Abstract: We recently reported that the activation of NF-kappaB and AP-1 was suppressed in monocytes infected with measles virus, but not in infected epithelial cells. This cell-type-specific suppression of the inflammatory response represents a potential for measles virus to evade host immune system. In the current study, we examined the suppression mechanism of lipopolysaccharide (LPS)-induced, namely Toll-like receptor 4 (TLR4)-mediated, activation of NF-kappaB and AP-1 in measles virus-infected monocytic cells. In the infected cells, LPS treatment failed to induce the formation of active protein kinase complex containing TAK1, TAB2 and tumor necrosis factor receptor-associated factor 6 (TRAF6), dissociate from TLR complexes containing Interleukin-1 receptor-associated kinase 1 (IRAK1). Ubiquitin-modifying enzyme A20, which is a host negative feedback regulator of NF-kappaB, was dramatically up-regulated in infected monocytic cells, but not in infected epithelial cells. Suppression of A20 expression by siRNA restored LPS-induced signaling in infected cells. Measles virus phosphoprotein (P protein) expression was necessary and sufficient for the induction of A20. P protein interacted indirectly with a negative regulatory motif in the A20 gene promoter, and released the suppression of A20 transcription, independent of the activation of NF-kappaB.

Abstract: We screened 457 Haemophilus influenzae strains isolated in Japan during 2002 to 2004 and identified 12 fluoroquinolone-resistant strains (2.6%). The resistant strains were divided into three genotypes (eight, three, and one of each type). These were isolated from patients over 58 years of age. Several fluoroquinolone-resistant clones appeared to have invaded the population of elderly patients in a particular area, Sapporo city.

Abstract: Recent studies have suggested a link between iron-deficiency anemia and Helicobacter pylori infection. In the current study, strains of H. pylori derived from patients with iron-deficiency anemia showed enhanced Fe ion uptake and Fe ion-dependent rapid growth compared with those from patients with non-iron-deficiency anemia. H. pylori with enhanced Fe ion-uptake ability may be a causative factor for iron-deficiency anemia.

Abstract: We have characterized the cell surface properties of three mutant series of Pseudomonas aeruginosa that show various defects in their lipopolysaccharide (LPS) core region. The deepest rough mutants of each series used in this study lacked completely rhamnose and glucose, and contained only galactosamine and alanine as LPS outer core constituents. However, rough mutants other than the deepest rough mutants showed high cell surface hydrophobicity compared to the corresponding parental strains, the deepest rough mutants showed less hydrophobicity than other rough mutants. The reactivity of an anti-lipid A monoclonal antibody with the deepest rough mutants was markedly higher than that with other counterparts. The deepest rough mutants tended to be more susceptible to antibiotics, such as gentamicin and polymyxin B, than the corresponding parental strains and other rough mutants. The above evidence indicates that neutral sugar, namely rhamnose and glucose, residues of the LPS outer core region play a critical role in the cell surface properties of P. aeruginosa.

Abstract: Alloiococcus otitidis is a recently discovered bacterium frequently associated with otitis media. However, no study is available as to whether A. otitidis has a pathogenic role and induces local immune response in the middle ear as a true pathogen. Whole bacterial sonicate of A. otitidis was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a nitrocellulose membrane. Then, Western blot analysis was performed with supernatant of the middle ear effusions from children with A. otitidis-positive otitis media. SDS-PAGE of the bacterial sonicate showed several protein bands, designated A1-A11. Western blot analysis revealed the presence of IgG, secretory IgA, IgG2, and IgM against A. otitidis in the middle ear effusions. Absorption of the specimens with sonicates of other major middle ear pathogens did not alter the reactivity of antibodies against the alloiococcal antigens. The results suggest that specific local immune response against A. otitidis is induced during middle ear infection of the organism as a true pathogen. A5, A6 or A11 is expected to be a main antigenic determinant. This is the first report to show evidence of local antibody response against A. otitidis and to disclose antigenic components of A. otitidis.

Abstract: A major cause of the high morbidity and mortality associated with measles infection is attributed to virus-mediated immunosuppression. In this report, we present evidence for a novel strategy of immunosuppression by the measles virus. We observed a marked suppression of lipopolysaccharide (LPS)-induced IL-8, RANTES, TNF-alpha and IL-6 production and NF-kappaB activation in human monocytic cell lines persistently infected with measles virus. This effect was not observed in human epithelial cells lines persistently infected with measles virus. There were no significant differences in expression levels of Toll-like receptors (TLRs) and their associated molecules, or other intracellular signaling molecules of the NF-kappaB signaling pathway in measles-virus-infected monocytic cells compared to uninfected cells. Infected monocytic cells exhibited decreased LPS-induced DNA binding of NF-kappaB and phosphorylation of JNK, namely activation of transcription factors NF-kappaB and AP-1. NF-kappaB was constitutively activated in human epithelial cells persistently infected with measles virus, and LPS treatment resulted in further activation. The cell-type-specific suppression of NF-kappaB activation represents a potential strategy of escape from the host immune system by measles virus via induced immunological silencing in infected cells.

Abstract: Helicobacter pylori is recognized as an etiologic agent of gastroduodenal diseases. Among toxic substances produced by H. pylori, LPS exhibits extremely low endotoxic activity as compared to the typical LPSs, such as that produced by Escherichia coli. We found that the LPS-low-responder stomach cancer cell line MKN28, which expresses Toll-like receptor 4 (TLR4) at extremely low levels, showed similar levels of interleukin-8 (IL-8) induction by H. pylori or E. coli LPS preparations. Weak IL-8 induction by H. pylori LPS preparations was suppressed by expression of a dominant negative mutant of TLR2 but not of TLR4. Data from luciferase reporter analysis indicated that cotransfection of TLR2-TLR1 or TLR2-TLR6 was required for the activation induced by H. pylori LPS preparations. In conclusion, the H. pylori LPS preparations significantly induce an inflammatory reaction via the receptor complex containing TLR2-TLR1 or TLR2-TLR6 but not that containing TLR4. The TLR2-TLR1 complex was preferentially recognized by the H. pylori LPS preparations over the TLR2-TLR6 complex. Whereas the magnitude of response to H. pylori LPS preparation was markedly less than that to E. coli LPS preparation in LPS-high-responder cells strongly expressing TLR4, it was comparable to that of E. coli LPS in low-responder cells expressing negligible amount of TLR4.

Abstract: Our previous study demonstrated that the frequency of penicillin-resistant Streptococcus pneumoniae (PRSP) was lower in our district than in districts in other Japanese studies. In this study, we investigated the prevalence of erythromycin resistance. The susceptibility to erythromycin and the distribution of the macrolide-resistance genes, mefA and ermB, were examined in S. pneumoniae isolates from the upper respiratory tracts of children in four cities in the Sapporo district, Hokkaido prefecture, Japan. Of the 156 isolates, 27 (17.3%) were erythromycin-sensitive, 6 (3.9%) were erythromycin-intermediately resistant, and 123 (78.9%) were erythromycin-resistant. Fifty-nine (37.8%) had the mefA gene, 89 (57.1%) had the ermB gene, and 129 (82.7%) had the mefA and/or the ermB gene. The ermB-positive isolates tended to show high resistance to erythromycin. Erythromycin-resistant isolates and the macrolide-resistance genes were often present in infants or younger children. The frequency of erythromycin-resistant isolates in the four cities was very high, ranging from 76.3% to 83. 3%, as high as the national average. Although erythromycin-resistant isolates generally tend to show cross-resistance to penicillin, the frequency of PRSP was very low in this study, as compared with other Japanese studies. Erythromycin resistance was frequently recognized not only in PRSP but also in penicillin-sensitive S. pneumoniae (PSSP) as well. In Japan, erythromycin resistance may have already become widespread, even in local areas where penicillin resistance is not especially prevalent.

Abstract: Recurrent otitis media are frequently intractable during childhood. It is unclear whether recurrent otitis media is caused by etiological bacteria colonization or by new infections. Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis were isolated from the nasopharynx of 7 otitisprone and 2 non-prone children with recurrent otitis media. Plural bacterial species and strains were found in all children while affected by otitis media. The same strain was repeatedly isolated from all otitisprone children even after administration of antibiotics but was not from the non-prone children. Antibiotic susceptibility did not differ significantly among the same repeatedly isolated strains. This pilot study suggests that the etiological bacteria tend to colonize and is hard to eliminate in otitis-prone children.

Abstract: The pathogenesis of severe acute respiratory syndrome (SARS) is poorly understood and cytokine dysregulation has been suggested as one relevant mechanism to be explored. We compared the cytokine profile in Caco2 cells after infection of SARS coronavirus (SARS-CoV) with other respiratory viruses including respiratory syncytial virus (RSV), influenza A virus (FluAV), and human parainfluenza virus type 2 (hPIV2). Interferon (IFN) system (production and response) was not suppressed by SARS-CoV infection. Therefore, SARS-CoV replication was suppressed by pretreatment with IFN. SARS-CoV and RSV induced high levels of IL-6 and RANTES compared with FluAV and hPIV2. Induction level of suppressor of cytokine signaling-3 (SOCS3) by SARS-CoV was significantly lower than that by RSV in spite of the significant production of IL-6. Toll-like receptors 4 and 9, which correlate with the induction of inflammatory response, were upregulated by SARS-CoV infection. Collectively, overinduction of inflammatory cytokine and dysregulation of cytokine signaling may contribute to the immunopathology associated with "severe" inflammation in SARS.

Abstract: We evaluated the specific IgG antibodies against heat shock proteins (HSPs) in cerebrospinal fluids (CSF) from patients with multiple sclerosis (MS). ELISA was employed to examine IgG antibodies against ten HSPs (HSP27, alphaA and alphaB crystallins, HSP60, CCT, Mycobacterium bovis HSP65, Escherichia coli GroEL, HSP70, HSC70 and HSP90) in CSF from 30 patients with MS, and 25 patients with motor neuron diseases (MND). Significantly higher antibody titers against HSP70 and HSC70 proteins were found in CSF obtained from patients with MS as compared with MND independent of CSF total protein, IgG concentrations and IgG indices, respectively. The antibody titers against HSP70 were indicated to be significantly higher in the progressive cases than in cases of remission. The results suggest that IgG antibodies against specific types of HSPs especially HSP70 family proteins (HSP70 and HSC70) in CSF may play an important role in the pathophysiology of MS through the modification of immune response and cytoprotective functions of molecular chaperons.

Abstract:

Abstract: Auto-antibodies against heat shock proteins (HSPs) are frequently found in the sera of patients with rheumatic and other autoimmune diseases. However, it is unclear whether these auto-antibodies play a role in the pathophysiology and etiology of these diseases. We found that a murine anti-HSP60 mAb enhanced the production of IL-8 and tumor necrosis factor-alpha induced by human HSP60 in the human monocytic cell lines THP-1 and U937, and human peripheral blood monocytes. Similar enhancement was observed with the combination of human HSP70 protein and a murine anti-HSP70 mAb. The enhancing effects were also observed for F(ab')2 fragment, but not for monovalent Fab fragment. This suggests that the enhancement is due to cross-linking of HSP by the anti-HSP antibodies. The induction of IL-8 was dramatically suppressed by the transfection of a dominant-negative mutant of Toll-like receptor 4. We also found that sera from patients with rheumatic autoimmune diseases, which contained higher anti-HSP60 auto-antibody titers than sera from healthy donors, significantly enhanced the IL-8 production induced by human HSP60 in THP-1 cells. We propose that auto-antibodies against HSPs have the potential to play a pathogenic role in rheumatic autoimmune diseases by enhancing inflammatory reactions.

