activity was totally recovered in the eluted antibodies. By contrast, although pre- and post-IVIG plasma samples contained antibodies to SpeA, these antibodies did not block the activity of this superantigen. Nonspecific immunomodulatory activity of IVIG was ruled out because neither the IVIG nor the affinity-purified antibodies significantly inhibited the response to the polyclonal T-cell mitogen phytohemagglutinin A. These data provide direct evidence that the neutralizing activity in IVIG, and in patient plasma samples following IVIG treatment is mediated by antibodies to superantigens and indicate that the quality rather than the quantity of these antibodies may be more clinically relevant.

Abstract: To study MHC class II-dependent and -independent SAg2 activation and the relative importance of CD80/CD28 costimulation, staphylococcal enterotoxin A (SEA) was presented to T cells as a fusion protein containing the Fab fragment of an mAb directed against the CA215 glycoprotein. Chinese hamster ovary (CHO) cells transfected with HLA-DR4, CA215, and CD80, individually or in combinations, were used as presenting cells. A strong T cell proliferation was obtained when C215Fab-SEA fusion proteins were presented by CHO-DR/CD80 or CHO-CA215/CD80 double transfectants, whereas only low levels of proliferation were seen in the absence of CD80. Large amounts of IL-2, IFN-gamma, and TNF were produced in addition to an increase in IL-2 mRNA as a result of CD80 costimulation. Only approximately 50% of the SEA-reactive T cells responded by expression of IL-2 receptor chains and by blast formation when activated with SEA in the absence of MHC class II. Reverse transcription-PCR-assisted repertoire analysis of SEA-reactive TCR V beta families showed that the CA215-dependent activation involved an expansion of fewer TCR V beta families compared with MHC class II-dependent activation. One-half of the six analyzed TCR V beta families were expanded independently of class II. This indicates that MHC class II has only a partial influence on the TCR V beta repertoire imprinted by SAg. This finding redefines the role of MHC class II in SAg presentation. It is suggested that MHC class II molecules are selected as SAg-binding molecules mainly as a suitable targeting receptor for professional APC expressing costimulatory molecules such as CD80 and CD86.

Abstract: BACKGROUND. Tumor necrosis factor-alpha (TNF-alpha) has been implicated as a mediator of the systemic manifestations associated with acute pancreatitis. The purpose of this study was to show that TNF-alpha expression in pancreatitis is a primary response and is not the result of endotoxemia. METHODS. Severe acute pancreatitis was induced in germ-free rats, which have no source of endogenous endotoxin, by ductal infusion of artificial bile. Control animals underwent sham operation and ductal infusion of saline solution. TNF-alpha levels were measured by the WEHI bioassay. Endotoxin was measured by the Limulus assay. RESULTS. TNF-alpha levels remained low in the sham group (mean, 24.6 +/- 8.0 pg/ml) but were significantly elevated in normal rats with pancreatitis (181 +/- 26.8 pg/ml; p < 0.001 versus sham group) and in germ-free rats with pancreatitis (213 +/- 90 pg/ml; p < 0.002 versus sham group). No endotoxin was detected in any of the experimental rats. CONCLUSIONS. Our results indicate that TNF-alpha levels are elevated in acute pancreatitis despite the absence of endotoxin, indicating a primary role of TNF-alpha in this disease.

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Abstract: The V beta repertoire of T cells of patients with gram-positive group A streptococcal (GAS) and non-GAS infections was analyzed to seek evidence for the role of superantigens in streptococcal toxic shock syndrome. No evidence of V beta overexpression but a consistent pattern of depletion of V beta 1, V beta 5.1, and V beta 12 was observed in patients with severe GAS infections. This pattern of V beta depletion was not observed in patients with nonsevere GAS infections or with severe non-GAS gram-positive infections. T cells from patients with severe GAS infections showed evidence of apoptosis; no apoptosis was found when there was no evidence of V beta depletion. There was no correlation with streptococcal M or T serotype or known spe genes. The depletion of specific V beta-bearing T cells in patients with severe GAS infections supports the role of a superantigen in these infections. The in vivo pattern of V beta specificity implicates a novel superantigen(s) in this disease.

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Abstract: The recent discovery of the mode of interaction between a group of microbial proteins known as superantigens and the immune system has opened a wide area of investigation into the possible role of these molecules in human diseases. Superantigens produced by certain viruses and bacteria, including Mycoplasma species, are either secreted or membrane-bound proteins. A unique feature of these proteins is that they can interact simultaneously with distinct receptors on different types of cells, resulting in enhanced cell-cell interaction and triggering a series of biochemical reactions that can lead to excessive cell proliferation and the release of inflammatory cytokines. However, although superantigens share many features, they can have very different biological effects that are potentiated by host genetic and environmental factors. This review focuses on a group of secreted pyrogenic toxins that belong to the superantigen family and highlights some of their structural-functional features and their roles in diseases such as toxic shock and autoimmunity. Deciphering the biological activities of the various superantigens and understanding their role in the pathogenesis of microbial infections and their sequelae will enable us to devise means by which we can intervene with their activity and/or manipulate them to our advantage.

Abstract: Tumor necrosis factor-alpha (TNF alpha) is postulated to be a mediator of the systemic complications associated with acute pancreatitis. Neutralization of TNF alpha with monoclonal antibody ameliorates the morbidity and mortality associated with acute pancreatitis in a rat model. Although high levels of TNF alpha are measurable in peripheral blood in acute pancreatitis, specific sites of TNF alpha production in this disease have not been described. In this study we show that induction of pancreatitis causes up-regulation of TNF alpha messenger RNA (mRNA) at a distant organ site, the spleen. Hemisplenectomies were performed in male Sprague-Dawley rats prior to induction of pancreatitis by pancreatic duct infusion of artificial bile. Completion hemisplenectomies were then performed at 30 min, 1 hr, and 2 hr after pancreatitis induction. Quantitation of TNF alpha mRNA in the hemispleens before and after pancreatitis using a semiquantitative reverse transcriptase-polymerase chain reaction method revealed an 80-fold increase in amount of TNF alpha mRNA by 2 hr after induction of pancreatitis. By contrast, control rats receiving a sham operation showed no significant increase in TNF alpha mRNA expression after infusion of the pancreatic duct with saline. The increase in TNF alpha mRNA production was associated with increased serum TNF alpha product levels and was independent of endotoxin. We conclude that severe acute pancreatitis in the rat model is associated with significant up-regulation of TNF alpha mRNA in splenic mononuclear cells. These data provide evidence that the local events of acute pancreatitis can induce up-regulation of TNF alpha mRNA at a distant site and suggest a possible mechanism of pathogenesis of the systemic manifestations of this disease.