Abstract: OBJECTIVE: Interferons (IFNs)-inducible myxovirus resistance protein A (MxA) has recently been used as an indirect marker of neutralizing antibody against IFN in patients with multiple sclerosis (MS). On the other hand, MxA inhibits the replication of viruses by means of modifying cellular function, including apoptotic pathway. Our objective is to investigate the genetic and pathological role of MxA in patients with MS. METHODS: We examined SNPs of MxA promoter region in 67 patients with MS. Moreover, to elucidate the functional roles of SNPs, we conducted Luciferase assay with pGL3-basic vector including patient-derived or artificially mutated MxA promoter region. RESULTS: A significantly higher frequency of the haplotype with -88T and -123A, which correlates with over-expression of MxA, was observed in MS. Moreover, we elucidated novel findings showing that nt -88 played a leading part with type I IFNs and that nt -123 played the same role independently without type I IFNs, respectively. CONCLUSION: SNPs on MxA promoter region may play an important role in the pathophysiology of MS and provide a novel strategy for the therapeutic resolutions of MS.

Abstract: Evaluation of beta-lactam susceptibility and polymerase chain reaction (PCR)-based genotyping of penicillin-binding proteins (PBP) 1A, 2B, and 2X were performed for Streptococcus pneumoniae isolates from children with otolaryngological infectious disease in the Sapporo district, Hokkaido Prefecture, Japan. Of 174 S. pneumoniae isolates, 14 (8%) were penicillin-resistant S. pneumoniae (PRSP), 87 (50%) were penicillin-intermediately-resistant, and 73 (42%) were penicillin-sensitive. Seventy-six (44%) had alterations in all of the three genes examined (pbp1a, pbp2b, and pbp2x), 81 (47%) had alterations in one or two of the genes, and 17 (10%) had no alterations. Isolates with alterations in all three genes showed low susceptibility to penicillin, while, in contrast, isolates with no alteration showed relatively high susceptibility to penicillin. Similar relationships were observed for other beta-lactams. The prevalence of PRSP in our study ranged from 5% to 12.8% (average, 8%), and there was much variation in the prevalence of PBP gene alterations among the cities. The results suggest that local differences in patterns of PBP gene alterations can be observed even at the district level. PCR-based genotyping of PBP genes is rapid, convenient, and useful to investigate genetic susceptibility to beta-lactams. Further, not only nationwide or prefectural surveys but also local surveillance at the district level is important for determining antimicrobial susceptibility status in daily practice.

Abstract: The rate of propagation of influenza virus in human adenocarcinoma Caco-2 cells was found to negatively correlate with the concentration of fetal bovine serum (FBS) in the culture medium. Virus replicated more rapidly at lower FBS concentrations (0 or 2%) than at higher concentrations (10 or 20%) during an early stage of infection. Basal and interferon (IFN)-induced levels of typical IFN-inducible anti-viral proteins, such as 2',5'-oligoadenylate synthetase, dsRNA-activated protein kinase and MxA, were unaffected by variation in FBS concentrations. But promyelocytic leukemia protein (PML) was expressed in a serum-dependent manner. In particular, the 65 to 70 kDa isoform of PML was markedly upregulated following the addition of serum. In contrast, other isoforms were induced by IFN treatment, and weakly induced by FBS concentrations. Immunofluorescence microscopy indicated that PML was mainly formed nuclear bodies in Caco-2 cells at various FBS concentrations, and the levels of the PML-nuclear bodies were upregulated by FBS. Overexpression of PML isoform consisting of 560 or 633 amino acid residues by transfection of expression plasmid results in significantly delayed viral replication rate in Caco-2 cells. On the other hand, downregulation of PML expression by RNAi enhanced viral replication. These results indicate that PML isoforms which are expressed in a serum-dependent manner suppress the propagation of influenza virus at an early stage of infection.

Abstract: Mumps virus (MuV) has been shown to antagonize the antiviral effects of interferon (IFN) through proteasome-mediated complete degradation of STAT1 by using the viral V protein (T. Kubota et al., Biochem. Biophys. Res. Commun. 283:255-259, 2001). However, we found that MuV could inhibit IFN signaling and the generation of a subsequent antiviral state long before the complete degradation of cellular STAT1 in infected cells. In MuV-infected cells, nuclear translocation and phosphorylation of STAT1 and STAT2 tyrosine residue (Y) at 701 and 689, respectively, by IFN-beta were significantly inhibited but the phosphorylation of Jak1 and Tyk2 was not inhibited. The transiently expressed MuV V protein also inhibited IFN-beta-induced Y701-STAT1 and Y689-STAT2 phosphorylation, suggesting that the V protein could block IFN-beta-induced signal transduction without the aid of other viral components. Finally, a substitution of an alanine residue in place of a cysteine residue in the C-terminal V-unique region known to be required for STAT1 degradation and inhibition of anti-IFN signaling resulted in the loss of V protein function to inhibit the Y701-STAT1 and Y689-STAT2 phosphorylation.

Abstract: Recent studies have speculated on the possible role of the mother in transmitting Helicobacter pylori infection to their children. In an attempt to either prove or disprove this supposition, we investigated the rates of infection of children born to H. pylori-positive mothers from birth to 5 years of age using serology and the stool antigen test. When infection of the children did occur, the strains from the children were compared to those of their mothers using DNA analysis. Sixty-nine of the 350 pregnant mothers (19.7%) had a positive serology for H. pylori. Fifty-one children underwent serological examinations and stool antigen tests at 4 to 6 days after birth, followed by 1, 3, and 6 months. They were continuously given the stool antigen test at 4- to 6-month intervals until the age of 5 years. Gastric juice samples were collected from the infected children and their mothers for culture and DNA analyses using a random amplified polymorphic DNA fingerprinting method. None of the 51 children acquired H. pylori infection during the first year of life. Of the 44 children enrolled in a 5-year follow-up study, five (11%) acquired H. pylori infection. They acquired the infection at the age of 1 year 2 months, 1 year 3 months, 1 year 6 months, 1 year 8 months, and 4 years 4 months. Random amplified polymorphic DNA fingerprinting confirmed that the strains of the five children exhibited DNA fingerprinting patterns identical to those of their mothers. These findings suggest that mother-to-child transmission is the most probable cause of intrafamilial spread of H. pylori.

Abstract: We showed previously that infection of herpes simplex virus type 1 (HSV-1) rapidly induced the suppressor of cytokine signaling-3 (SOCS3), a host negative regulator of the JAK/STAT pathway, in the amnion cell line FL. Thus, HSV-1 suppresses the interferon (IFN) signaling pathway at the step of IFN-induced phosphorylation of janus kinases during an early infection stage. In the present study, we examined SOCS3 induction by HSV-1 infection in several types of human cell lines. FL cells and the T-cell line CCRF-CEM strongly induced SOCS3 during HSV-1 infection. The virus rapidly propagated in both cell lines and produced a lytic infection. On the other hand, the monocytic cell lines U937 and THP-1, and the B-cell line AKATA showed neither SOCS3 induction nor suppression of IFN-induced STAT1 phosphorylation during HSV-1 infection. These cell lines resulted in a persistent or prolonged infection, which continuously produced a low titer of infectious virus. The induction of SOCS3 by HSV-1 should occur via STAT3 activation immediately after HSV-1 infection. SOCS3 induction was inhibited by the addition of a Jak3 inhibitor WHI-P131. Treatment with WHI-P131 or transfection of antisense oligonucleotides specific for SOCS3 dramatically suppressed replication of HSV-1 in FL cells. The suppression of viral replication by WHI-P131 was released in the presence of neutralizing anti-IFN-alpha and anti-IFN-beta antibodies. In conclusion, suppression of IFN signaling by HSV-1-induced SOCS3 is required for efficient replication and lytic infection of HSV-1. The SOCS3 induction varied among cell lines, indicating that it is an important factor determining the cell type specificity of efficient HSV-1 replication.

Abstract: We examined antibodies against 10 heat shock proteins (HSPs) in cerebrospinal fluids (CSF) and sera from patients with Guillain-Barré syndrome (GBS). Significantly higher IgG antibody titers against HSP27, HSP60, HSP70 and HSP90 family, including mycobacterial HSP65 and Escherichia coli GroEL, were found in CSF from GBS patients as compared with motor neuron disease. Serum IgG antibodies against each HSP showed no difference between GBS patients and normal controls. GBS seems to be induced by reactive autoimmune responses frequently triggered by infections. The CSF antibodies against HSPs may modify the immune responses and/or cell-protective functions of HSPs in the pathophysiology of GBS.

Abstract: The etiology of Kawasaki disease (KD) remains unknown, although some infectious organism has been suggested as the cause. Recent studies suggest that some bacterial toxins with superantigen activity are involved in its pathogenesis, but no specific bacterial toxin has yet been identified. Throat swabs for bacterial culture were obtained from 21 patients with KD and 20 with other febrile illnesses as controls. Mitogenic activity in culture supernatants obtained from individual bacterial strains was measured by lymphocyte proliferation assay. Sixty-one bacterial strains were isolated from KD patients, and 62 strains from control patients. There was no apparent difference in bacterial species in the throat flora between KD patients and febrile controls. Moreover, total and individual mitogenic activity of strains from KD patients was no greater than that of strains from febrile controls. The bacterial superantigen activity of throat flora may not play a major role in the pathogenesis of KD.

Abstract: We investigated the induction of interleukin-8 (IL-8) by bacterial lipopolysaccharide (LPS) and peptidoglycan (PGN) in the bladder cancer cell lines T24, 5637, UM-UC-3, and HT1197. T24 and 5637 cells strongly induced IL-8 after stimulation with LPS or PGN in a dose- and time-dependent manner, whereas UM-UC-3 and HT1197 cells did so very weakly. The expression of CD14 at the mRNA, total cellular protein, and cell surface protein levels differed among these cell lines, but the expression levels of Toll-like receptors 2 and 4 (TLR2 and TLR4) were not significantly different. The CD14 expression levels were found to correlate with the inducibility of IL-8 by LPS or PGN. Treatment of T24 and 5637 cells with phosphatidylinositol-specific phospholipase C to eliminate CD14 from the cell surface dramatically suppressed the induction of IL-8. On the other hand, UM-UC-3 cells transfected with CD14 cDNA expressed membrane-anchored CD14 and showed more efficient induction of IL-8 by LPS stimulation than untransfected controls. These results suggest that the presence of the membrane-anchored, but not the soluble, form of CD14 is a strong factor in IL-8 induction in bladder epithelial cells in response to bacterial components. The presence of the membrane-anchored form of CD14 may thus be a determinant for the inflammatory response of uroepithelial cells.

Abstract: We identified fluoroquinolone-resistant Streptococcus pneumoniae strains among 670 clinical isolates isolated from 1999 to 2003 in Hokkaido prefecture, Japan. All eleven stains were resistant to ciprofloxacin and levofloxacin. Furthermore, ten strains were also resistant to fluoroquinolones that are more effective with gram-positive bacteria, namely tosufloxacin, sparfloxacin, and gatifloxacin. Nucleotide sequence analysis of the quinolone-resistance determining region (QRDR) of the quinolone target genes coding for topoisomerase i.v. subunits (parC and parE) and DNA gyrase subunits (gyrA and gyrB). Eight stains, which showed higher resistance, had resistance mutations in two genes (gyrA and parC, or gyrA and parE), and other three strains had one resistance mutation in parC. The mutation patterns were varied between the strains. Data from random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) indicated that eleven strains were identified as ten independent clones. Lines of evidence indicated that genetic mutations leading to fluoroquinolone resistance occur sporadically rather through the spreading of a particular resistant strain. Notably, the fluoroquinolone-resistant strains were only isolated from adults, particularly from patients more than 60 years of age (9/60 strains; 15.0%). Resistant strains were not found in 574 strains isolates from patients under 20 years of age. This may be due to the fact that fluoroquionolones other than norfloxacin are not applicable to children in Japan.

Abstract: We showed previously that herpes simplex virus type 1 (HSV-1) suppresses the interferon (IFN) signaling pathway during the early infection stage in the human amnion cell line FL. HSV-1 inhibits the IFN-induced phosphorylation of Janus kinases (JAK) in infected FL cells. In the present study, we showed that the suppressor of cytokine signaling-3 (SOCS3), a host negative regulator of the JAK/STAT pathway, is rapidly induced in FL cells after HSV-1 infection. Maximal levels of SOCS3 protein were detected at around 1 to 2 h after infection. This is consistent with the occurrence of HSV-1-mediated inhibition of IFN-induced JAK phosphorylation. The HSV-1 wild-type strain VR3 induced SOCS3 more efficiently than did mutants that are defective in UL41 or UL13 and that are hyperresponsive to IFN. Induction of the IRF-7 protein and transcriptional activation of IFN-alpha4, which occur in a JAK/STAT pathway-dependent manner, were poorly induced by VR3 but efficiently induced by the mutant viruses. In contrast, phosphorylation of IRF-3 and transcriptional activation of IFN-beta, which are JAK/STAT pathway-independent process, were equally well induced by the wild-type strain and the mutants. In conclusion, the SOCS3 protein appears to be mainly responsible for the suppression of IFN signaling and IFN production that occurs during HSV-1 infection.

Abstract: Collectins, including surfactant proteins A (SP-A) and D (SP-D) and mannose binding lectin (MBL), are the important constituents of the innate immune system. Mycobacterium avium, a facultative intracellular pathogen, has developed numerous mechanisms for entering mononuclear phagocytes. In this study, we investigated the interactions of collectins with M. avium and the effects of these lectins on phagocytosis of M. avium by macrophages. SP-A, SP-D, and MBL exhibited a concentration-dependent binding to M. avium. The binding of SP-A to M. avium was Ca(2+)-dependent but that of SP-D and MBL was Ca(2+)-independent. SP-A and SP-D but not MBL enhanced the phagocytosis of FITC-labeled M. avium by rat alveolar macrophages and human monocyte-derived macrophages. Excess mannan, zymosan, and lipoarabinomannan derived from the M. avium-intracellular complex, significantly decreased the collectin-stimulated phagocytosis of M. avium. Enhanced phagocytosis was not affected by the presence of cycloheximide or chelation of Ca(2+). The mutated collectin, SP-A(E195Q, R197D) exhibited decreased binding to M. avium but stimulated phagocytosis to a level comparable to wild-type SP-A. Enhanced phagocytosis by cells persisted even after preincubation and removal of SP-A or SP-D. Rat alveolar macrophages that had been incubated with SP-A or SP-D also exhibited enhanced uptake of (125)I-mannosylated BSA. Analysis by confocal microscopy and flow cytometry revealed that the lung collectins up-regulated the cell surface expression of mannose receptor on monocyte-derived macrophages. These results provide compelling evidence that SP-A and SP-D enhance mannose receptor-mediated phagocytosis of M. avium by macrophages.

Abstract: The authors examined antibodies against native vacuolating cytotoxin (VacA) of Helicobacter pylori in CSF from 12 patients with Miller-Fisher syndrome (MFS). The VacA protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis was carried out. Eight of 12 MFS patients had a specific immunoglobulin G antibody against VacA in the CSF. There is sequence homology between VacA and some membrane ion transport proteins, raising the possibility that A-VacA-Ab involves the ion channels in the node of Ranvier in some patients with MFS.

Abstract: In order to establish infection to host cells, viruses suppress or escape from the host immune response against microorganisms by various strategies. Interferon (IFN) system is an important contributor of innate immunity. IFN is induced by viral infection, and it promotes antiviral state through induction and/or activation of the effector molecules. Many viruses possess the suppression or inhibition mechanisms for the anti-viral effector molecules, whereas they also perform inhibition of IFN signaling pathway, JAK/STAT pathway. We consider that latter is a most effective strategy counteracting IFN function, because the signaling pathway is an entrance of the system. The strategies counteracting JAK/STAT pathway are varied among virus species. Viruses perform (i) production of IFN-binding protein, (ii) degradation of JAK/STAT components, (iii) suppression of activation (phosphorylation) of the components, (iv) inhibition of nuclear translocation of activated transcription factor, and (v) induction of host JAK/STAT negative regulator. Here, we present these strategies, especially our recent resulta of HSV1, mumps virus, and measles virus. For example, HSV1 induces a host JAK/STAT negative regulator SOCS3 (suppressor of cytokine signaling-3). Mumps virus V protein promotes degradation of both STAT-1 and STAT-3. Measles virus freezes the flexibility of IFN-alpha receptor complex by the action of viral proteins, C and V.

Abstract: We examined the macrolide susceptibility and the presence of macrolide-resistance genes in 780 Streptococcus pneumoniae strains that were isolated and collected at trunk hospitals and commercial clinical laboratories in Hokkaido prefecture, Japan, between 1999 and 2003. Of the 780 strains, 57.0% and 49.6% were found to bear the macrolide-resistance genes erm(B) and mef(A), respectively, while 87.9% had either or both of these genes. The mef(A)-positive strains were more frequently found in patients who were younger than 10 years (43.4%) compared to patients who were 10 years or older (30.3%), whereas the erm(B)-positive strains were similarly frequent in both groups (57.2% vs 54.9%). Strains that were extremely resistant to erythromycin (> or = 256 microg/ml) were frequently found in strains isolated at trunk hospitals but were rarely found in strains that had been collected at commercial clinical laboratories. In conclusion, the high frequency of emergence of macrolide resistance in S. pneumoniae strains was similar to reports from other areas of Japan and other east Asian countries. However, the distribution of resistant genes to macrolides and the distribution of the minimum inhibitory concentrations (MICs) differed depending on patients' ages and depending on whether the strains were isolated at trunk hospitals or commercial clinical laboratories.

Abstract: Natural infection with measles virus (MeV) is initiated when the virus reaches epithelial cells in the respiratory tract, oropharynx, or conjunctivae. Human epithelial cells infected with MeV frequently show growth suppression. In this study, we investigated the possible mechanisms for this suppression. The bronchiolar epithelial cell A549 showed growth arrest in G(0)/G(1) following MeV infection or treatment with gamma interferon (IFN-gamma). IFN regulatory factor-1 (IRF-1) was upregulated during MeV infection, although A549 did not produce IFN-gamma. Cells of the cervical squamous cell line SiHa persistently infected with various strains of MeV displayed slower growth than uninfected SiHa cells, although the growth rates varied depending on the MeV strain. Transfection of antisense-oriented IRF-1 cDNA released the MeV-infected SiHa cells from growth suppression. Although these infected cells did not produce IFN-gamma and suppressed IFN-alpha/beta-induced Jak1 phosphorylation, Jak1 was constitutively phosphorylated. The growth rates negatively correlated with levels of both IRF-1 expression and constitutively phosphorylated Jak1. These results indicate that MeV upregulates IRF-1 in a manner that is independent of IFN but dependent on the JAK/STAT pathway. This induction of IRF-1 appears to suppress cell growth, although the extent seems to vary among MeV strains.

Abstract: Pulmonary surfactant proteins A (SP-A) and D (SP-D), members of the collectin family, play important roles in the innate immune system of the lung. Here, we show that SP-A but not SP-D augmented phagocytosis of Streptococcus pneumoniae by alveolar macrophages, independent of its binding to the bacteria. Analysis of the SP-A/SP-D chimeras, in which progressively longer carboxyl-terminal regions of SP-A were replaced with the corresponding SP-D regions, has revealed that the SP-D region Gly(346)-Phe(355) can be substituted for the SP-A region Leu(219)-Phe(228) without altering the SP-A activity of enhancing the phagocytosis and that the SP-A region Cys(204)-Cys(218) is required for the SP-A-mediated phagocytosis. Acetylated low density lipoprotein significantly reduced the SP-A-stimulated uptake of the bacteria. SP-A failed to enhance the phagocytosis of S. pneumoniae by alveolar macrophages derived from scavenger receptor A (SR-A)-deficient mice, demonstrating that SP-A augments SRA-mediated phagocytosis. Preincubation of macrophages with SP-A at 37 degrees C but not at 4 degrees C stimulated the phagocytosis. The SP-A-mediated enhanced phagocytosis was not inhibited by the presence of cycloheximide. SP-A increased cell surface localization of SR-A that was inhibitable by apigenin, a casein kinase 2 (CK2) inhibitor. SP-A-treated macrophages exhibited significantly greater binding of acetylated low density lipoprotein than nontreated cells. The SP-A-stimulated phagocytosis was also abolished by apigenin. In addition, SP-A stimulated CK2 activity. These results demonstrate that SP-A enhances the phagocytosis of S. pneumoniae by alveolar macrophages through a CK2-dependent increase of cell surface SR-A localization. This study reveals a novel mechanism of bacterial clearance by alveolar macrophages.

Abstract:

Abstract: Interferon-gamma (IFN-gamma) induced cell death in five oral squamous cell carcinoma (SCC) lines. Cell death was specific to IFN-gamma treatment and did not occur with either IFN-alpha or TNF-alpha. IFN-gamma did not induce typical apoptotic phenotype in cells, such as morphological changes and DNA ladder formation. Caspase-3 was partially activated by IFN-gamma. Protein levels of molecular chaperones were examined in cells treated with IFN-gamma. Among these, levels of heat shock protein 27 (Hsp27) were specifically reduced upon IFN-gamma treatment of oral SCC cells. Recombinant clones overexpressing Hsp27 were more resistant to IFN-gamma-induced cell death than parent cells. Conversely, cells expressing a dominant-negative mutant of Hsp27, in which three serine residues (15, 78 and 82) were replaced by glycine, were hypersensitive to the effects of IFN-gamma and exhibited a typical apoptotic phenotype. Pretreatment of cells with IFN-gamma enhanced apoptotic cell death induced by cisplatin. Our data suggest that IFN-gamma suppresses Hsp27 expression in oral SCC cells and blocks the inhibitory effects of this molecular chaperone on apoptotic cell death. Moreover, IFN-gamma initiates the transition of oral SCC cells to the proapoptotic and/or aborted apoptotic state. Hsp27 plays a crucial role in the inhibition of apoptosis of oral SCC cells. Our findings highlight the importance of employing IFN-gamma in combination with certain anticancer drugs as treatments for oral cancer. We suggest that Hsp27 plays a significant role in the IFN-gamma-induced sensitization of oral SCC cells to anticancer drugs.