Abstract: A soluble HLA ELISA for the detection of donor specific anti-HLA class I IgG antibodies was developed and compared with microlymphocytotoxicity. Donor sHLA was prepared from donor blood or purified blood lymphocytes and captured onto monoclonal antibody coated ELISA plates. After incubation of captured HLA with test serum, bound IgG antibodies were detected using a peroxidase-conjugated anti-human IgG antibody. Serum samples from patients on waiting lists to receive kidney transplants were tested by lymphocytotoxicity (AHG protocol) and/or sHLA ELISA in four different laboratories using HLA preparations from eight organ donors. Concordant crossmatch results were obtained for 854 (99%) of 864 ELISA crossmatches. In contrast, concordant results were obtained for 234 (91%) of 256 lymphocytotoxicity crossmatches. Interlaboratory reproducibility of ELISA results was 99%. In contrast, interlaboratory reproducibility of lymphocytotoxicity assay results was 78%. Endpoint titrations of serum specimens containing anti-HLA antibodies demonstrated equivalent sensitivity of ELISA and AHG lymphocytotoxicity crossmatch and similar sensitivity of ELISA and flow cytometry crossmatch. Specimens tested positive by lymphocytotoxicity without DTT treatment but negative with DTT treatment were tested negative by ELISA. Comparison of lymphocytotoxicity and ELISA crossmatch results showed an agreement of 94%. This demonstrates that detection of anti-donor HLA class I antibodies by ELISA is a reliable alternative to microlymphocytotoxicity testing.

Abstract: The CD28 molecule expressed on the surface of T cells plays a pivotal role in transducing costimulatory signals necessary for cell activation. CD28 coligation enhances tyrosine phosphorylation and phosphoinositol 3-kinase association in responsive cells. CD28 cross-linking has also been reported to activate inositol phospholipid turnover and to cause release of intracellular calcium. Here we examine the effects of CD28 cross-linking on early activation of protein kinase C (PKC). We have reported recently that either PMA or CD28 cross-linking synergizes with signals delivered by superantigen and cytokines to induce the proliferation of APC-depleted T cells. Unlike PMA, CD28 cross-linking alone failed to induce an increase in membrane-associated PKC activity. However, PKC activation was seen in resting T cells when CD28 was cross-linked in the presence of superantigen plus APC-derived supernatant, which by themselves had no effect on PKC activity. Inhibition of PKC activity using calphostin C blocked the response of pure T cells to superantigen in the presence of either autologous APC, PMA, or CD28 cross-linking. This effect was specific; it was only seen when calphostin C was added within the first hour of stimulation. Assays of [Ca2+]i levels showed that CD28 cross-linking augmented and prolonged the rise in [Ca2+]i induced in T cells by superantigen and APC-derived cytokines. In the presence of superantigen, the proliferative response of T cells costimulated by CD28 cross-linking was cyclosporin A-sensitive, whereas in the presence of PMA, CD28 cross-linking conferred resistance to cyclosporin A. Both the phosphorylation of phospholipase C gamma 1 at tyrosine and the rise in [Ca2+]i induced by CD28 cross-linking in preactivated T cells were blocked by herbimycin A. Herbimycin A treatment also blocked the ability of CD28 cross-linking to induce a rise in [Ca2+]i in resting T cells. We conclude that CD28 costimulatory signals augment superantigen-induced TCR signals by converging onto common TCR effector pathways involving the activation of phospholipase C gamma 1 and PKC and by generating a cyclosporin A-sensitive pathway.

Abstract: Recent studies have suggested that T cells play a critical role in the pathogenesis of psoriasis. Guttate psoriasis is a well-defined form of psoriasis frequently associated with streptococcal throat infection. This study tested the hypothesis that T cells in acute guttate psoriasis skin lesions may be activated by streptococcal superantigens. Peripheral blood as well as lesional and perilesional skin biopsies were analyzed for T cell receptor V beta repertoire using monoclonal antibodies against 10 different V beta families. Skin biopsies from all patients with acute guttate psoriasis, but not skin biopsies from patients with acute atopic dermatitis or inflammatory skin lesions induced in normal subjects with sodium lauryl sulfate, demonstrated selective accumulation of V beta 2+ T cells (P < 0.05). The expansion of V beta 2+ T cells occurred in both the CD4+ and the CD8+ T cell subsets. Sequence analysis of T cell receptor beta chain genes of V beta 2-expressing T cells from skin biopsies of patients with guttate psoriasis showed extensive junctional region diversity that is more compatible with a superantigen rather than a conventional (nominal) antigen-driven T cell response. All streptococcal isolates from patients with guttate psoriasis secreted streptococcal pyrogenic exotoxin C, a superantigen known to stimulate marked V beta 2+ T cell expansion. These data support the concept that acute guttate psoriasis is associated with superantigenic stimulation of T cells triggered by streptococcal superantigen(s).

Abstract: Human lymphocyte methionine adenosyltransferase (HuLy MAT) consists of heterologous subunits alpha and beta. The cDNA sequence of the alpha subunit of HuLy MAT from Jurkat leukemic T cells was identical to that of the human kidney alpha subunit and highly homologous to the sequence of the extrahepatic MAT from other sources. The 3'-untranslated sequence was found to be highly conserved, suggesting that it may be important in regulating the expression of MAT. The extrahepatic alpha subunit unit of MAT was found to be expressed also in human liver, and no differences were found in the sequence of the alpha subunit from normal and malignant T cells. The sequence of two unspliced introns found in the cDNA clones from the Jurkat library enabled us to isolate genomic clones harboring the human extrahepatic alpha subunit gene and to localize it to the centromere on chromosome arm 2p, an area that corresponds to band 2p11.2. Expression of the alpha subunit cDNA in Escherichia coli yielded two peptides with the immunoreactivity and mobilities of authentic alpha/alpha' subunits from HuLy. The Km of the recombinant alpha subunit was 80 microM, which is 20-fold higher than found for the (alpha alpha')x beta y holoenzyme purified from leukemic lymphocytes and 4-10-fold higher than found for the normal lymphocyte enzyme. The data suggest that the alpha/alpha' subunits mediate the enzyme catalytic activity and that the beta subunit may be a regulatory subunit of extrahepatic MAT.

Abstract: BACKGROUND. Tumor necrosis factor (TNF) is rapidly gaining recognition as one of the early, critical mediators of several inflammatory conditions, most notably endotoxic shock. The purposes of this study were to determine whether TNF levels are raised in severe acute pancreatitis, thus pointing to its role as a potential mediator of the inflammatory process, and to determine the possible sites of production and uptake. METHODS. TNF levels were measured during a 2-hour period in a rat model of acute pancreatitis by using an antegrade infusion of artificial bile. TNF levels were measured with a bioassay. RESULTS. TNF levels increased proportionately with time and serum amylase level, reaching a mean value of 2700 pg/ml at 2 hours compared with sham operated rats (mean, 125 pg/ml) (p < 0.001). TNF levels in nonoperated controls were undetectable. These measurements were found to be independent of endotoxin production. In addition, selective sampling from the portal vein, hepatic vein, and femoral artery showed hepatic degradation of TNF (p < 0.005), indicating that the liver may play an important role in protecting the host from multiple organ failure. CONCLUSIONS. Our results showed that TNF levels are elevated in acute pancreatitis and may suggest a role for this cytokine in the pathogenesis of the disease.