Abstract: Viral infection modulates the regulation of apoptosis in host cells. Here, we report a novel mechanism by which human cells infected with mumps virus become susceptible to apoptosis caused by extracellular stresses. Mumps virus stimulates proteasome-dependent degradation of STAT-1 by action of viral accessory protein V, resulting in a severe decrease in STAT-1 protein in infected cells. We exposed mumps virus-infected and uninfected cells to heat and chemical stress. The infected cells failed to acquire resistance to apoptotic stimuli (thermotolerance) after exposure to these mild stresses. The induction of HSP27 by stress exposure was dramatically suppressed in the infected cells, but HSP70 induction was not affected. STAT-1 was required for transcriptional activation of the HSP27 gene, but not for the HSP70 gene, and cDNA transfection of STAT-1 in mumps virus-infected cells restored thermotolerance. Phosphorylated heat shock factor-1 (HSF-1) and STAT-1 phosphorylated on neither tyrosine nor serine residues were co-transported to the nucleus in response to stress. Furthermore, overexpression of unphosphorylatable mutants of STAT-1 also restored thermotolerance in mumps virus-infected cells. These lines of evidence indicate that the induction of HSP27 by stress requires STAT-1 in addition to the activated HSF-1. Furthermore, STAT-1 required for the induction of HSP27 worked independent to its phosphorylation. Thus, HSP27-dependent thermotolerance is suppressed by mumps virus infection through the destruction of STAT-1. The lack of thermotolerance should allow the infected cells to be eliminated by apoptosis and might be a host defense against viral infection.

Abstract: To establish infections, viruses use various strategies to suppress the host defense mechanism, such as interferon (IFN)-induced antiviral state. We found that cells infected with a wild strain of measles virus (MeV) displayed nearly complete suppression of IFN-alpha-induced antiviral state, but not IFN-gamma-induced state. This phenomenon is due to the suppression of IFN-alpha-inducible gene expression at a transcriptional level. In the IFN-alpha signal transduction pathway, Jak1 phosphorylation induced by IFN-alpha is dramatically suppressed in MeV-infected cells; however, phosphorylation induced by IFN-gamma is not. We performed immunoprecipitation experiments using antibodies against type 1 IFN receptor chain 1 (INFAR1) and antibody against RACK1, which is reported to be a scaffold protein interacting with type I IFN receptor chain 2 and STAT1. These experiments indicated that IFNAR1 forms a complex containing the MeV-accessory proteins C and V, RACK1, and STAT1 in MeV-infected cells but not in uninfected cells. Composition of this complex in the infected cells altered little by IFN-alpha treatment. These results indicate that MeV suppresses the IFN-alpha, but not IFN-gamma, signaling pathway by inhibition of Jak1 phosphorylation. Our data suggest that functional disorder of the type I IFN receptor complex is due to "freezing" of the receptor through its association with the C and/or V proteins of MeV.

Abstract: The 47-kDa heat shock protein (HSP47) is an endoplasmic reticulum molecular chaperone that assists in the maturation of collagen molecules and whose expression is known to be upregulated in lesions of fibrotic diseases. We examined the levels of HSP47 protein and autoantibodies to HSP47 in the sera of patients with rheumatic autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, and mixed connective tissue disease (MCTD) by enzyme-linked immunosorbent assay and immunoblot analysis. Patients with idiopathic pulmonary fibrosis (IPF) were assessed as an example of non-autoimmune fibrotic disease. HSP47 antigen and autoantibody levels are significantly elevated in the sera of the rheumatic autoimmune disease patients, but not in the sera of the IPF patients. The sera of the MCTD patients showed particularly high levels of HSP47 antigen relative to healthy controls (1.99+/-0.22 vs 0.41+/-0.07 ng/ml). Autoantibodies to HSP47 were also in high levels in the sera of MCTD patients. These results suggest that simultaneous occurrence of systemic inflammation and upregulation of HSP47 caused leakage of HSP47 from fibrotic lesions into the peripheral blood, and the leaked antigen induced high titer of autoantibodies to HSP47. The high levels of HSP47 antigen and autoantibody may be useful blood markers of MCTD.

Abstract: We identified and genetically characterized seven fluoroquinolone-resistant Streptococcus pneumoniae strains among 293 clinical strains isolated from 1999 to 2001 in Japan. The resistant strains were isolated only from adults, and 7 of 31 isolates (22.6%) were from patients more than 20 years old. Resistant strains were not found in 262 isolates from children under age 10.

Abstract: Constitutive levels of production of STAT-1 were reduced by 10 h postinfection (p.i.) and significantly lost by 24 h p.i. in FL cells acutely infected with mumps virus (MuV). This result was consistent with that observed in previous studies and experiments with cells persistently infected with MuV (FLMT cells). There was a marked decrease in the amount of STAT-1 in cells expressing MuV accessory protein V (MuV-V). Furthermore, single amino acid substitutions in the Cys-rich region of V protein (Vc189a, Vc207a, and Vc214a) showed that each cysteine residue plays an important role in the decrease in STAT-1 production, but substitution of a histidine residue at amino acid position 203 had no effect. These events and the resultant suppression of the alpha interferon (IFN-alpha) response were confirmed by a luciferase reporter gene assay with five tandem repeats of the IFN-alpha-stimulated response element as an enhancer element of the firely luciferase gene. STAT-1 production was restored and detectable in FLMT cells treated with a proteosome inhibitor, such as MG132 or lactacystin. In the presence of MG132, ubiquitination of STAT-1 and the interaction of MuV-V with STAT-1 were demonstrated in FLMT cells by immunoprecipitation with anti-STAT-1 antibody. The same results for the interaction and ubiquitination were obtained in experiments with an expression vector for a C-terminal deletion mutant of STAT-1. The truncated STAT-1 molecules were degraded in the presence of MuV-V. Therefore, the C-terminal region (transcriptional activation and Src homology 2 domains) of STAT-1 is not necessary for its degradation caused by MuV-V. Our data suggest that MuV-V promotes ubiquitination and degradation of STAT-1.

Abstract: It has been reported that mumps virus protein V or the C-terminal Cys-rich region of protein V (Vsp) is associated with blocking of the interferon (IFN) signal transduction pathway through a decrease in STAT-1 production. The intracellular target of the V protein was investigated by using a two-hybrid screening system with Vsp as bait. Full-length V protein and Vsp were able to bind to RACK1, and the interaction did not require two WD domains, WD1 and WD2, in RACK1. A significant interaction between V protein and RACK1 was also demonstrated in cells persistently infected with mumps virus (FLMT cells), and the formation of the complex was not affected by treatment with IFN. On the other hand, in uninfected cells, STAT-1 was associated with the long form of the beta subunit of the alpha IFN receptor, and this association was mediated by the function of RACK1 as an adaptor protein. Immunoprecipitation and glutathione S-transferase pull-down experiments revealed that the association of RACK1 or mumps virus V protein with the IFN receptor was undetectable in mumps virus-infected cells. Furthermore, RACK1 interacted with mumps virus V protein with a higher affinity than STAT-1 did. Therefore, it is suggested that mumps virus V protein has the ability to interact strongly with RACK1 and consequently to bring about the disruption of the complex formed from STAT-1, RACK1, and the IFN receptor.

Abstract: The chaperonin-containing t-complex polypeptide 1 (CCT) is a cytosolic molecular chaperone composed of eight subunits that assists in the folding of actin, tubulin and other cytosolic proteins. We show here that the content of particular subunits of CCT within mammalian cells decreases concomitantly with the reduction of chaperone activity during cell cycle arrest at M phase. CCT recovers chaperone activity upon resumption of these subunits after release from M phase arrest or during arrest at S phase. The levels of alpha, delta and zeta-1 subunits decreased more rapidly than the other subunits during M phase arrest by colcemid treatment and recovered after release from the arrest. Gel filtration chromatography or native (nondenaturing) PAGE analysis followed by immunoblotting indicated that the alpha and delta subunit content in the 700- to 900-kDa CCT complex was appreciably lower in the M phase cells than in asynchronous cells. In vivo, the CCT complex of M-phase-arrested cells was found to bind lower amounts of tubulin than that of asynchronous cells. In vitro, the CCT complex of M phase-arrested cells was less active in binding and folding denatured actin than that of asynchronous cells. On the other hand, the CCT complex of asynchronous cells (a mixture of various phases of cell cycle) exhibited lower alpha and delta subunit content and lower chaperone activity than that of S-phase-arrested cells obtained by excess thymidine treatment. In addition, turnover (synthesis and degradation) rates of the alpha and delta subunits in vivo were more rapid than those of most other subunits. These results suggest that the content of alpha and delta subunits of CCT reduces from the complete active complex in S phase cells to incomplete inactive complex in M phase cells.

Abstract: It has been reported that interferon (IFN)-alpha/gamma signal transduction pathway is blocked in several cell lines persistently infected with mumps virus (MV) through decrease of STAT-1alpha. Expression of the MV structural V protein (MV-V) or C terminal CYS-RICH region of the V protein (MV-Vsp) inhibited the establishment of the antivirus state induced by IFN, but not by expression of the MV-P protein. Suppression of IFN-induced STAT-1alpha, STAT-2, and IRF-9 (p48) induction was also recognized in the cells transfected with expression vector of the MV-V (pTM-V) or MV-Vsp (pTM-Vsp) protein, even though it was in the absence of the other virus protein. It is supposed that the cysteine-rich domain of V protein (Vsp) is involved in the suppression of the IFN signal transduction pathway.

Abstract:

Abstract: The chaperonin-containing t-complex polypeptide 1 (CCT) is a hetero-oligomeric molecular chaperone that assists in the folding of actin, tubulin, and other cytosolic proteins. We recently reported that the expression level of CCT is closely correlated with growth rates of mammalian cultured cells. Here we examine the levels of CCT subunits and other molecular chaperones in tumor tissues of patients with hepatocelluar and colonic carcinoma, and compare them with nontumor tissues in the same patients. Expression levels of CCTbeta in tumor tissues was significantly higher than in nontumor tissues in all patients with hepatocellular carcinoma (n = 15) and 83% of patients with colonic carcinoma (n = 17). The increased level of CCT expression in colonic cancer cells was confirmed by immunohistochemistry with anti-CCTbeta antibody. The levels of CCTbeta were highly correlated (r = 0.606) with those of the proliferating cell nuclear antigen (PCNA), which was used as an indicator of cell growth. CCTalpha gave similar results, although the correlation with PCNA levels was weaker. Other cytosolic and endoplasmic reticulum chaperones also showed higher expression in significant numbers of tumor tissues but less frequently than that observed with CCT. These results suggest that CCT is up-regulated in rapidly proliferating tumor cells in vivo to effectively produce proteins required for growth, and may serve as a useful tumor marker because it is widely distributed in the cytosol.

Abstract: We examined the influence on the interferon (IFN) signaling pathway of infection with herpes simplex virus type 1 (HSV-1) strain VR3. Data from reporter gene assays showed that expression of both type I and type II IFN-inducible genes was dramatically suppressed during the early stage of HSV-1 infection (2 to 3 h postinfection). During these periods, phosphorylation levels of janus kinases (JAKs) and STATs did not increase after treatment of HSV-1-infected FL cells with IFN-alpha or IFN-gamma, although cellular protein levels of the JAKs and the STATs were not significantly changed. In contrast, the inhibitory effect of HSV-1 on phosphorylation of STAT1 was not observed in U937 cells, which show resistance to steady-state accumulation of RNA for HSV-1 immediate-early genes. The phosphorylation of STAT1 in FL cells was not inhibited by infection with a UV-inactivated virus. These results indicate that viral gene expression or viral protein production is necessary for the inhibition of phosphorylation by HSV-1.

Abstract: The chaperonin containing t-complex polypeptide 1 (CCT) is a heterooligomeric molecular chaperone that assists in the folding of actin, tubulin, and other cytosolic proteins. We show here that degradation of CCT in mammalian cells is inhibited by a proteasome-specific inhibitor, lactacystin. When CCT synthesis was inhibited by growth arrest of cells, the decrease in CCT levels was much slower in the presence of lactacystin than in its absence. Pulse-chase experiments indicated that degradation of CCT is inhibited 2- to 2.5-fold by addition of lactacystin. In addition, CCT degradation rate in ts85 cells that produce thermolabile ubiquitin-activating enzyme E1 was reduced 3-fold at the nonpermissive temperature compared to the degradation at the permissive temperature. These results indicate that the ubiquitin-proteasome system is involved in CCT degradation.

Abstract: The chaperonin containing TCP-1 (CCT) is a molecular chaperone consisting of eight subunit species and assists in the folding of actin, tubulin and some other cytosolic proteins. We examined the stress response of CCT subunit proteins in mammalian cultured cells using chemical stressors that cause accumulation of unfolded proteins. Levels of CCT subunit proteins in HeLa cells were coordinately and transiently upregulated under continuous chemical stress with sodium arsenite. CCT subunit levels in several mammalian cell lines were also upregulated during recovery from chemical stress caused by sodium arsenite or a proline analogue, L-azetidine-2-carboxylic acid. Several unidentified proteins that were newly synthesized and associated with CCT were found to increase concomitantly with CCT subunits themselves and known substrates during recovery from the stress. These results suggest that CCT plays important roles in the recovery of cells from protein damage by assisting in the folding of proteins that are actively synthesized and/or renatured during this period.

Abstract: The chaperonin containing t-complex polypeptide 1 (CCT) is a molecular chaperone assisting in the folding of proteins in eukaryotic cytosol, and the Ccta (encoding the alpha subunit of CCT)/t-complex polypeptide 1 gene encodes the alpha subunit of CCT. We show here that transcription of the mouse Ccta gene is regulated by selenocysteine tRNA gene transcription activating factor (Staf) family zinc-finger transcription factors ZNF143 and ZNF76. Reporter gene assay using HeLa cells indicated that the Ccta gene promoter contains two 18-base pair-long cis-acting elements with similar sequences at -70 and -20 base pairs (designated CCT alpha subunit gene transcription activating element 1 (CAE1) and CAE2, respectively). By yeast one-hybrid screening of CAE1-binding factors, we isolated human ZNF143, which is known to activate transcription of selenocysteine tRNA and small nuclear RNA genes. DNA binding domains of ZNF143 and ZNF76 produced in E. coli recognized CAE1 and CAE2 elements in electrophoretic mobility shift assay. HeLa cell nuclear extract contained a protein that specifically binds to CAE1 and CAE2 and recognized by anti-ZNF143 antibody. Transcription from a minimal Ccta promoter containing CAE2 element in HeLa cells was enhanced by overexpression of full-length ZNF143 and ZNF76 but inhibited by that of their DNA binding domains alone. These results demonstrate that the Staf family proteins control transcription of at least one of the chaperone-encoding genes besides that of tRNA and small nuclear RNA genes. These RNA and chaperone genes are suggested to be coregulated to facilitate synthesis of mature proteins during active cell growth.

Abstract: Chaperonin CCT containing t-complex polypeptide 1 is a cytosolic molecular chaperone that assists in the folding of actin, tubulin, and other proteins and is a member of the 60-kDa heat shock protein (Hsp60) family. We examined antibody titers against human CCT and other Hsp60 family members in the sera of patients with rheumatic autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematodes, Sjögren syndrome, and mixed connective tissue disease. Autoantibody titers against not only human mitochondrial Hsp60 but also CCT were significantly higher in the sera of patients with rheumatic autoimmune diseases than in healthy control sera. Although immunoglobulin G (IgG) titers against Escherichia coli GroEL were high in all the groups of sera tested, no significant differences in anti-GroEL responses were detected between patients and healthy controls. IgG titers against mycobacterial Hsp65 showed a similar pattern to titers of autoantibodies recognizing GroEL. Immunoabsorption experiments demonstrated that most of the autoantibodies recognizing CCT were cross-reactive with mitochondrial Hsp60, E coli GroEL, and mycobacterial Hsp65. Although most of the anti-Hsp60 IgG recognized CCT, anti-GroEL (or antimycobacterial Hsp65) IgG contained antibodies specific for GroEL (or mycobacterial Hsp65) in addition to antibodies cross-reactive with CCT and Hsp60. Results from immunoblot analyses, together with weak (15% to 20%) amino acid sequence identities between CCT and the other Hsp60 family members, suggested that CCT-reactive autoantibodies recognize conformational epitopes that are conserved among CCT and other Hsp60 family members.

Abstract: Past studies have shown that the cell surface lipopolysaccharides (LPSs) of the ubiquitous human gastric pathogen Helicobacter pylori (a type 1 carcinogen) isolated from people residing in Europe and North America express predominantly type 2 Lewis x (Le(x)) and Le(y) epitopes and, infrequently, type 1 Le(a), Le(b), and Le(d) antigens. This production of Lewis blood-group structures by H. pylori LPSs, similar to those found in the surfaces of human gastric cells, allows the bacterium to mimic its human niche. In this study, LPSs of H.pylori strains extracted from patients living in China, Japan, and Singapore were chemically and serologically analyzed. When compared with Western H.pylori LPSs, these Asian strains showed a stronger tendency to produce type 1 blood groups. Of particular interest, and novel observations in H.pylori, the O-chain regions of strains F-58C and R-58A carried type 1 Le(a) without the presence of type 2 Le(x), strains R-7A and H607 were shown to have the capability of producing the type 1 blood group A antigen, and strains CA2, H507, and H428 expressed simultaneously the difucosyl isomeric antigens, type 1 Le(b) and type 2 Le(y). The apparent proclivity for the production of type 1 histo-blood group antigens in Asian H.pylori LPSs, as compared with Western strains, may be an adaptive evolutionary effect in that differences in the gastric cell surfaces of the respective hosts might be significantly dissimilar to select for the formation of different LPS structures on the resident H.pylori strain.

Abstract: We examined the levels of antibody titers against Helicobacter pylori antigens, three types of lipopolysaccharides (LPSs), recombinant CagA antigen, recombinant VacA antigen and partially purified cellular antigens in the sera of Japanese volunteers. The three types of LPSs are LPS carrying the highly antigenic epitope, LPS carrying the weakly antigenic epitope and rough LPS, classified on the basis of antigenicity in humans. IgG titers against all H. pylori antigens tested were significantly different between gastroduodenal patients and healthy adults without H. pylori infection. IgG titers against LPS carrying the weakly antigenic epitope, rough LPS and VacA antigen, as well as IgA titers against the partially purified cellular extract were significantly higher in gastroduodenal patients than in H. pylori-positive healthy adults. However, IgG titers against LPS carrying the highly antigenic epitope, CagA antigen or the partially purified cellular extract showed no significant difference between patients and H. pylori-positive healthy adults. The results indicated that increases in IgG titers against VacA antigen and the weakly antigenic and core epitopes of LPS, and in IgA titer against the partially purified cellular extract, were associated with disease state and may be useful in identifying active infection of H. pylori.

Abstract: We have purified lipopolysaccharides (LPS) from 10 Helicobacter pylori clinical isolates which were selected on the basis of chemotype and antigenic variation. Data from immunoblotting of the purified LPS with sera from humans with H. pylori infection and from absorption of the sera with LPS indicated the presence of two distinct epitopes, termed the highly antigenic and the weakly antigenic epitopes, on the polysaccharide chains. Among 68 H. pylori clinical isolates, all smooth strains possessed either epitope; the epitopes were each carried by about 50% of the smooth strains. Thus, H. pylori strains can be classified into three types on the basis of their antigenicity in humans: those with smooth LPS carrying the highly antigenic epitope, those with smooth LPS carrying the weakly antigenic epitope, and those with rough LPS. Sera from humans with H. pylori infection could be grouped into three categories: those containing immunoglobulin G (IgG) antibodies against the highly antigenic epitope, those containing IgG against the weakly antigenic epitope, and those containing both specific IgGs; these groups made up about 50%, less than 10%, and about 40%, respectively, of all infected sera tested. In other words, IgG against the highly antigenic epitope were detected in more than 90% of H. pylori-infected individuals with high titers. IgG against the weakly antigenic epitope were detected in about 50% of the sera tested; however, the antibody titers were low. The two human epitopes existed independently from the mimic structures of Lewis antigens, which are known to be an important epitope of H. pylori LPS. No significant relationship between the reactivities toward purified LPS of human sera and a panel of anti-Lewis antigen antibodies was found. Moreover, the reactivities of the anti-Lewis antigen antibodies, but not human sera, were sensitive to particular alpha-L-fucosidases. The human epitopes appeared to be located on O-polysaccharide chains containing endo-beta-galactosidase-sensitive galactose residues as the backbone. Data from chemical analyses indicated that all LPS commonly contained galactose, glucosamine, glucose, and fucose (except one rough strain) as probable polysaccharide components, together with typical components of inner core and lipid A. We were not able to distinguish between the differences of antigenicity in humans by on the basis of the chemical composition of the LPS.

Abstract: The chaperonin-containing TCP-1 (CCT) is a hetero-oligomeric molecular chaperone that mediates protein folding in the cytosol of eukaryotes. Eight (or nine in testis) subunit species are assembled in the CCT hexadecamer complex. We have cloned seven CCT subunit genes, Cctb, Cctd, Ccte, Cctz-1, Cctz-2 (testis specific), Ccth and Cctq, from mouse genomic DNA libraries, in addition to the Ccta and Cctg genes reported previously, and the entire nucleotide sequences of these DNA clones were determined. These genes are approximately 15-20 kb in length except for Cctz-2 which is longer than 35 kb, and all the Cct genes consist of 11-16 exons. Primer extension analyses of testis RNA indicate one to several potential transcription start sites 50-150 bp upstream from the translation start codon of each Cct gene. There are several possible Sp1-binding sequences, but no obvious TATA box was observed around the potential start sites. From 5'-flanking regions to the first introns, the Cct genes are rich in CpG dinucleotides. In reporter gene assays using these regions, five of eight Cct genes showed strong transcriptional activity comparable with the combination of SV40 promoter and enhancer in HeLa cells. We also show, by Western and Northern blot analyses, that CCT expression levels vary widely among different tissues but the expression patterns are very similar among the eight subunit species. It is likely that expression levels of the eight different subunits are tightly co-regulated to maintain a constant ratio of these subunits which constitute the CCT hexadecamer complex with a fixed subunit arrangement.