Abstract: Tumor necrosis factor (TNF) is an inflammatory cytokine that may be an important mediator in the development of the systemic sequelae associated with severe acute pancreatitis. The purpose of this study was to determine whether the neutralization of TNF-alpha with a polyclonal antibody could ameliorate selected biochemical parameters of severe pancreatitis in a rat model. Pancreatitis was induced by an antegrade injection of artificial bile into the bile duct. Forty rats were randomized into 4 groups: no surgery (controls), saline infusion to bile duct (sham), placebo treatment in animals with pancreatitis (placebo + Px), and pretreatment with a polyclonal antibody (PAb) in animals with pancreatitis (PAb + Px). Serum TNF-alpha, amylase, calcium, hematocrit, glucose, and ascites volume were measured 2 hours after bile duct infusion. Pretreatment with the PAb produced a significant improvement in all parameters when compared with pancreatitis animals treated with placebo (p < 0.001). In addition, TNF-alpha, which was elevated in animals with pancreatitis, was reduced significantly in treated animals (p < 0.001). These results suggest that TNF-alpha may be an important mediator in the evolution of the systemic manifestations of severe acute pancreatitis.

Abstract: The M protein of Streptococcus pyogenes plays a major role in the virulence of these bacteria. Members of the M protein superfamily are characterized by the presence of tandem segments of repeated amino acid sequences. The NH2-terminal end of the M proteins is a hypervariable region that harbors the type-specific epitopes of the molecule. Pepsin cleaves the molecule into a highly conserved carboxyl terminal half and a variable amino terminal portion referred to as pep M. In some individuals, infection with certain serotypes of group A streptococci is followed by autoimmune disorders such as rheumatic fever and acute glomerulonephritis. The serotypes of M protein that show a high degree of association with acute rheumatic fever are referred to as rheumatogenic serotypes. We have reported that one such serotype, type 5, is a superantigen to human T cells, specifically stimulating T cells bearing V beta 2, V beta 4, and V beta 8 elements. Here we extend our studies by examining other rheumatogenic serotypes for superantigenic properties. Studies with types 6, 18, 19, and 24 M proteins revealed that they are all superantigens to human T cells. The specificity to V beta 4 was shared by the rheumatogenic M proteins tested; however, each pep M serotype has its unique characteristic set of V beta specificity and these are distinct from those reported for the streptococcal pyrogenic exotoxins. The non-rheumatogenic serotype, pep M2, only stimulated V beta 2-bearing T cells. This study establishes that the structurally related M proteins represent a family of streptococcal superantigens analogous to the structurally related family of staphylococcal enterotoxin superantigens.

Abstract: Streptococcal pyrogenic exotoxin F (SpeF), previously referred to as mitogenic factor, is a newly described potent mitogen produced by group A streptococci. To investigate whether this protein belongs to the family of microbial superantigens, we analyzed the cellular and molecular requirements for its presentation to T cells and compared it with the known streptococcal superantigen pyrogenic exotoxin A (SpeA) and the nonspecific polyclonal T-cell mitogen phytohemagglutinin (PHA). SpeF and SpeA were efficiently presented by autologous antigen-presenting cells (APCs) and an allogeneic B lymphoma cell line, Raji. In contrast, the monocytic cell line U937, which does not express major histocompatibility complex (MHC) class II molecules, failed to present SpeF as well as SpeA but supported the response to PHA. Thus, the presentation of SpeF by APCs was class II dependent but not MHC restricted. The requirement for HLA class II was further supported by the ability of anti-HLA-DQ monoclonal antibody to block the SpeF-induced proliferative response by 75 to 100%. Paraformaldehyde (PFA) fixation of autologous APCs resulted in an impaired ability of SpeF and SpeA to induce optimal T-cell proliferation. In contrast, fixation of Raji cells did not affect the induced proliferation. The stimulatory effect of PHA remained unaffected by both the use of PFA-fixed APCs and the addition of the HLA class II-specific monoclonal antibodies. The addition of a supernatant enriched in interleukin 1 and interleukin 6 to fixed autologous APCs resulted in an increased SpeF-induced response; thus, the impairment was not due to a requirement for processing, but, rather, costimulatory factors produced by metabolically active APCs were needed. SpeF was found to preferentially activate T cells bearing V beta 2, 4, 8, 15, and 19, as determined by quantitative PCR. The data presented clearly show that SpeF is a superantigen. We also studied the prevalence of the speF gene in clinical isolates by Southern blot analyses, and the gene could be detected in 42 group A streptococcal strains, which represented 14 serotypes.

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Abstract: Two peaks of methionine adenosyltransferase (MAT) activity from human erythrocytes were partially purified on a DEAE-cellulose column. Using anti-MAT antibodies, a 60 kDa form of MAT, referred to as rho was identified in peak I. Although rho represented the major MAT protein in crude erythrocyte extracts, the enzyme was very labile and accounted for only 6% of the total MAT activity. Peak II enzyme was stable, and consisted of the previously described catalytic alpha (53 kDa) subunit and the beta subunit (38 kDa), both of which are found in activated human lymphocytes and leukemic cells of lymphoid origin. Mature normal and polycythemic erythrocytes contained predominantly rho as the major MAT protein, while nucleated erythrocytes and reticulocytes contained predominantly the lambda (68 kDa), the major form found in resting human lymphocytes. Human erythroleukemic cells (HEL 92.1.7) contained the alpha, alpha' and beta subunits of MAT, and in this regard was indistinguishable from MAT found in activated lymphocytes and leukemic cells of lymphoid origin (Jurkat). Since rho was generated during the incubation of extracts from resting lymphocytes, which contain predominantly lambda, in the absence of protease inhibitors; the rho form of MAT appears to be derived from the lambda form by proteolytic cleavage. The data indicate that distinct forms of MAT are present at different stages of erythrocyte maturation and reveal the presence of a new form of MAT with reduced activity compared to previously described forms.

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Abstract: The CD28 pathway functions to provide costimulatory signals that are required for TCR-mediated activation of T cells. The role of this pathway in superantigenic stimulation of resting human T cells was investigated in the presence and absence of APC using a streptococcal superantigen, pep M5. Anti-B7/BB1 mAb inhibited the response of T cells when pep M5 was presented by APC. In the absence of APC, cross-linking CD28 by anti-CD28 mAb provides signals that synergize with APC-derived cytokines and with superantigen resulting in T cell proliferation. Anti-HLA-DR, -DQ mAb blocked the response of T cells to pep M5 presented by APC but had no effect on the response of purified T cells to the superantigen costimulated via CD28 cross-linking. These data show that the CD28 pathway is important for superantigenic stimulation of T cells and that signaling through this pathway can substitute for the APC-associated costimulatory activity that is essential for T cell stimulation. Moreover, the results are consistent with the notion that, in the presence of appropriate costimulation, pep M5 can directly interact with T cells and induce them to proliferate.