Abstract: The chaperonin containing t-complex polypeptide 1 (CCT) is a heterooligomeric molecular chaperone assisting in the folding of actin, tubulin, and other cytosolic proteins. The expression levels of CCT subunits varied among seven mouse cell lines tested but showed a close correlation with growth rate. Both the CCT protein and mRNA levels in the human promyelolytic cell HL60 decreased concomitant with growth arrest during differentiation. More rapid decrease in CCT level occurred when the mouse interleukin (IL)-3-dependent myeloid DA3 cells were starved for IL-3. Readdition of IL-3 caused rapid resumption of CCT synthesis during synchronous growth: the maximum CCT protein and mRNA levels were observed at G(1)/S transition through early S phase. The turnover rate of CCT was nearly constant regardless of growth. Gel filtration and immunoprecipitation analyses indicated that CCT in vivo is associated with tubulin at early S phase, but not at G(0)/G(1) phase. These results demonstrated that CCT expression is strongly up-regulated during cell growth especially from G(1)/S transition to early S phase and is primarily controlled at the mRNA level. CCT appears to play important roles for cell growth by assisting in the folding of tubulin and other proteins.

Abstract: The chaperonin containing TCP-1 (CCT) is a eukaryotic molecular chaperone consisting of eight subunit species and assists in the folding of cytosolic proteins. We show here that all eight mouse CCT subunit genes contain sequences called heat shock elements for binding heat shock transcription factors (HSFs) by electrophoretic mobility shift assays and that these genes are transcriptionally activated by HSFs in reporter gene assays using HeLa cells transiently overexpressing HSFs. These results suggest that HSF1 and/or HSF2 play a role in Cct gene expression.

Abstract: We have evaluated the use of proteinase K (PK)-treated cells isolated from Helicobacter pylori as lipopolysaccharide (LPS) antigens in an immunoblot assay and an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of H. pylori infection. The sera from patients with chronic gastritis, gastric ulcer, duodenal ulcer or gastric cancer, and from healthy adults with or without H. pylori infection were assayed with three commercial serodiagnostic kits (HM-CAP, Helico-G, and G.A.P. II) and novel methods relying on the use of PK-treated cells. The PK-treated cells used in these assays were selected on the basis of their possibility to possess a common epitope in the O-polysaccharides of H. pylori, which is known to be highly immunogenic in humans. Of the sera from these patients, 71-94% were positive with the commercial kits, 97% with immunoblot assay, and 90% with ELISA. On the other hand, of the healthy adults infected with H. pylori, 72-97% were positive with the commercial kits, 86% with immunoblot assay, and 72% with ELISA. PK-treated cells that did not contain the common epitope were unsuitable as an antigen for immunoblot assay or ELISA. Furthermore, the reactivity of these sera reacted specifically with H. pylori PK-treated cells but not with LPSs from other gram-negative bacteria, such as Campylobacter, Proteus, Bordetella, and Salmonella. These results demonstrate that the serological assays relying on the use of H. pylori PK-treated cells possessing a highly antigenic epitope are potentially useful as a serodiagnostic test for H. pylori infection.

Abstract: We have examined the antibody response to Helicobacter pylori lipopolysaccharides (LPS) in humans. We used sera from patients with gastroduodenal diseases and healthy adults infected or not infected with H. pylori. Data from the experiments for antibody binding to LPS suggested that the polysaccharide chains from many H. pylori strains showed high immunogenicity in humans. Sera from most (above 70%) H. pylori-infected individuals contained immunoglobulin G (IgG) antibodies against the polysaccharide region highly immunogenic H. pylori LPS. The IgG titers of individual serum samples that reacted strongly with highly immunogenic LPS were quite similar (r2 = 0.84 to 0.98). The results suggest wide distribution among H. pylori strains of a highly antigenic epitope in the polysaccharide moieties of their LPS. Also, the similarity in the titers of individual serum samples against highly immunogenic LPS points to the existence of epitopes sharing a common structural motif. However, some strains showed low antigenicity, even those with polysaccharide-carrying LPS. The dominant subclass of IgG that reacted with the highly immunogenic LPS was IgG2, which was preferentially raised against polysaccharide antigens. Recently, a structure that mimics that of the Lewis antigens was identified in the O-polysaccharide fraction of H. pylori LPS; however, no correlation between antigenicity of the polysaccharide chain in humans and the presence of Lewis antigens was found. The IgA and IgM titers against H. pylori LPS seemed to be mostly nonspecific and directed against lipid A. In a few cases, however, sera from individuals infected with H. pylori gave strong IgA and IgM titers against the highly immunogenic polysaccharide. In conclusion, the LPS of many H. pylori strains possess an antigenic epitope in their polysaccharide regions that is immunogenic in humans. However, our results show that the antigenic epitope is unlikely to be immunologically related to structures mimicking Lewis antigens.

Abstract: We have examined the antibody response to Helicobacter pylori lipopolysaccharide (LPS) during natural infection in humans. The sera of over 70% of H. pylori-infected individuals were found to contain immunoglobulin G antibodies against the LPS fractions isolated from smooth strains of H. pylori but not against those derived from rough strains, as determined by enzyme-linked immunosorbent assay. These results taken together with the immunoblot data indicated that the polysaccharide region of H. pylori LPS is antigenic in humans. However, the antigenicity of the polysaccharide varied, depending on the strain. We found that smooth H. pylori strains isolated from the tumors of patients with gastric cancer showed significantly lower antigenicity than smooth strains derived from patients with chronic gastritis and gastric and duodenal ulcers. The results suggest that the levels of antigenicity of the polysaccharide region of H. pylori LPS in humans correlate with the nature of the gastroduodenal diseases and that they allow a particular distinction to be made between gastric cancer and other gastroduodenal diseases, especially chronic gastritis.

Abstract: We have examined the reactivity of monoclonal antibodies (MAbs) specific for Lewis antigens (Le(x), Le(y), Le(a), and Le(b)) with Helicobacter pylori lipopolysaccharides (LPS) by immunoblot analysis and enzyme-linked immunosorbent assay (ELISA). Sixty-eight strains of H. pylori were isolated from patients with chronic gastritis, gastric and duodenal ulcers, and gastric cancer in Japan. The cells were treated with proteinase K, and the resulting fractions were used as a source of LPS for the immunoassays. In the immunoblot analysis, 28 isolates (41%) and 29 isolates (42%) reacted with anti-Le(x) and anti-Le(y) MAbs, respectively, while 4 isolates (6%) and 7 isolates (10%) reacted with anti-Le(a) and anti-Le(b) MAbs. On the other hand, in ELISA, the number of isolates that reacted with anti-Le(x) MAbs fell significantly to 21 isolates (30%) but the number of isolates that reacted with the other anti-Lewis antigen MAbs remained relatively unchanged. These data show that the immunoblotting technique is more sensitive than the ELISA technique for the detection of immunocomplexes of anti-Le(x) MAbs and components of H. pylori LPS. Furthermore, human serum was found to react with the synthetic Lewis antigens regardless of the status of the individual's H. pylori infection. This means that humans may naturally possess antibodies against Lewis antigens in the absence of H. pylori infection.

Abstract: A hybridoma cell line producing a human anti-lipid A monoclonal antibody (mAb), FKF-IF3 (IgM (k)) was obtained by cell fusion of Epstein-Barr virus-transformed cells and mouse myeloma. The mAb bound to not only Gram-negative bacterial lipid A, but also to polysaccharide portions of Pseudomonas aeruginosa lipopolysaccharides (LPS). The mAb seemed to recognize two distinct regions of P. aeruginosa LPS other than lipid A, namely the outer core regions of some serotype strains and the O-polysaccharide region of serotype A strains. The mAb cross-reacted with N-acetyl-beta-glucosamine-conjugated bovine serum albumin, N-acetyl-beta-galactosamine-conjugated bovine serum albumin, myosin and actin, but not with other autoantigens such as ss- and ds-DNA, cardiolipin and glycosaminoglycans. The mAb conferred protective activity against a mouse pseudomonal infection model. The evidence suggested that the mAb was a naturally occurring polyspecific antibody that participated in defense against pseudomonal infections.

Abstract: Pseudomonas aeruginosa PML14e is a mutant strain, isolated from strain PML14 (Homma serotype I), that is resistant to all types of R-pyocins. PML14e completely lacked glucose and rhamnose as components of the lipopolysaccharide (LPS) outer core region. Whereas the O-polysaccharide attachment site on the LPS core was considered to be absent, PML14e was agglutinable with anti-serotype-I antibodies. The O-polysaccharide of PML14e was recovered in the supernatant after ultracentrifugation of the aqueous layer from a hot phenol/water extraction. Chromatographic behaviour and chemical analysis indicated that the PML14e O-polysaccharide was not linked to the lipid A. 1H-NMR spectroscopy indicated that the structure of the PML14e O-polysaccharide was the same as that of the O-polysaccharide from PML14. The above evidence indicated that the O-polysaccharide is expressed on the cell surface of the mutant strain PML14e as the lipid A-free form. To examine the nature of the cell surface, the accessibility of monoclonal antibodies (mAbs) against cell surface antigens was tested by enzyme-linked immunosorbent assay. An anti-lipid A mAb and an anti-outer-membrane protein mAb, the epitopes for which are considered to be exposed on rough strains, bound to a greater extent to the PML14e cells than to two other LPS-core-defective rough mutants, PML14b and PML14d. Whereas these mutants appeared to have lesser defects in the LPS core, they expressed less O-polysaccharide than PML14e. The results indicated that the epitopes exposed on rough strains, such as lipid A and outer-membrane proteins, were mainly hindered by covalently linked core oligosaccharide rather than by the O-polysaccharide chain.

Abstract: Polymorphonuclear leukocytes (PMN) are directly involved in development of ischemic myocardial injury. Adhesion of PMN to endothelial cells is an initial step that triggers a sequential process leading to acute inflammatory responses. Interaction between P-selectin and its oligosaccharide ligand, sialyl Lewis x (sLex), plays an important role in the early stage of the adhesion. To examine the role of P-selectin in various animal disease models especially in rats, we have cloned rat E- and P-selectin cDNAs and established monoclonal antibodies against these rat selectins. In this report, we describe the generation and characterization of anti-rat P-selectin antibodies (ARPs). These antibodies detect cell surface P-selectin on thrombin-stimulated rat platelets. More importantly, intravenous administration of ARP2-4 reduced infarction developed after 30 min of ischemia followed by 24 h of reperfusion in a rat myocardial injury model. In addition, similar protective effect was also observed by administration of a sLex-oligosaccharide. These results indicate that cell adhesion mediated via P-selectin is involved in the development of ischemia and reperfusion injury in rat heart.

Abstract: P-selectin (GMP-140, CD62P) is a member of the selectin family of cell adhesion molecules expressed on the cell surface of platelets and endothelial cells, which mediates leukocyte adhesion via carbohydrate ligands. We now report that a monoclonal antibody (mAb), recognizing the ninth consensus repeat domain of the human P-selectin molecule, enhanced the adhesion activity of neutrophils to platelets, whereas the monovalent Fab fragment of the mAb did not exert this effect. The enhancement by the mAb was thought to result from cross-linking of the P-selectin molecules. The results indicated that cross-linking adjacent to the transmembrane domain of P-selectin enhanced the cell adhesion activity.

Abstract: Strains of Pseudomonas aeruginosa isolated from patients with cystic fibrosis (CF) never spread systemically. This may be due to serum sensitivity since these strains are very sensitive to complement-mediated bactericidal activity. A serum-resistant mutant, P. aeruginosa TUM3 HSR, was obtained from serum-sensitive strain TUM3 from a CF patient in order to clarify the mechanism of failure of systemic spread. LPS profiles on silver-stained gels and immunological analysis revealed that a long O-polysaccharide side chain was overproduced on the LPS molecules of TUM3 HSR as compared with the LPS of TUM3. The clearance rate from the bloodstream in mice was compared in the two strains. The number of TUM3 bacteria in 1 ml of blood, 10 min after injection into the tail vein, significantly decreased from 1.7 x 10(8) to 3.7 x 10(5) c.f.u. ml-1. In contrast, TUM3 HSR was not eliminated during the same period (decrease from 1.9 x 10(8) to 3.4 x 10(7) c.f.u. ml-1). Interestingly, these isogenic strains were not killed by 40% murine serum, probably reflecting immaturity of the complement-mediated killing system in mice. These results pointed to a correlation between LPS structure and blood clearance rate in mice. This was confirmed by examining blood clearance kinetics using the smooth-LPS strain Salmonella typhimurium LT2 and LPS-deficient mutants derived from it. S. typhimurium LT2 resisted blood clearance while the LPS-deficient mutants were cleared rapidly. None of the S. typhimurium strains were killed by murine serum. The number of P. aeruginosa TUM3 and S. typhimurium LPS-deficient mutants trapped in the liver following injection into the peripheral circulation was greater than that of their counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Pseudomonas aeruginosa is routinely serotyped in Japan by using the Homma scheme. The serotypes (O serotypes) are based on the chemical structure of the O-polysaccharide portion of the lipopolysaccharide (LPS). However, the nature of the Homma serotype M antigen has remained obscure because strains classified as serotype M usually have the rough phenotype. I characterized the target antigen of serotype M. The results of Western blotting (immunoblotting) showed that commercially available typing monoclonal antibody (MAb) against serotype M specifically bound to outer membrane protein (Opr) G and that typing rabbit antiserum specific for serotype M mainly contained antibodies against Oprs F and H2. These Oprs were distributed among all P. aeruginosa strains tested, including the serotype standard, serotype M and nontypeable strains, and a series of LPS-core-defective mutants derived from strain PAC1. However, the rough mutants derived from strain PAC1 agglutinated with the anti-serotype M antibodies, whereas the smooth strains did not. LPS preparations from serotype M strains possessed few or no polysaccharide chains. These strains had higher levels of binding activity with anti-serotype M MAb, as well as with anti-lipid A MAb, which specifically bound to the cell surface of the rough-natured gram-negative bacterial strains with high activity. The anti-serotype M antiserum also contained rough-LPS-specific antibodies, but the epitope was distributed among only a few strains. The results suggested that the Oprs acted as the serotype M antigen and that LPS did not. In conclusion, the rough strains agglutinated with anti-Opr antibodies and were distinguished as serotype M from the smooth strains of other serotypes, because the antibodies were accessible to the cell surface lacking O polysaccharides. I supposed that Homma serotype M is an index of the rough nature of P. aeruginosa strains rather than one of the O serotypes.

Abstract: phi CTX is a temperate phage of Pseudomonas aeruginosa harbouring the ctx gene that encodes cytotoxin (CTX). We identified phi CTX as an R pyocin-related phage, by serological and molecular analysis, based on the findings that the infectivity of the phage was inhibited with the antisera directed R pyocins and R pyocin-related phages and that the phi CTX genome showed DNA homology to the genome of PS17 (a representative of the R pyocin-related phages) as well as to the pyocin R2 genes. Another new CTX-converting, R pyocin-related phage named PS21 was isolated from a CTX-producing strain of P. aeruginosa, suggesting the distribution of the ctx gene by certain members of R pyocin-related phage family.

Abstract: A temperate phage, phi CTX, is a cytotoxin-converting phage of Pseudomonas aeruginosa. In this study, we characterized the lipopolysaccharide (LPS) structures of phi CTX-resistant mutants derived from phi CTX-sensitive strains. phi CTX infectivity was neutralized by LPS preparations derived from sensitive strains but not by those from resistant strains. phi CTX-resistant mutants had lower-molecular-weight rough (R)-type LPS than the parental strains and lacked the reactivity of some anti-LPS core monoclonal antibodies. Some LPS core components were lacking or significantly decreased in the resistant mutants. These results suggested that a receptor site of the cytotoxin-converting phage phi CTX was the LPS core region and that especially L-rhamnose and D-glucose residues in the outer core were involved in phage binding. The host range of phi CTX was nearly O-serotype dependent, probably because of the diversity of the LPS core structure among P. aeruginosa strains. phi CTX bound to most strains of Homma serotypes A, G, and I but not to strains of serotypes B and E. Furthermore, we found that a genetic locus specifying phi CTX sensitivity (and consequently participating in the biosynthesis of part of the LPS core) existed in or near the locus participating in the determination of O-serotype specificity (somA), which has been mapped between leu-10 and eda-9001. phi CTX, as well as anti-LPS core monoclonal antibodies, will be a good tool for structural characterization of the P. aeruginosa LPS core region.

Abstract: Epitopes for Pseudomonas aeruginosa O11-specific human monoclonal antibodies (mAbs) and O11 serotyping antisera have been characterized. These mAbs recognized the O-polysaccharide portion of the lipopolysaccharide. The structure of the O-polysaccharide of O11 has been reported to be comprised of trisaccharide repeating-units as follows: -->3)-alpha-L-FucpNAc-(1-->1)-beta-D-FucpNAc-(1--> 2)-beta-D-Glcp-(1-->. (FucpNAc, 2-acetamido-2,6-dideoxygalactopyranoside.) Data from inhibition studies of binding in enzyme-linked immunosorbent assays and cell-agglutination assays, using monosaccharides and periodate-oxidized O-polysaccharide showed that the glucose residue, especially the C-3-C-6 segment and the beta-anomeric configuration, in the polysaccharide is essential for the epitopes of all anti-O11 mAbs; however, the detailed epitope specificities were different from one another. Furthermore, epitopes for serotyping antisera of O11 seemed to be similar to those for the human mAbs.

Abstract: The O-polysaccharide chain (PS-1), released by mild acidic treatment of the LPS of V. anguillarum V-123 (serogroup JO-2), a pathogenic bacterium of marine and estuarine fish, consists of 2-amino-2-deoxy-D-galacturonic acid, 2-amino-2,6-dideoxy-D-glucose (D-quinovosamine), and 4-amino-4,6-dideoxy-D-glucose (D-viosamine) N-acylated with 2,4-dihydroxy-3,3,4-trimethylpyroglutamic acid. Strong-acid hydrolysis of PS-1 afforded alpha-GalNA-(1----4)-alpha-GalNA-(1----3)-QuiN (A1) and alpha-GalNA-(1----3)-QuiN (A2), and hydrolysis with hydrogen fluoride gave N-acetylated A1 and 4-amino-4,6-dideoxy-D-glucose N-acylated by 2,4-dihydroxy-3,3,4-trimethylpyroglutamic acid. Mild treatment of PS-1 with alkali removed the N-formyl substituents and Smith degradation of the product gave alpha-QuiNAc-(1----3)-beta-VioNAcyl-(1----3)-alpha-GalNAc A-(1----3)-2,3,4- trihydroxybutanoic acid (S1) and S2 in which the carboxyl group of the GalNAcA residue was amidated. Thus, the repeating unit of the O-polysaccharide is----3)-alpha-GalNAcA(amino)-(1----4)-alpha-GalNFoA-(1----3 )- alpha-QuiNAc-(1----3)-beta-VioNAcyl-(1----in which the N-Acyl group is 2,4-dihydroxy-3,3,4-trimethylpyroglutamic acid and Fo is formyl.

Abstract: The core region of Pseudomonas aeruginosa lipopolysaccharide (LPS) was analysed by four LPS-core-specific human monoclonal antibodies (mAbs; FK-2E7, MH-4H7, OM-1D6 and NM-3G8). Reactivity of these mAbs to about 180 P. aeruginosa strains was tested. FK-2E7 bound to strains of Homma serotype E and I at a frequency of about 90%, to strains of serotype M at about 50%, and to strains of serotype A and G at about 30%. MH-4H7 bound to P. aeruginosa strains of serotype A, F, G, H, K and M at a high frequency (45-87%), but did not bind to any strains of serotype B, C, E and I. OM-1D6 and NM-3G8 bound to P. aeruginosa strains in a nearly serotype-specific manner. OM-1D6 reacted with all strains of serotype G so far tested, and a few strains of serotype M. Furthermore, L-rhamnose in the LPS core of serotype G was an immunodominant sugar recognized by OM-1D6 as an epitope. NM-3G8 bound to only a few strains of serotype B and M. The variable reactivity of these mAbs suggests that antigenic heterogeneity of the LPS core is somewhat related with (O-polysaccharide-based) serotype. Among these mAbs, MH-4H7 and OM-1D6 showed a high level of protective activity against P. aeruginosa in an experimental infection model using normal mice. In vivo protective activity was shown to be closely related to in vitro binding activity to whole cells as determined by agglutination and flow cytometry, but not ELISA.

Abstract: Mouse monoclonal antibodies (MAbs) against Pseudomonas aeruginosa exotoxin A (Ex-A) were established, and 4 of 20 MAbs were extensively studied for analysis of the structure-function relationship of Ex-A. IN vivo experiments demonstrated that MAb Ex-3C7 protected mice either injected with Ex-A or infected with Ex-A-producing P. aeruginosa from death caused by Ex-A at the highest rate, followed by MAbs Ex-4F2 and Ex-8H5, in that order. MAb Ex-2A10 failed to rescue the mice. MAb Ex-3C7 (immunoglobulin G1 [IgG1]) inhibited incorporation of Ex-A into target cells and strongly neutralized cytotoxicity in cell culture but did not inhibit an enzymatic activity of Ex-A, ADP-ribosyltransferase, at all. The MAb also bound Ex-A, even at a low pH of 4, and recognized amino acid residues 241 to 297 (domain Ia/II), suggesting that MAb Ex-3C7 can interfere with the conformational change and/or processing of Ex-A by keeping a complex of Ex-A and antibody stable at low pH in the phagolysosome. MAb Ex-4F2 (IgG1), which recognizes residues 550 to 590 (domain III), strongly inhibited Ex-A incorporation and neutralized cytotoxicity in cell culture but only weakly inhibited ADP-ribosyltransferase. MAb Ex-8H5 (IgG1), which recognizes residues 591 to 613 (domain III), also inhibited cytotoxicity in cell culture, but weakly. In contrast to the above three MAbs, MAb Ex-2A10 (IgG2b) greatly inhibited ADP-ribosyltransferase but showed no inhibition of Ex-A incorporation and no neutralizing activity against cell toxicity. A line of evidence indicates that (i) domain Ia/II plays an important role in the pathogenesis of Ex-A and (ii) MAbs that inhibit an intracellular postbinding process, such as conformational change, processing, and translocation of Ex-A in target cells, can display potent inhibitory activity against cytotoxicity in vivo, as well as in cell culture, and would be a good candidate for therapy of pseudomonal infections.