Abstract: The requirements for T-cell activation by the streptococcal superantigen (SAg), pepsin-extracted M protein from type 5 streptococci (pep M5), were studied by monitoring Ca2+ influx and cell proliferation. Cells from a pep M5-specific T-cell line showed no change in intracellular Ca2+ levels in response to pep M5 when added alone or with freshly isolated autologous antigen-presenting cells (APC). However, after being incubated with pep M5 overnight, the APC secreted soluble factors that together with pep M5 induced a marked increase in intracellular Ca2+ levels in pep M5-specific T cells or freshly isolated, purified T cells. Removal of the SAg from the overnight APC-derived supernatants resulted in loss of the Ca(2+)-mobilizing activity, which was restored within seconds of addition of SAg, suggesting that both the SAg and the soluble factors synergize to induce the Ca2+ influx. Induction of cell proliferation required additional signals inasmuch as the activated APC-derived supernatant failed to synergize with pep M5 to induce the proliferation of purified T cells and required the presence of phorbol myristate acetate for this activity. Metabolically inactive, fixed APC were impaired in their ability to present pep M5 to T cells. Presentation of pep M5 by fixed APC was, however, restored when the APC-derived soluble costimulatory factors were added to the culture. Our data suggest that pep M5-induced activation of T cells is dependent on APC-derived soluble factors and an APC membrane-associated costimulatory molecule(s). These interactions may be important in regulating the in vivo responses to M proteins, could contribute to the severity or progression of infections with Streptococcus pyogenes, and may influence the susceptibility of individuals to its associated nonsuppurative autoimmune sequelae.

Abstract: We undertook this study to determine the quality, quantity, and temporal relationship of pep M5-induced cytokine release. The ability of pep M5 to stimulate interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) production by a T-cell-depleted, monocyte- and B-cell-enriched cell population was dependent on the presence of T cells. The requirement for T cells could be met by addition of exogenous gamma interferon (IFN-gamma). In the presence of IFN-gamma, pep M5 induced the release of TNF-alpha, IL-1, and IL-6, TNF-alpha levels peaked at 24 h, while IL-1 and IL-6 levels peaked at 48 h. pep M5 induced T cells to produce IFN-gamma, which may have accounted for the ability of the super antigen to induce the production of IL-1, IL-6, TNF-alpha, and TNF-beta by peripheral blood mononuclear cells (PBMC). The addition of excess IFN-gamma to cultures of pep M5 and PBMC did not further increase the release of these cytokines at 24 and 48 h but resulted in sustained higher levels at 72 h. Interestingly, TNF-beta production occurred only in the presence of pep M5 and exogenous IFN-gamma. The ability of pep M5 to induce cytokine production was compared with that of a potent super antigen, staphylococcal enterotoxin B (SEB). SEB was a 2- to 14-fold-more-potent inducer of IFN-gamma production. Furthermore, the profile of cytokine released by PBMC in response to this super antigen mimicked that seen with pep M5 in the presence of exogenous IFN-gamma. In conclusion, pep M5 induces the production of cytokines that are involved in immune regulation and inflammation. These cytokines also play a major role in human T-cell responses to this super antigen.

Abstract: A double-blind, randomized, prospective study was undertaken to determine if the dose of OKT3 used for induction immunosuppression following kidney and kidney-pancreas transplantation affected clinical outcomes. Twenty-five patients were randomized in each group. Five patients in each group received a combined kidney/pancreas transplant. All patients received sequential quadruple immune suppression (azathioprine and methylprednisolone, followed by oral prednisone and cyclosporine A), regardless of randomization to the standard (5 mg) or low-dose (2 mg) OKT3 group. OKT3 was administered for 7 to 14 days. The dose of OKT3 was adjusted to ascertain the clearance of peripheral positive CD3 lymphocytes. The mean cumulative OKT3 dose for the standard dose group was 52.0 mg versus 23.4 mg for the low-dose group (P < 0.00001). Dosage increases were necessary for 29% of the standard dose and 32% of the low-dose patients. The side effect score for the standard versus low-dose group was not statistically different (0.79 +/- 0.58 v 0.84 +/- 0.68), except for chills, which occurred more frequently in the low-dose-treated patients (P = 0.003). Anti-OKT3 antibodies developed with similar frequency in both dosage groups, with 8% exhibiting titers of 1:500 or greater at the end of treatment. Kidney graft survival was 96% for the standard dose and 92% for the low-dose group. The overall incidence of rejection was similar in both groups; however the low-dose group did experience an increase in early rejection episodes. The incidence of major and minor viral and bacterial infections was also similar for both dosage groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Superantigens interact with specific V beta elements of the T cell receptor and consequently activate all T cells bearing those elements. The ability of superantigens to stimulate T cells depends on the presence of APC that express MHC class II molecules on their surface. The question we are addressing is: do superantigens have to be seen in context of MHC class II molecules, or can they be recognized directly by T cell-receptor elements? We have previously shown that the APC requirement for the stimulation of T cells by the streptococcal superantigen, pep M5, can be bypassed by the addition of PMA and cytokines or by crosslinking CD28 molecules. Here we asked if the response of APC-depleted T cells to this superantigen is V beta-restricted and whether in the presence of PMA and cytokines the specificity of pep M5 to V beta elements is altered. We provide evidence that in the absence of APC, but in the presence of PMA and cytokines, the specificity of pep M5 to V beta elements is identical to that observed when APC are present, with V beta 2, V beta 4, and V beta 8 being significantly expanded. In addition, we ruled out the possibility that the response is due to a minor contamination with APC or to the expression of DR molecules on T cells because anti-HLA class II monoclonal antibodies did not block the reconstituted response, whereas they totally abrogated the response in the presence of APC. We conclude that pep M5 does not have to complex with MHC class II molecules in order to interact with specific V beta elements. In addition, we propose that the inhibitory effects of the anti-class II antibodies when APC are present may be due to preventing pep M5 from binding and activating APC, thereby blocking the production of costimulatory molecules necessary for T cell activation by this superantigen.

Abstract: Ten overlapping synthetic peptides, spanning the entire amino acid sequence of pepsin-extracted type 5 M protein, (pep M5), were used to delineate regions within this molecule that are important for the mitogenic activity of this streptococcal superantigen. Two of the overlapping peptides, SM5-8 and SM5-10, blocked the response of T cells to pep M5 in a dose-dependent manner. Neither peptide was toxic to T cells, but SM5-8 exhibited nonspecific inhibition of the T-cell response to either superantigen or polyclonal mitogens. In contrast, the inhibition by SM5-10 was pep M5-specific. Analysis of TCR V beta gene usage of the pep M5 response in the presence and absence of SM5-10 revealed that this peptide preferentially blocks the expansion of pep M5-specific V beta elements. The degree of inhibition of V beta expansion varied from one individual to the other, reflecting their TCR repertoire. SM5-10 did not significantly inhibit the response of T cells to other streptococcal superantigens. The data suggest that the region represented by SM5-10 of the pep M5 is important for superantigenic activity. In addition, the ability of this synthetic peptide to inhibit the response to pep M5 provides evidence that the mitogenic response is indeed mediated by this superantigen and is not a result of contamination with other streptococcal superantigens.

Abstract: Methionine adenosyltransferase (MAT), a key enzyme in metabolism, catalyzes the synthesis of one of the most important and pivotal biological molecules, S-adenosyl-methionine. In every organism studied thus far, MAT exists in multiple forms; most are encoded by related, but distinct genes. Molecular and immunological studies revealed the presence of considerable conservation in the structure of MAT from different species; however, the various MAT isozymes differ in their physical and kinetic properties in ways that allow them to be regulated differently. Recent studies suggest that human MAT is composed of nonidentical subunits that can assume multiple states of aggregation, each with different kinetic characteristics. The tissue distribution of MAT isozymes and the ability of cells within the same tissue to switch between the different forms of MAT suggest that this mode of regulation is important for cellular function and differentiation. Therefore, understanding the regulation and structure-function relationship of this fascinating enzyme should help us clarify its role in biology and may provide us with tools to effectively manipulate its activity in clinical situations such as cancer, autoimmunity and organ transplantation.

Abstract: A number of streptococcal products, including the streptococcal pyrogenic exotoxin (SPE) types A, B, and C as well as a 22-kDa fragment of M type 5 protein (pep M5), are potent stimulants of human T-lymphocyte blastogenesis and belong to the newly designated family of superantigens. The V beta usage of human T cells stimulated with these toxins was investigated by using the polymerase chain reaction. We demonstrate that SPE A, B, and C as well as pep M5 stimulate the proliferation of T cells in a dose-dependent manner. pep M5 stimulates cells bearing V beta 2, 4, and 8 elements of the T-cell receptor (TCR), whereas SPE A stimulates TCR V beta 2-, 12-, 14-, and 15-bearing cells. SPE B stimulated only cells expressing TCR V beta 8 elements, while SPE C stimulated cells expressing V beta 1, 2, 5.1, and 10. These studies reveal that the preferential usage of particular V beta elements is distinct for these different superantigens, which may be important in the pathogenesis of various streptococcal diseases.

Abstract: M protein from type 5 group A streptococci has been identified as a member of the family of polyclonal T cell activators termed superantigens because it preferentially stimulates T cells bearing specific V beta elements of the T cell receptor (TCR). In this study the molecular and cellular requirements for presentation of this protein to T cells were investigated. Only accessory cells (AC) expressing class II major histocompatibility complex (MHC) molecules were capable of supporting T cell activation in response to a 22 kDa fragment of M protein (pep M). Despite the need for class II elements, processing of pep M5 by the antigen-presenting cells (APC) was not required for T cell proliferation induced by pep M5. Fixation of APC by paraformaldehyde (PF) treatment impaired their ability to induce optimal T cell proliferation in response to pep M5 without significantly affecting interleukin (IL-2) production. In contrast, PF-fixation of cells from the B cell lymphoma line, Raji, did not affect their ability to present pep M5 to human T cells. Addition of rIL-1 and IL-6 to PF-treated APC restored pep M5-induced blastogenesis. Our data suggest that pep M5 directly associates with HLA class II molecules forming a complex that can induce IL-2 production but not optimal proliferation by T cells. Additional signals provided by the AC are required to trigger optimal T cell proliferation in response to this superantigen.

Abstract: We have recently reported that methionine adenosyltransferase (MAT) in resting human peripheral blood T-cells is primarily present in the form of a precursor which we named lambda. This protein decreases upon cell stimulation, as both MAT activity and the amount of the catalytic alpha/alpha' subunits of the enzyme increase. When resting cells are activated by phytohemagglutinin, the decrease in lambda and increase in alpha/alpha' occurs after interleukin 2 (IL-2) production and before DNA synthesis. The human T-leukemia cell line, Jurkat, is unique in its ability to produce IL-2 in response to exogenous stimuli such as T-cell mitogens and therefore provides a convenient model for studying biochemical reactions involved in T-cell activation. In this study the regulation of MAT activity and S-adenosylmethionine (AdoMet) in resting and activated Jurkat cells was investigated. Here we report that MAT activity in unstimulated Jurkat cells is about 10- and 3-fold higher than the activity in resting and activated peripheral blood mononuclear cells, respectively. Activation of Jurkat cells with phytohemagglutinin resulted in increased IL-2-production, but not an increase in MAT activity. Identical results were obtained using freshly isolated cells from acute lymphoblastic leukemia patients. AdoMet utilization and pool size were approximately 3- and 10-fold higher, respectively, in Jurkat cells compared to peripheral blood mononuclear cells, and both parameters were unaffected by phytohemagglutinin stimulation. Jurkat MAT was determined to be structurally indistinguishable from enzyme from T- or B-leukemia cells but was different from resting, normal T-cells in that it lacked the lambda form. Furthermore, unlike MAT in resting T-cells, the relative amounts of the alpha, alpha', and beta subunits of the enzyme did not change throughout the course of IL-2 induction. We conclude that AdoMet metabolism and MAT activity in Jurkat cells are constitutively high and that induction of IL-2 synthesis in these cells is independent of changes in AdoMet synthesis or turnover. The lack of the lambda form and the difference in MAT regulation between leukemic T-cells and peripheral blood mononuclear cells may be exploited in the design of specific chemotherapeutic agents.

Abstract: Activation of resting human peripheral blood T lymphocytes by the lectin phytohemagglutinin results in an increase in methionine adenosyltransferase (MAT) activity, accompanied by an increase in the amount of the alpha/alpha' catalytic subunits of the enzyme. In contrast, the amount of the noncatalytic beta subunit remains constant throughout the course of the response. Using both polyclonal antibodies to the holoenzyme and monoclonal antibodies to the alpha/alpha' subunits, we detected a cross-reactive 68-kDa protein, which we refer to as lambda. This protein is present in high abundance in resting T cells but decreases upon cell stimulation, as both MAT activity and the amount of the catalytic alpha/alpha' subunits increase. The decrease in lambda and increase in alpha/alpha' occurs after interleukin-2 production and before DNA synthesis. lambda virtually disappears when the cells are actively dividing. Several continuous T cell lines (HPB-ALL, MOLT-4, and Jurkat) as well as a freshly isolated T cell leukemia (ALL-2) had no detectable lambda. The Km for L-methionine for enzyme from resting peripheral blood mononuclear cells was 19-23 microM, which is 3-8-fold higher than purified MAT from fresh leukemic cells or enzyme from Jurkat cells, both of which have a Km of 3.5-3.8 microM. Kinetic analysis of enzyme activity from activated peripheral blood mononuclear cells suggested the presence of two forms of enzyme catalyzing the synthesis of AdoMet. After separation of lambda from the alpha and beta subunits by hydrophobic chromatography, it was determined that lambda has MAT activity but that it is significantly less active than the form containing the alpha subunit. It therefore appears that in resting T cells MAT is sequestered as a less active form. We hypothesize that lambda is a precursor to the catalytic subunits of human lymphocyte MAT and propose that the transition from lambda to alpha/alpha' may be important in the response of T cells to mitogenic signals.