Abstract: We have established seven murine hybridoma cell lines which produce monoclonal antibodies (mAbs) against Pseudomonas aeruginosa elastase. The seven mAbs recognized at least six different epitopes on the elastase molecule. All mAbs inhibited both enzymatic activities of elastase and protease, in which elastin fluorescein and hide powder azure were used as substrates, respectively. One of them, mAb E-4D3, strongly neutralized enzymatic activities of peptidase in which furylacryloyl-glycyl-leucinamide was used as a substrate, as well as of elastase and protease. In contrast, the other six mAbs did not neutralize peptidase activity at all. The Ki value for furylacryloyl-glycl-leucinamide of E-4D3, as well as its Fab fragment, was comparable to those for metalloprotease inhibitors such as phosphoramidon and Zincov inhibitor. The binding of mAb E-4D3 was inhibited by phosphoramidon and Zincov inhibitor, but not by metal chelators such as EDTA and o-phenanthroline. A line of evidence suggests that mAb E-4D3 directly interacts with active site and highly neutralizes enzymatic activity of P. aeruginosa elastase. Data of Western blotting and ELISA suggest that mAb E-4D3 is likely to recognize an elastase molecule in a conformation-dependent manner as an epitope. In contrast, the neutralizing activity of the other mAbs against elastase and protease seems to be caused by a low accessibility of an enzyme to insoluble and high-molecular-mass substrates through the binding and steric hindrance of the mAbs to an enzyme.

Abstract: Three stable hybridoma cell lines, IN-2A8, IN-5D6, and ZI-3A8, that secrete human monoclonal antibodies (MAbs) specific for b-type flagella of Pseudomonas aeruginosa were established by fusing peripheral blood lymphocytes from healthy volunteers with murine myeloma P3X63-Ag8.653 cells. The immunoglobulin M MAbs reacted specifically with flagellin (Mr, 52,000) by Western blotting (immunoblotting) analysis and bound specifically to clinical isolates belonging to Homma serotypes A, B, H, I, and M at frequencies of 58, 50, 46, 30, and 35%, respectively, but did not bind to any serotype E or G isolates. Overall, the MAbs bound to 31% of the clinical isolates. MAb IN-2A8 strongly protected burned mice challenged with P. aeruginosa bearing b-type flagella from death following parenteral administration of 0.1 microgram per mouse. This MAb also inhibited P. aeruginosa colony spreading in soft agar at a concentration of more than 1 microgram/ml but only slightly enhanced opsonophagocytosis by human polymorphonuclear leukocytes. A line of evidence suggests that the potent in vivo activity of MAb IN-2A8 in the burned-mouse model is likely to be caused by its inhibition of bacterial motility after binding to flagella.

Abstract:

Abstract: The protective activity against experimental Pseudomonas aeruginosa infection of a human monoclonal antibody, MH-4H7, which is thought to recognize L-rhamnose and its neighboring residues in the outer core region of P. aeruginosa lipopolysaccharide and which binds to strains of Homma serotypes A, F, G, H, K, and M, was studied in normal, burned, and leukopenic mice. MH-4H7 at doses of 0.1 to 1.0 micrograms per mouse (5 to 50 micrograms/kg) was effective against serotype A, F, G, H, and K clinical isolates of P. aeruginosa tested in normal mice but not against strains of serotype M, B, E, or I. The 50% protective doses were calculated to be 0.01 and 0.1 micrograms per mouse against challenge with serotype G strains and 3 to 8 micrograms per mouse against challenge with serotype A strains. MH-4H7 promoted macrophage-mediated opsonophagocytosis of serotype A, F, G, H, and K strains but not of serotype M strains. The opsonophagocytic activity, expressed as the reduction rate of viable bacteria in the presence of MH-4H7, macrophages, and complement, was higher against serotype G strains (more than 90%) than against serotype A strains (60 to 80%) and serotype F, H, and K strains (50 to 86%). It was correlated with the protective activity but not with the binding intensity of MH-4H7 to the organisms. In addition, burned and leukopenic mice as well as normal mice infected with serotype G strains recovered from a very low dosage of MH-4H7. Thus, a monoclonal antibody directed to the outer core region of P. aeruginosa lipopolysaccharide was effective against infection with a wide range of O-serotype strains of P. aeruginosa.

Abstract: The human monoclonal antibody MH-4H7 recognizes the lipopolysaccharide outer core region of some Pseudomonas aeruginosa strains and in of some Pseudomonas aeruginosa strains and in particular strongly binds to strains of Lányi serotype 04. In this paper, we report that this monoclonal antibody also reacts with Escherichia coli O26 LPS. However, our results suggest that the previous reported immunological cross reaction between P. aeruginosa 04 and E. coli O26 strains (which was observed by using antisera against heat-stable antigens) is not due to the similarity of the O-polysaccharides.

Abstract: We have established a human--mouse heterohybridoma cell line producing a human monoclonal antibody TS-3G2 (IgG gamma 1, K). This monoclonal antibody specifically bound to O-polysaccharides belonging to plural Pseudomonas aeruginosa Homma serotypes, A and H, in contrast to serotype-specific monoclonal antibody which exclusively bound to strains belonging to a single specific serotype. The binding affinity for serotype A strains was higher than that for serotype H strains. Competitive enzyme immunoassay experiments with O-polysaccharide preparations derived from IID 1001, NCTC 8505 (serotype A) and IID 1009 (serotype H) and their derivatives demonstrated that the N-acetyl-L-galactosaminuronic acid residue in O-polysaccharide was essentially involved in the epitope for TS-3G2. Furthermore, a 6-deoxy-hexosamine residue neighboring the reducing terminal of N-acetyl-L-galactosaminuronic acid residues was also concerned with the epitope to some extent. In the experimental infection model of normal mice, the monoclonal antibody TS-3G2 showed a protective activity against both strains of serotype A and H.

Abstract: S. Sawada and co-workers reported that a monoclonal antibody (MAb), E87, interacted with about 80% of Pseudomonas aeruginosa isolates, and they separated a rhamnose-rich polysaccharide as the probable antigen for MAb E87 from P. aeruginosa IFO 3080 (S. Sawada, T. Kawamura, Y. Masuho, and K. Tomibe, J. Infec. Dis. 152:1290-1299, 1985). In the present study, the rhamnose-rich polysaccharide was shown to be structurally and immunologically identical to the D-rhamnan of P. aeruginosa IID 1008 (S. Yokota, S. Kaya, S. Sawada, T. Kawamura, Y. Araki, and E. Ito, Eur. J. Biochem. 167:203-209, 1987). Furthermore, a set of enzymes responsible for the formation of GDP-rhamnose (probably in a D-form) from GDP-D-mannose was found in the 100,000 x g supernatant fractions obtained from all of nine P. aeruginosa strains reactive against MAb E87. The result strongly supports a possibility that lipopolysaccharides having a D-rhamnan chain widely occur as the common antigen among various P. aeruginosa isolates.

Abstract: Structural studies were carried out on an acidic O-polysaccharide released by mild acid treatment from the lipopolysaccharide of Pseudomonas aeruginosa IID 1001 (ATCC 27577), which is serologically related to the serotypes Habs O3, Lanyi O1, and Wokatsch O13 in other serological classifications of Pseudomonas aeruginosa. The composition and data from structural analyses including H-NMR and C-NMR measurements, methylation, and Smith degradation showed that the structure of the IID 1001 O-polysaccharide was coincident with that of the Habs O3 and Lanyi O1 antigens (or Wokatsch O13). However, whereas solvolysis of the O-antigen of Habs O3 as well as that of Lanyi O1 by hydrogen fluoride has been reported to yield a reducing trisaccharide, GlcNAc(alpha 1----4)GalNAcA(alpha 1----3)Bac2NAc4Nacyl (acyl represents a 3-hydroxybutanoyl group), hydrogen fluoride hydrolysis of IID 1001 O-polysaccharide yielded a nonreducing trisaccharide with the reducing terminal bacillosamine residue linked at C-1 to the hydroxyl group of its N-acyl substituent, 3-hydroxybutanoic acid. These results, in combination with mass spectral data, led to the most likely structure for the tetrasaccharide repeating unit of the IID 1001 O-polysaccharide, (formula; see text) in which the location of N-acyl groups on bacillosamine residues differs from that in the O-antigens of Habs O3 and Lanyi O1 (or Wokatsch O13).

Abstract: Structural studies were carried out on a rhamnose-rich polysaccharide isolated from the O-polysaccharide fraction of lipopolysaccharide in Pseudomonas aeruginosa IID 1008 (ATCC 27584) after destruction of the major O-specific chain by alkaline treatment. The isolated polysaccharide contained rhamnose, 3-O-methyl-6-deoxyhexose, glucose, xylose, alanine, galactosamine and phosphorus in a molar ratio of 67:6.9:4.3:2.1:1.1:1.0:4.1. Data from analysis involving Smith degradation, methylation, 1H-NMR spectroscopy and optical rotation measurement showed that the polysaccharide was built up of three moieties, a rhamnan chain composed of about 70 D-rhamnose residues, the core chain and an oligosaccharide chain comprising 3-O-methyl-6-deoxyhexose, xylose, rhamnose and probably glucose. The repeating unit of the rhamnan chain was indicated to have the following structure:----3)D-Rha(alpha 1----3)D-Rha(alpha 1----2)D-Rha(alpha 1----. This structure is identical with that proposed previously for the repeating unit of the side chain of lipopolysaccharide from plant pathogenic bacteria Pseudomonas syringae pv. morsprunorum C28 [Smith, A.R.W., Zamze, S.E., Munro, S.M., Carter, K. J. and Hignett, R.C. (1985) Eur. J. Biochem. 149, 73-78].

Abstract: Structural studies were carried out on the O-polysaccharide fraction obtained from the lipopolysaccharide of Pseudomonas aeruginosa IID 1008 (ATCC 27584). The O-polysaccharide comprises L-rhamnose, N-acetyl-D-quinovosamine, N-acetyl-D-galactosaminuronic acid, and N-formyl-D-galactosaminuronic acid. The characterization of oligosaccharide fragments resulting from acid hydrolysis, Smith degradation and alkaline degradation of the O-polysaccharide, together with 1H-NMR and 13C-NMR spectroscopic data of the polysaccharide, led to the following structure for the repeating units: ----3)Rha(alpha 1----4)GalNAcA(alpha 1----4 GalNFoA(alpha 1----3)QuiNAc(alpha 1----. Almost all of the carboxyl groups of the N-acetylgalactosaminuronic acid residues and about half of the same groups of the N-formylgalactosaminuronic acid residues were in an amide form.