Abstract: S-Adenosylmethionine (AdoMet), inorganic pyrophosphate (PPi) and inorganic phosphate (Pi) are potent product inhibitors of AdoMet synthetase and have been postulated to play a role in increasing AdoMet levels and turnover in peripheral blood mononuclear cells (PBM) after stimulation with phytohemagglutinin (PHA). Measurements of these metabolites in PHA-stimulated PBM showed the expected 2- to 3-fold increases in AdoMet after 8 h, and smaller increases in PPi and Pi. Since the kinetic model requires substantial decreases in PPi and Pi in response to PHA, product inhibition cannot explain the observed changes in AdoMet metabolism in this system. A 2.5-fold increase in AdoMet synthetase catalytic activity was found in crude extracts of PBM within 8 h of PHA-stimulation and probably accounts for increased cellular levels and utilization of AdoMet. Immunochemical analyses with a monoclonal antibody specific for the alpha/alpha' subunits of human lymphocyte AdoMet synthetase showed that these increases in catalytic activity were not associated with increases in immunoreactive protein. The ratio of catalytic activity to immunoreactivity in stimulated cells was 4-fold higher than in unstimulated controls and almost identical to that found in extracts from the human B-lymphocyte line WI-L2. Unstimulated PBM appear to contain substantial amounts of AdoMet synthetase alpha/alpha' subunit with reduced or absent catalytic activity, which can be activated by PHA-stimulation.

Abstract: A synthetic 48-bp oligonucleotide specifying the N-terminal 15 amino acids of M protein of Streptococcus pyogenes type 5 (plus a CTA codon, to terminate translation of genes with the insert in reverse orientation) was inserted by blunt-end ligation at the site of the 48-bp EcoRV deletion in the Salmonella flagellin gene in plasmid pLS408 (S. M. C. Newton, C. O. Jacob, and B. A. D. Stocker, Science 244: 70-72, 1989). The resulting plasmid was transferred from Escherichia coli via a restriction-negative Salmonella typhimurium strain into an aromatic-compound-dependent, flagellin-negative live-vaccine strain of Salmonella dublin to produce strain SL7127, which was motile. Expression of the inserted epitope in flagellin and its exposure at the flagellar filament surface were shown by immunoblotting and by the reaction of flagellate bacteria (immobilization, immunogold labeling) with antibody raised by injection of the corresponding synthetic peptide, S-M5(1-15). Rabbits immunized by injection of the live-vaccine strain with flagella composed of the chimeric flagellin or by injection of concentrated flagella from such bacteria developed antibodies reactive in an enzyme-linked immunosorbent assay with peptide S-M5(1-15) and with the large peptic-digest peptide pepM5. These antibodies were opsonic for type 5 streptococci. Mice that were given parenteral live SL7127 (six doses, each 1 x 10(6) to 2 x 10(6), over 8 weeks) developed titers of ca. 12,800 for the M5-specific peptides and opsonizing activity for type 5 streptococci but not for type 24 streptococci. Sera from mice similarly immunized with a control live vaccine strain without an insert in the flagellin gene did not react with the M5-specific antigens. All of the five mice given the control strain, without an insert, died after challenge with type 5 streptococci or type 24 streptococci; by contrast, four of the five mice given strain SL7127, with an insert, survived the M5 challenge, but none of the five challenged with the type 24 strain survived. Therefore, our study shows that an M protein epitope can be expressed in the context of an unrelated protein and maintain its immunogenicity. Furthermore, we demonstrate that mice can be protected against a Streptococcus pyogenes type 5 challenge by immunization with a Salmonella live vaccine with flagella made of flagellin with an insert carrying a protective epitope of M5 protein but without the cross-reactive epitopes of the complete protein.

Abstract: Isolated islet allografts survive indefinitely in the abdominal testis of nonimmunosuppressed diabetic rats. The predominant feature of these testes is that the presence of Sertoli cells, but not Leydig cells, is required for extended survival of the islet allografts. Sertoli cells cultures were therefore established in vitro and we examined the effects of the conditioned media on Con A--stimulated spleen lymphocyte proliferation. These studies revealed that a product(s) secreted by Sertoli cells inhibits Con A-stimulated lymphocyte proliferation in a dose-dependent manner. The synthesis of this product is both temperature-dependent, occurring predominantly at 37 degrees C, and hormone-dependent, requiring the presence of follicle stimulating hormone, in the culture medium. We further examined the mechanism of inhibition of lymphocyte proliferation and showed that Sertoli cell-enriched media inhibit the production of IL-2 in a dose-dependent manner. Furthermore, the finding that the addition of exogenous IL-2 is not able to reverse this inhibition indicates that the Sertoli cell-enriched media inhibit both IL-2 production and IL-2 responsiveness of T lymphocytes.

Abstract: M proteins, the major virulence factor of group A streptococci, have been implicated in the pathogenesis of acute rheumatic fever (ARF) and other streptococcal related autoimmune diseases. A 22-kD fragment of M type 5 protein is a potent stimulant of human T cells and has recently been shown by our laboratory to belong to the newly designated family of superantigens. Using flow cytometry and the polymerase chain reaction, we demonstrate that this molecule reacts with subsets of human T cells expressing specific T cell receptor (TCR) V beta elements, namely V beta 2, 4, and 8. We employed similar techniques to analyze the TCR V alpha usage of pep M5-stimulated T cells. These studies revealed that the preferential usage of particular V alpha elements is not specific for the superantigen; rather, it may reflect the repertoire of the individual being tested. The expansion of a large number of T cells bearing specific TCR V beta sequences by M protein may account for its role in mediating the pathogenesis of post-streptococcal diseases. Furthermore, the preferential usage of TCR V alpha elements in certain individuals may be an important factor that predisposes them to development of self-reactivity.

Abstract: Rabbit antisera evoked by type 5 streptococcal M protein (M5) were screened by enzyme-linked immunosorbent assay (ELISA) for immunological cross-reactivity with purified rabbit muscle phosphorylases a and b. Of 10 pep M5 antisera tested, 3 showed significant cross-reactivity with both forms of the enzyme. ELISA inhibition studies using one of the pep M5 antisera showed that all of the phosphorylase b antibodies were inhibited by pep M5, the immunogen, and phosphorylase b, the ELISA antigen. All of the antibodies were also inhibited by pep M6 and pep M19, but not by pep M24, indicating that the cross-reactive epitopes were shared by multiple serotypes of M protein. Western blot (immunoblot) analyses showed that pep M5 antisera reacted strongly with the subunit of phosphorylase b. In addition, purified phosphorylase partially inhibited the binding of pep M5 antibodies to a 95-kilodalton protein of human myocardium. One of the three cross-reactive pep M5 antisera inhibited the enzymatic activity of phosphorylase a in a dose-related fashion, reaching a maximum inhibition of 75%. The enzymatic activity in the presence of antibody was totally restored when the antiserum was first incubated with pep M5.

Abstract: Lipoteichoic acid isolated from Streptococcus faecalis or Streptococcus pyogenes caused direct activation of the respiratory burst in human peripheral blood monocytes. This activity appears to be related to the ability of lipoteichoic acid to bind to the monocyte membrane and trigger the polarization of receptors (capping).

Abstract: A steady-state kinetic analysis of human S-adenosylmethionine synthetase indicates that the reaction is Bi Ter with ordered addition of ATP and L-methionine and release of S-adenosylmethionine as the first product. Pyrophosphate and phosphate are then released randomly. I-Parabolic inhibition by phosphate with respect to ATP indicates that this product must bind to more than one site. A model in which phosphate binds to the pyrophosphate site gives a rate equation that is consistent with the kinetic data. Values have been determined for those constants in the equation that are large enough to evaluate, and the in vitro kinetic behavior of S-adenosylmethionine synthetase can be predicted at substrate and product concentrations that are expected intracellularly. Inhibition by combinations of products, especially pyrophosphate and phosphate, is synergistic. Of particular interest is the ability of pyrophosphate and phosphate to increase the sensitivity of the enzyme to inhibition by S-adenosylmethionine. This phenomenon may play a role in regulating steady-state cellular concentrations of S-adenosylmethionine.

Abstract: The requirement for co-stimulatory molecules in T-cell stimulation by mitogens and superantigens in the absence of antigen-presenting cells (APC) was investigated. Phytohemagglutinin (PHA) induced interleukin (IL)-2 receptor (IL-2R) expression on purified T-cells, but proliferation occurred only when exogenous IL-2 was added. In contrast, the proliferative response to a pepsin-extracted type 5 M-protein from Streptococcus pyogenes (pep M5), a recently identified superantigen, required signals provided by phorbol 12-myristate 13-acetate (PMA), IL-1 and IL-6. pep M5 alone did not induce IL-2R expression; however, when combined with PMA, IL-1 and IL-6, IL-2R was expressed. Differences were also observed in the response of the leukemic T-cell line, Jurkat, to PHA and pep M5. Soluble PHA, but not pep M5, induced IL-2 production by these cells in the presence of PMA. Cross-linking by its specific antibody or adsorption of pep M5 to microtiter plates was required to activate Jurkat cells. Both PHA and pep M5 induced Ca2+ mobilization in Jurkat cells; however, only PHA induced a rise in intracellular Ca2+ in purified T-cells, whereas pep M5 was unable to induce this activity unless IL-1, IL-6 and PMA were added. Our data provide biochemical evidence that mitogenic and superantigenic stimulation of T-cells is different.

Abstract: M proteins that define the serotypes of group A streptococci are powerful blastogens for human T lymphocytes. The mechanism by which they activate T cells was investigated and compared with the conventional T cell mitogen phytohemagglutinin, and the known superantigen staphylococcal enterotoxin B. Although major histocompatibility complex (MHC) class II molecules are required for presentation, there is no MHC restriction, since allogeneic class II molecules presented the bacterial protein to human T cells. Type 5 M protein appears to bind class II molecules on the antigen-presenting cells and stimulate T cells bearing V beta 8 sequences. Our results indicate that this streptococcal M protein is a superantigen and suggest a possible mechanism of its role in the pathogenesis of the postinfectious autoimmune sequelae.

Abstract: Although the physical and kinetic properties of S-adenosylmethionine (AdoMet) synthetases from different sources are quite different, it appears that these enzymes have structurally or antigenically conserved regions as demonstrated by studies with AdoMet synthetase specific antibodies. Polyclonal anti-human lymphocyte AdoMet synthetase crossreacted with enzyme from rat liver (beta isozyme), Escherichia coli and yeast. In addition, polyclonal anti-E. coli enzyme and antibodies to synthetic peptides copying several regions of the yeast enzyme reacted with the human gamma and rat beta isozymes. Antibodies to yeast SAM1 encoded protein residues 6-21, 87-113 and 87-124 inhibited the activity of human lymphocyte AdoMet synthetase, while antibodies to residues 272-287 had no effect on the enzyme activity. Our results suggest that these conserved regions may be important in enzyme activity.

Abstract: Stimulation of T cells by superantigens has been reported to be dependent on the presence of APC where binding to class II molecules is a prerequisite to recognition by the TCR. We examined the response of human T cells and a leukemic T cell line, Jurkat to the superantigen, streptococcal M protein. We show that immobilized or cross-linked streptococcal M protein stimulates Jurkat cells (V beta 8), but not normal purified human T cells, to produce IL-2. Activation of purified T cells by this superantigen required costimulatory signals provided by PMA, IL-1, and IL-6. These cytokines and growth factors alone can induce IL-2 production by T cells; however, proliferation occurred only in the presence of superantigen, which together with PMA, IL-1, and IL-6 induced the expression of IL-2R alpha on T cells. Similar results were obtained when the response of purified T cells to another known superantigen, staphylococcal enterotoxin B were examined, indicating that this phenomenon is not unique to M protein. Superantigens interact with a large number of T cells with particular V beta, and thus provide excellent models for studies of the role of biochemical events and signal transduction in T cell activation. Understanding these events may also explain the pathogenesis of autoimmune diseases associated with certain superantigens, such as streptococcal M protein that is thought to be involved in rheumatic fever and rheumatic heart disease.

Abstract: Purified polypeptide fragments of certain surface M proteins of group A streptococci stimulate blastogenesis and the differentiation of cytotoxic T lymphocytes of normal human lymphocytes. The biochemical basis of lymphocyte stimulation by a type M5 protein polypeptide fragment (pep M5) was investigated. Optimal blastogenic doses of pep M5 or phytohemagglutinin stimulated the phosphorylation of several cellular proteins. However, pep M5 but not phytohemagglutinin induced the phosphorylation of 28- and 35-kDa proteins. The 28-kDa protein was shown to be phosphorylated only at serine residues, whereas the 35-kDa protein was phosphorylated only at tyrosine residues. Stimulation of peripheral blood lymphocytes with pep M5 caused a two-fold increase in the CD8+ and CD4+ 4B4+ subpopulations of T lymphocytes. The phosphorylation of the 28-kDa protein appeared to be confined to the CD4+ T cell subpopulation.

Abstract: Purified group A streptococcal M proteins, pep M5 and pep M6, bearing heart cross-reactive epitopes were compared with pep M24, which lacks such epitopes, in their ability to induce functional differentiation of human T lymphocytes. Lymphocytes activated by pep M5 and pep M6 demonstrated cytotoxic activity against cultured heart cells, whereas pep M24-activated cells differentiated into suppressor T cells, which specifically blocked cytotoxic T lymphocytes against cultured human myocardial cells and not NK cell activity against K562 cells. Pep M5 and not pep M24 induced an increase in the number of CD4, 4B4, helper/inducer T cells. In addition, these M proteins appear to induce different biochemical changes in T lymphocytes. Both pep M5 and pep M24 induced the phosphorylation of a 35-kDa cytoplasmic protein; however, only pep M5 induced the phosphorylation of a 28-kDa membrane protein, primarily in CD4 T cells. These data indicate that the virulent M protein Ag of group A streptococci may exert their effect on the human immune system via different mechanisms. Determining these mechanisms and the biochemical pathways involved in T cell differentiation triggered by these Ag may be important in understanding the pathogenesis of post-streptococcal diseases.

Abstract: A sensitive assay for measuring serine hydroxymethyltransferase activity has been developed, based on the binding of N5,N10-[14C]methylene tetrahydrofolate (THF) to DEAE-cellulose paper. The complete assay requires THF, pyridoxal 5'-phosphate, [14C]serine, and enzyme. The reaction is stopped by streaking an aliquot of the reaction mixture onto a square of DEAE-cellulose paper, washing the paper with water to remove unreacted serine, drying the paper, and counting the bound N5,N10-[14C]methylene-THF. To determine that the labeled product was N5,N10-methylene-THF, unlabeled formaldehyde, which exchanges with the labeled methylene carbon, was added after the product had accumulated; 2 min after the addition of formaldehyde the amount of labeled product was reduced by 50%, and by 85% after 10 min. In addition, glycine, which reverses the reaction, and hydroxylamine, which reacts with the methylene carbon, reduced the number of counts bound to the paper. Binding of product to the filter is proportional to both enzyme concentration and assay time. No counts were retained on phosphocellulose filters. This assay represents a new and simple method for measuring serine hydroxymethyltransferase activity, which can be used to measure enzyme activity in tissue homogenates and for screening large numbers of samples.

Abstract: Methionine adenosyltransferase (MAT) activity, and the concentration of its reaction product, S-adenosylmethionine (AdoMet), were measured in the lenses of rats of different ages; ranging from 1 day of age to over 1 yr. The highest specific activity of MAT was found in the lenses of the one day old rats (sp. ac. 0.327 units/mg-1 protein). After 1 week the specific activity had dropped to 0.067, and by 6 weeks had declined to adult levels (0.02 units/mg-1 protein). AdoMet concentrations were measured by HPLC in perchloric acid extracts. The highest concentration of AdoMet was found in the lenses of day-old rats (48.2 microM), and gradually declined with increasing age, reaching 5.5 microM in the oldest rats. In addition, the specific activity of MAT was found to be higher in the lens epithelium than in the cortex plus nucleus. The specific activity of MAT is almost an order of magnitude higher in the lens epithelial fraction (0.099 units mg-1 protein) than in the combined cortex plus nucleus fraction (0.011 units mg-1 protein).

Abstract: The effects of the specific antigen M5 protein of group A streptococci on AdoMet synthetase activity and AdoMet levels in peripheral blood (PB) lymphocytes were studied and were compared with the effects of the nonspecific polyclonal T cell mitogen PHA. M5 protein stimulated AdoMet synthetase activity, whereas PHA had a biphasic effect with an early inhibitory effect and a later stimulatory effect on AdoMet synthetase activity. S-Carbamyl-L-cysteine (SCC), an inhibitor of human lymphocyte AdoMet synthetase, reduced AdoMet levels and inhibited the blastogenic response of PB lymphocytes to both M5 protein and PHA. Inhibition of the response to M5 protein was stronger than that to PHA. However, the inhibitory effects of SCC were totally reversible by washing the cells. It is our hypothesis that such differences in the biochemical events triggered by specific antigen as opposed to a polyclonal mitogen may determine the direction of the functional differentiation of T lymphocytes.

Abstract: Methionine adenosyltransferase (MAT) has been partially purified from rat lenses using a combination of ammonium sulfate fractionation and hydrophobic chromatography on phenyl Sepharose columns. The partially purified enzyme resembles purified Type II MAT from non-hepatic tissues. The Km for methionine is 3.0 microM, and the Km for ATP is 80 microM. The enzyme is activated by potassium ions (25-50 mM), and inhibited by higher concentrations of potassium. A divalent cation (magnesium or manganese) is essential for activity. The Vmax with magnesium is about five times higher than with manganese, but the optimal manganese concentration is around 2.0 mM, compared with 10-20 mM for magnesium. The enzyme is active over a broad pH range, with optimal activity between pH 7.0 and 8.0. The enzyme is inhibited by all three of its products, phosphate, pyrophosphate, and S-adenosylmethionine. Individually phosphate and pyrophosphate are weak inhibitors, but in combination they show a marked synergistic inhibitory effect. Tripolyphosphate is also an effective inhibitor. The inhibition of the enzyme by the cataractogenic agent, dimethylsulfoxide, further confirmed the similarity to Type II MAT.

Abstract: S-Adenosylmethionine synthetase has been purified to apparent homogeneity from human chronic lymphocytic leukemia cells. Equilibrium sedimentation studies and denaturing polyacrylamide gel electrophoresis indicate that the native enzyme has a molecular weight of 185,000 and a subunit composition of either alpha alpha' beta 2, alpha 2 beta 2, or alpha' 2 beta 2, where alpha, alpha', and beta are polypeptide chains of molecular weight 53,000, 51,000, and 38,000. The alpha and alpha' subunits appear to be the same polypeptide and presumably differ by some kind of post-translational modification. Stoichiometric studies show that the expected products S-adenosylmethionine, pyrophosphate, and orthophosphate are generated in equimolar amounts. The enzyme exhibits linear kinetics with respect to substrate dependency and product inhibition, except for orthophosphate which shows parabolic noncompetitive inhibition with respect to ATP. Initial velocity studies of substrate dependence and product inhibition indicate a steady state mechanism that is ordered Bi Ter with ATP adding before L-methionine and S-adenosylmethionine as the first product released. Pyrophosphate and orthophosphate, however, appear to be released by a random mechanism. Free Mg2+ is an essential activator with a half-maximal effect at 1.0 mM. The Km and Kia for ATP are 31 microM and 84 microM, and the Km for L-methionine is 3.3 microM. The enzyme also has tripolyphosphatase activity which is stimulated by S-adenosylmethionine.

Abstract: Rabbit antiserum to rat peritoneal exudate (PE) macrophage (M phi) antigens was prepared and its reactivity with cell surface proteins of M phi, granulocytes, and lymphocytes was studied by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). A total of 14 membrane antigens were identified of which three were found to be expressed only by M phi and granulocytes. By one-dimensional analysis, a protein with an approximate m.w. of 105,000 was present on splenic and PE M phi and on splenic lymphocytes. Two-dimensional analysis revealed that this band was heterogeneous and contained at least three species, one of which was restricted to expression on M phi and granulocytes. A second protein of 150,000 daltons was resolved into two species by two-dimensional analysis. Both of these species were present on M phi and granulocytes but not on lymphocytes. Both the 105,000- and 150,000-dalton proteins were glycosylated. Because the 105,000- and 150-000-dalton proteins expressed by M phi were also expressed by granulocytes, is is likely that these are differentiation antigens whose expression is a characteristic property of cells within both monocytoid and myeloid lineages. All three 105,000-dalton species and one of the two 150,000-dalton species were detected on mouse M phi, indicating their expression is not unique to the rat.

Abstract: