Abstract: Kiwellin, an allergenic protein formerly isolated from green kiwi fruit, has been identified as the most abundant component of the gold kiwi species. A protein named KiTH, showing a 20 kDa band on reducing SDS-PAGE and 100% identity with the C-terminal region of kiwellin, has been identified in the extract of the ripe green species. In vitro treatment of purified kiwellin with the protease actinidin from green kiwi fruit originated KiTH and kissper, a recently described pore-forming peptide. Primary structure analysis and experimental evidence suggest that kiwellin is a modular protein with two domains. It may undergo in vivo proteolytic processing by actinidin, thus producing KiTH and kissper. When probed with sera recognizing kiwellin from green kiwi fruit, KiTH showed IgE binding, with reactivity levels sometimes different from those of kiwellin. The IgE-binding capacity of kiwellin from gold kiwi fruit appears to be similar to that of the green species.
Abstract: The diagnosis and therapy of allergic disorders are usually performed with crude extracts which are a heterogeneous mixture of proteins with different allergenic potency. The knowledge of the allergenic composition is a key step for diagnostic and therapeutic options. Parietaria judaica pollen represents one of the main sources of allergens in the Mediterranean area and its major allergens have already been identified (Par j 1 and Par j 2). In addition, inhibition studies performed using a calcium-binding protein (CBP) from grass pollen (Phl p 7) showed the presence of a homologue of this cross-reactive allergen in the Parietaria extract. Screening of a cDNA library allowed us to isolate a 480bp cDNA containing the information for an 87 AA long protein with high level of homology to calcium-binding proteins from other allergenic sources. It was expressed as a recombinant allergen in Escherichia coli and purified by affinity chromatography. Its expression allowed us to study the prevalence of this allergen in a population of allergic patients in southern Europe. Immunoblotting and inhibition studies showed that this allergen shares a pattern of IgE epitopes in common with other 2-EF-hand calcium-binding proteins from botanically non-related species. The immunological properties of the Pj CBP were investigated by CD63 activation assay and CFDA-SE staining. In conclusion, DNA recombinant technology allowed the isolation, expression and immunological characterization of a cross-reactive calcium-binding protein allergen from Parietaria judaica pollen.
Abstract: BACKGROUND: Immunoglobluin E (IgE)-mediated hypersensitivity to natural rubber latex (NRL) is a major problem in allergy practice. Currently, the use of skin prick tests (SPTs) with latex extracts and specific IgE detection for the diagnosis of NRL allergy in suspected patients is directed to identification of risk factors. Many cases of NRL allergy remain undiagnosed due to misreporting of symptoms by the patients or lack of proper questions asked by the physician. MATERIALS AND METHODS: A total of 6,126 subjects referred for respiratory symptoms underwent SPTs with NRL. Positive subjects were resurveyed for exposure to NRL, and specific IgE for NRL extracts and recombinant molecules was determined. Immunoblots of NRL extracts were performed to identify IgE patterns. RESULTS: Forty-six of 3,930 sensitized subjects had a positive SPT with NRL, displaying a prevalence of NRL sensitization of 0.75% for the general and 1.2% for the sensitized population. Eleven out of 46 (23.9%) subjects could be defined as NRL asymptomatic, whereas 35 (76.1%) developed symptoms upon exposure to NRL. Specific IgE to NRL was detected for 22 (75.86%) of 29 tested sera. Seventeen out of 22 (77%) sera displayed specific IgE to recombinant allergens with most reactions to Hev b 5, Hev b 6.01 and Hev b 6.02. Immunoblots of NRL extract fractions with patients' sera showed heterogeneous patterns. CONCLUSIONS: SPTs with NRL extract should be routinely performed in patients with respiratory symptoms. Hev b 5, Hev b 6.01 and Hev b 6.02 are the most important allergens, but further characterization of NRL extracts is needed to identify novel allergens and to clarify the role of crossreactive carbohydrate determinants.
Abstract: This study was aimed at showing the capacity of probiotic VSL#3 to hydrolyze wheat flour allergens. Hydrolysis was investigated either by the use of baker's yeast bread treated with digestive enzymes and VSL#3, an experimental design that mimicked the activity of probiotics during gut colonization, or by the use of VSL#3 as a starter for dough fermentation, an experimental design that mimicked the predigestion of wheat flour proteins during food processing. Albumins, globulins, and gliadins extracted from wheat flour and chemically acidified and started dough and total proteins extracted from breads were analyzed by immunoblotting with pooled sera from patients with an allergy to wheat. Hydrolysis of wheat flour proteins was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2DE). Mass spectrometry matrix-assisted laser desorption and ionization-time of flight was used to identify some immunoglobulin E (IgE)-binding proteins. As shown by immunoblotting with sera from allergic patients, several IgE-binding proteins persisted after treatment of baker's yeast bread by pepsin and pancreatin. The signal of all these IgE-binding proteins disappeared after further treatment by VSL#3. As shown by SDS-PAGE and related immunoblotting and 2DE analyses, when VSL#3 was used as a starter for bread making, it caused a marked degradation of wheat proteins, including some IgE-binding proteins such as the putative transcription factor APFI and wheat alpha-amylase inhibitors. Indeed, the IgE-binding profile of the bread manufactured by VSL#3 was largely different from that of baker's yeast bread. The IgE-binding proteins that persisted in the bread made with VSL#3 were completely degraded by pepsin and pancreatin.
Abstract: Allergens are proteins or glycoproteins that are recognized by IgE produced by the immune system of allergic individuals. Until now around 1,500 allergenic structures have been identified and this number seems not have reached a plateau after 3-4 decades of research and the advent of molecular biology. Several allergen databases are available on Internet. Different aims and philosophies lead to different products. Here we report about main feature of web sites dedicated to allergens and we describe in more details our current work on the Allergome platform. The web server Allergome (www.allergome.org) represent a free independent open resource whose goal is to provide an exhaustive repository of data related to all the IgE-binding compounds. The main purpose of Allergome is to collect a list of allergenic sources and molecules by using the widest selection criteria and sources. A further development of the Allergome platform has been represented by the Real Time Monitoring of IgE sensitization module (ReTiME) that allows uploading of raw data from both in vivo and in vitro testing, thus representing the first attempt to have IT applied to allergy data mining. More recently, a new module (RefArray) representing a tool for literature mining has been released.
Abstract: BACKGROUND: Muscle relaxants represent the drugs most frequently involved in intraoperative anaphylaxis during surgical procedures. Our aim was to report the case of a delayed reaction to suxamethonium and analyze specific T cell lines with regard to their specificity, phenotype and cytokine profile. METHODS: We generated a drug-specific T cell line from a biopsy at the site of positive intradermal reactions and analyzed the immunophenotype, T cell receptor Vbeta domain expression and cytokine profile. RESULTS: T cells isolated from positive intradermal test reactions to suxamethonium showed a strict dose-dependent proliferation in response to drug-pulsed autologous antigen-presenting cells. The drug-specific CD4+ T cells were oligoclonal memory CD3+CD4+ T cells and expressed the skin homing receptors cutaneous lymphocyte antigen (CLA) and CCR4. Furthermore CD4+ suxamethonium-reactive T cell lines were IFN-gamma-positive and synthesized high levels of IFN-gamma and TNF-alpha. CONCLUSION: The study describes a delayed hypersensitivity to suxamethonium, driven by an oligoclonal T helper cell 1-skewed CD4+ memory T cell population, expressing the skin homing receptors CLA and CCR4.
Abstract: Immunotherapy for allergic diseases give rise to questions about when a decision must be taken to define the number of extracts to be used in a single treatment. This represents a long-lasting matter of debate between American and European allergists, which seems to be without real solution. Through the use of extract-based versus molecule-based diagnostic approaches we suggest a possible solution to this controversial issue. We used four model patients previously tested with allergenic extracts and later on selected on the basis of a panel of available allergenic molecules. Their reactivity patterns in term of extracts and molecules were compared and the decision for allergenic extract immunotherapy was made choosing either the 'classical' approach or the molecular approach. From our study it seems that molecules could offer the solution to the 'mixing' issue of allergenic extracts. This innovative approach seems to provide a solution for both the American and the European approach.
Abstract: The approaches used in both basic research and clinical work in allergology must be reconsidered in the light of information technology. Internet resources are further amplified by the parallel development of other new tools, such as molecular biology and nanotechnology. Information technology, molecular biology and nanotechnology are tightly related entities that fully express their power once applied to biomedical fields. Bioinformatics applied to allergological research simplifies the approach to the increasing wealth of knowledge.
Abstract: The Arg-Gly-Asp (RGD) motif is known to mediate cell adhesion to several extracellular matrix components as well as cell-cell interactions. In the present study, we investigated whether the RGDS peptide interferes with cell-cell recognition-based events such as allogeneic activation of PBMC and PBMC adhesion to human umbilical vein endothelial cells (HUVEC). We show here for the first time, to our knowledge, that RGDS significantly inhibits adhesion of activated PBMC to HUVEC; in addition, RGDS inhibits PBMC allogenenic activation in human mixed lymphocyte reaction assays. Caspases played a pivotal role in both events, because preventing their activation abolished or strongly reduced the observed inhibitory effect. The RGDS antirecognition effect was strongly increased by pretreatment of HUVEC with RGDS, which affected mostly T lymphocyte adhesion to HUVEC. These results indicate that PBMC allogeneic activation, as well as reciprocal recognition between activated PBMC and endothelial cells, are RGDS-dependent events that occur through a dual effect involving anti-adhesive and caspase-dependent mechanisms. These data suggest a potential role of RGDS in cell-mediated immunity, inflammation and organ transplantation.
Abstract: We studied the expression of adhesion molecules affecting recirculation and homing on peripheral blood CD4(+) T cells of patients with systemic sclerosis (SSc), in order to evaluate whether the distribution of tissue targeted subsets could reflect the participation of internal organs or the extent of cutaneous involvement [i.e. limited cutaneous (lc) and diffuse cutaneous (dc)]. Peripheral blood mononuclear cells (PBMC) from 51 patients with SSc and 19 sex- and age-matched controls were investigated by cytofluorimetric analysis for lymphocyte subpopulations carrying the following surface molecules: CD3, CD4, CLA, alpha4beta7 and alpha4beta1. Standard routine biochemistry and clinical examinations were also performed in all patients. We found that both alpha4beta1(+) and alpha4beta7(+) cells within the CD4(+) T cell population were significantly increased, while CLA(+) CD4(+) T cells were significantly reduced in SSc, compared to healthy donors. Significantly lower absolute numbers of alpha4beta7(+) cells were found in lc- compared to dc-SSc. Patients with oesophageal involvement had high numbers of alpha4beta7(+) cells, while those with nephritis also showed low levels of CLA(+) cells. Lung involvement was related directly to alpha4beta1(+) cell numbers and inversely to alpha4beta7(+) CD4 cell numbers. Taken together, our findings demonstrate that distinct CD4(+) T cell populations with selective homing properties show changes from normal distribution in SSc, and such changes are related to clinical expression and organ involvement in the course of the disease.
Abstract: Systemic sclerosis (SSc) is a connective tissue disorder characterized by excessive collagen deposition in the skin and internal organs. Several cytokines and chemokines have been implicated in the induction of fibrosis, but a definitive relationship between specific cytokines and organ involvement has not been established yet. Serum samples, PBMC and T cell lines (TCL) obtained from 54 patients affected by SSc and 20 healthy donors (HD) were examined by ELISA for Interferon-gamma (IFN-gamma ), interleukin (IL)-4, IL-6, IL-10, IL-18, Transforming growth factor (TGF)-beta1, Tumour necrosis factor (TNF)-alpha, sCD30, Macrophage derived chemokine (MDC), Monocyte chemoattractant protein (MCP)-1, Macrophage inflammatory protein (MIP)-1alpha and Regulated on activation normal T-cell expressed and secreted (RANTES). In all the SSc serum samples, we found significantly increased levels of IL6, TNFalpha and MCP-1 but reduced amounts of gamma-IFN and MDC. IL6, IL10, IL18, MIP-1alpha and TNFalpha measured in supernatants from PHA-stimulated PBMC and IL6, MCP-1 and RANTES in supernatants from stimulated TCL were also increased in patients. MDC was decreased in all the biological SSc sources studied. TGF-beta1, IL10, and sCD30 were produced at a significantly lower level by SSc TCL. Serum IL6 and sCD30 levels were significantly increased in dc-SSc patients compared to lc-SSc as were levels of MCP-1 produced by PBMC and IL10 from TCL. We observed a strict relationship between pulmonary fibrosis and IL10, MCP-1 (both from TCL) and serum IL6. Kidney involvement was related to serum MCP-1 levels and IL18 production from PBMC. Oesophageal involvement correlated with MDC production from PBMC and IL10 synthesis by TCL. We showed that IL-6, IL-10, MDC and MCP-1 are variably associated with internal organ involvement and allow the discrimination between limited and diffuse forms of the disease.
Abstract: BACKGROUND: Multiple drug allergy syndrome is a clinical condition characterized by reactions against more than one different class of, both pharmacologically and structurally, unrelated drugs. Scanty data are available to date about a multiple drug delayed hypersensitivity syndrome. Our aim was to report the case of a delayed reaction to both beta-methasone (beta-MT) and penicillin-G (pen-G) occurring in the same patient, and analyse beta-MT- and pen-G-specific T-cell Lines (TCLs) with regard to their specificity, phenotype and cytokine profile. METHODS: We generated two drug-specific TCLs from biopsies at the site of positive intradermal reactions, and analysed their immunophenotype, T-cell receptor Vbeta (TCR-Vbeta) domains expression and cytokine profile. RESULTS: We demonstrated the specificity of the T cells isolated from positive intradermal test reactions to pen-G and beta-MT through the strict dose-dependent proliferation in response to drug-pulsed autologous antigen presenting cells. Fluorescence activated cell sorter (FACS) analysis revealed a predominance of CD4+ cells in the inflammatory cell infiltrate of intradermal test with beta-MT, while a predominance of CD8+ T cells in the site of delayed reaction to pen-G was found. The drug specific CD4+ and CD8+ T cells were heterogeneous, with regard to TCR-Vbeta usage. CD8+ pen-G-TCL displayed a preferential T helper 2 (Th2) profile, while a substantially heterogeneous pattern of cytokine production characterized specific beta-MT TCL. CONCLUSION: The study describes the coexistence in the same patient of a delayed hypersensitivity to both penicillin G and beta-MT, driven, respectively, by pen-G-specificTh2-skewed CD8+ and beta-MT specificTh0 CD4+ T cells. This case further support the existence of a multiple drug allergy syndrome also for delayed hypersensitivity.
Abstract: Proinflammatory cytokines are deemed to play a pivotal role in the pathogenesis of multiple sderosis (MS). They provide signals for T-cell activation and inflammatory cell recruitment in the brain and might directly alter neuroglial and neuronal cell survival and function. We found that peripheral blood mononuclear cells (PBMCs) from MS patents spontaneously produce high levels of TNFalpha, TNFbeta, IFNgamma, and oncostatin M (oncM), a proinflammatory cytokine actng on cells of neural, vascular, hematopoietic, and lymphoid origin. Spontaneous production of these cytokines was significantly higher (p < 0.01) in PBMC short-term culture supernatants from MS patients than in blood donors (HC). On average, lectin-induced production of these cytokines by PBMC was higher in MS patents than in HC significantly so only for TNFalpha (p = 0.013). Determination of TNFalpha, TNFbeta IFNgamma, and oncM in corresponding sera showed that on average, oncM levels were higher in MS patients than in HC, though the results were not statistically significant whereas levels of TNFalpha, TNFbeta and IFNgamma were below the assay threshold in most patients. The finding that MS PBMCs are primed in vivo to produce and release high levels of proinflammatory cytokines suggests the presence of a basal activation of the immune system which, in turn, may play a role in the complex circuitry of molecular and cellular interactions responsible for neurologic damage in MS.
Abstract: Ataxia telangiectasia (A-T), a genetic disorder caused by the homozygous mutation of the ATM gene, frequently associates with variable degrees of cellular and humoral immunodeficiency. However, the immune defects occurring in patients with A-T are still poorly characterized. Here we show that the T-cell receptor (TCR) variable beta (BV)-chain repertoire of 9 A-T patients was restricted by diffuse expansions of some variable genes prevalently occurring within the CD4 subset and clustering to certain TCRBV genes (eg, 5.1, 11, 14, and 23). In addition, the study of the third complementarity-determining region (CDR3) showed, in all patients, significantly altered profiles in most BV genes examined suggesting diffuse oligoclonal expansions. The sequencing of TCR CDR3 regions revealed completely normal V(D)J coding joints and confirmed a reduced diversity of the antigen-receptor repertoire. The B-cell repertoire was similarly restricted and skewed by diffuse oligoclonal expansions with normal V(D)J joints. Thymic output, evaluated by measuring TCR rearrangement excision circles, was extremely low. The majority of peripheral T cells had the phenotype and the function of effector memory cells, indicating that in vivo they are able to respond normally by terminal differentiation to antigenic stimulation. These results indicate that ATM mutation limits the generation of a wide repertoire of normally functioning T and B cells.
Abstract: Metoclopramide hydrochloride (MCPH) is dopamine antagonist antiemetic drug that binds D2 receptor at the central nervous system and peripheral levels, which stimulates the upper gastrointestinal tract motility. It is often used in the management of some forms of nausea and vomiting (1-3). Occupational allergy to drugs is seldom reported. No case of occupational allergy to MCPH have been reported, to date. We present a case of airborne allergic asthma in a worker employed in the synthesis of Metoclopramide hydrochloride.
Abstract: Anisakis simplex (AS), a fish and cephalopodes parasite, may cause allergic reactions in humans on eating and/or handling contaminated fish. We present a case of occupational hypersensitivity to AS in a woman employed in a frozen-fish factory. She showed both generalised urticarial rash and asthmatic symptoms after work place exposure. All these symptoms immediately disappeared after work place exposure was ceased. The presence of a positive skin prick test and high specific IgE values confirmed a hypersensitivity to anisakis. This is the first case reported of both occupational generalised urticaria and allergic airborne asthma due to AS in the same patient. We suggest that AS could be an important cause of occupational asthma and/or urticaria in the fish industry.
Abstract: Chromosomal rearrangements observed in T-cell prolymphocytic leukemia involve the translocation of one T-cell receptor gene to either chromosome 14q32 or Xq28, deregulating the expression of cellular protooncogenes of unknown function, such as TCL1 or its homologue, MTCP1. In the human hematopoietic system, TCL1 expression is predominantly observed in developing B lymphocytes, whereas its overexpression in T cells causes mature T-cell proliferation in transgenic mice. In this study, using a newly generated monoclonal antibody against recombinant TCL1 protein, we extended our analysis mainly by immunohistochemistry and also by fluorescence-activated cell sorting and Western blot to a large tumor lymphoma data bank including 194 cases of lymphoproliferative disorders of B- and T-cell origin as well as reactive lymphoid tissues. The results obtained show that in reactive lymphoid tissues, TCL1 is strongly expressed by a subset of mantle zone B lymphocytes and is expressed to a lesser extent by follicle center cells and by scattered interfollicular small lymphocytes. In B-cell neoplasia, TCL1 was expressed in the majority of the cases, including lymphoblastic lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, follicular lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma (60%), and primary cutaneous B cell lymphoma (55%). TCL1 expression was observed in both the cytoplasmic and nuclear compartments, as confirmed by Western blot analysis. Conversely, TCL1 was not expressed in Hodgkin/Reed-Sternberg cells, multiple myelomas, marginal zone B-cell lymphomas, CD30+ anaplastic large cell lymphoma, lymphoblastic T-cell lymphoma, peripheral T-cell lymphoma, and mycosis fungoides. These data indicate that TCL1 is expressed in more differentiated B cells, under both reactive and neoplastic conditions, from antigen committed B cells and in germinal center B cells and is down-regulated in the latest stage of B-cell differentiation.
Abstract: Evidence indicates that, at least in the early stage, Kaposi's sarcoma (KS) is a cytokine-mediated disease and that it is consistently associated with a novel herpesvirus termed human herpesvirus-8 (HHV-8). To gain insights into the mechanisms by which cytokines and HHV-8 may cooperate in disease pathogenesis, we examined the phenotype, the Th1 (gamma-interferon [gamma IFN]) and Th2 (interleukin-4 [IL-4] cytokine profile and the presence of HHV-8 in peripheral blood mononuclear cells (PBMC), tumor-infiltrating lymphocytes (TIL), and spindle cell cultures derived from skin lesions of patients affected by classical KS (C-KS) and acquired immunodeficiency syndrome (AIDS)-associated KS (AIDS-KS). TIL and spindle cell cultures were examined at day 0 or after culture in conditioned media from activated T cells (TCM) that contain the same cytokines increased in KS tissues. No differences were found in the immunophenotype of PBMC from C-KS patients versus controls, except for AIDS-KS patients who showed a T-CD8+ expansion. However, a preferential infiltration of T-CD8+ cells was found in all KS lesions examined, which was maintained after culture of TIL in TCM. gamma IFN production was found in both PBMC and cultures derived from all KS examined; some IL-4 positive supernatants were found only in three AIDS-KS cases. Uninvolved skin did not show appreciable lymphocyte infiltration or cytokine production. The culture conditions of the lesional skin allowed also the appearance of adherent, spindle-like cells bearing markers of tissue macrophages. Finally, most or all of the PBMC, lesions, and macrophagic cell cultures from the skin lesions were found to be positive for HHV-8 infection by nested polymerase chain reaction (PCR). These findings indicate that patients with KS express a Th1 phenotype with a prevalent gamma IFN production, likely accounted for by the local T-CD8+ infiltration. By analogy with other viral infections (i.e., Epstein-Barr virus), this suggests that in loco recruitment of lymphoid cells and the subsequent gamma IFN production may be in response to or elicited by HHV-8 that was found in both PBMC and macrophagic cell cultures from the lesions of the same patients.
Abstract: We evaluated the relationship between cytokine profile and the expression of the lymphocyte activation gene-3 (LAG-3) in both T cell clones and polyclonal T cell lines; LAG-3 is a CD4-like protein whose expression is reportedly restricted to Th1/0 cells and dependent upon IFN-gamma. We found that, while LAG-3 was expressed only by CD4+ T cell clones producing IFN-gamma, most CD8+ clones producing IL-4 but not IFN-gamma (i.e., with a T cytotoxic-2-like profile) were LAG-3+. The intensity of LAG-3 expression by CD8+ clones correlated with the amount of released IFN-gamma, suggesting that this cytokine is not required for expression but rather for the up-regulation of LAG-3. Flow cytometric analyses of polyclonal T cell lines confirmed that LAG-3 could be expressed by both CD4+ and CD8+ cells that did not contain cytoplasmic IFN-gamma. In these cell lines, large proportions of CD4+ and CD8+ cells coexpressed LAG-3 and CD30, a putative marker of Th2-like cells. Overall, our data do not support the earlier suggestion that LAG-3 and CD30 are selective markers of T cells with type-1 and type-2 cytokine profiles, respectively.
Abstract: A combination of three beta, or C-C, chemokines, as well as IL-16, have been shown to inhibit HIV replication in vitro. Cellular antiviral factor is a more potent agent, and acts on all HIV strains. All are mainly, but not exclusively, produced by CD8+ T cells, both in HIV+ and healthy subjects. We studied the production of these HIV-suppressive factors in patients with HIV infection at different stages of disease. No difference in production by PBMC stimulated with PHA has been observed in asymptomatic HIV+, long-term nonprogressors (LTnP), and AIDS patients. When T cell line supernatants from these three groups were studied, no significant difference was found for C-C chemokines or IL-16 production, and viral suppression. However, T cell clones from LTnP secreted higher levels of all three chemokines, IL-16, and exerted a stronger inhibition on HIV replication. CD8+ clones showed a higher production than CD4+ clones. These clones were able to produce all antiviral factors irrespective of the secretion of type 1 or type 2 cytokines. The antiviral activities were not correlated, implying that viral suppression did not depend solely on C-C chemokines or IL-16. We postulate that all factors are needed to prevent HIV disease progression.
Abstract: The authors studied CD40 ligant (CD40L) expression and interleukin-10 (IL-10) production in 16 patients with common variable immunodeficiency (CVI). Mean CD40L expression, determined by using cytofluorimetry, and measured as the mean fluorescence intensity following stimulation of peripheral blood mononuclear cells (PBMC) with phorbol myristate acetate (PMA) and calcium ionophore in 12 patients, was comparable to that of controls. However, three CVI patients showed fluorescence intensity in stimulated cells below 2 standard deviations of normal donors' mean and two other patients had only a slight increase of stimulated versus unstimulated cells (< 10 channels). IL-10 production after stimulation of PBMC with both anti-CD3 or anti-CD3 plus PMA gave similar results in CVI patients and normal controls. In vitro stimulation of PBMC with anti-CD40 and various combinations of cytokines (IL-2, IL-4 and IL-10) induced IgG production above 100 ng/ml in one CVI patient out of 13 tested. The data suggest that alterations of IL-10 production are unlikely to play a major role in the pathogenesis of impaired IgG production in most CVI patients. CD40L appears to be normally expressed in two thirds of CVI patients, but it may be functionally defective.
Abstract: Centenarians, particularly healthy centenarians, constitute the example of successful aging and the study of their immune status can help to define the endpoint of the changes occurring throughout life. We characterized T cell clones (TCC) of two healthy centenarians, studying their phenotypes and production of representative Th1 and Th2 cytokines (IFN-gamma and IL-4) and compared them with TCC obtained by three young normal subjects; in all 180TCC were analyzed. In young donors, 35TCC were CD4+, 56CD8+ and 2 were alpha beta +CD4-CD8- (double negative). In centenarians, we obtained 46CD4+TCC, 38CD8+, 2CD4+CD8+ (double positive) and 1 gamma delta + double negative. Of the young subjects' TCC, 71% produced IFN-gamma but no IL-4 (Th1 pattern) and this prevalence decreased to 39% in TCC from the centenarians. The number of clones showing the opposite Th2 pattern was similar in young and aged donors (3 out of 93TCC and 2 out of 87TCC, respectively). The intermediate profile of TCC producing both IL-4 and IFN-gamma (Th0) was found in 25.8% of clones from young people, but it almost doubled to 58.6% in centenarians. The analysis shows that the Th profiles of CD8+TCC is nearly superimposable in the two groups, whereas a major shift from a Th1 to a Th0 pattern is presented by CD4+TCC. The balance provided by a majority of CD4+TCC showing a Th0 pattern may ensure both humoral and cell-mediated defences. In CD8+TCC, however, a Th1 pattern still is present, possibly for efficient generation of cytotoxic responses. These findings should be extended by studying other centenarians and elderly subjects.
Abstract: The association between an acquired form of hyper-IgM syndrome and a chronic hepatitis C virus (HCV) infection in a 71-year-old female patient is described. Both diseases were diagnosed at the age of 58 years. She was started on intramuscular and then intravenous immunoglobulin replacement therapy. HCV RNA was detected in 1992. The patient remained in well-balanced clinical condition until 1994, when total and specific anti-HCV IgM levels increased and the patient developed an IgM kappa monoclonal gammopathy. Adherent cells and B cells were HCV RNA positive, while T cells were HCV RNA negative. Anti-IgM reactivity was specifically directed to the core antigen of the HCV. The patient we describe showed a picture of a late-onset form of hypogammaglobulinemia with a progressive increase in IgM antibodies, possibly due to the concomitant HCV infection. It is possible that the immunodeficiency might also result from the HCV infection, with formation of specific antibodies belonging to the IgM class, and that the worsening of the clinical condition may be directly related to the persistent viral infection.
Abstract: PURPOSE: To determine the efficacy of percutaneous needle aspiration (PNA) with antibiotic therapy in treatment for pyogenic liver abscess (PLA). MATERIALS AND METHODS: One hundred fifteen patients (59 male; 56 female; age range, 16-86 years; mean age, 45.3 years) with 147 PLAs (mean diameter, 6.8 cm; range, 3-16 cm) underwent PNA with ultrasound (US) guidance and antibiotic therapy. Needle caliber (22-16 gauge) was tailored to PLA volume. If necessary, PNA was repeated every 3-7 days. RESULTS: Three hundred one PNAs were performed (range, 1-4 per patient; mean, 2.2 per patient). A single puncture was sufficient in 57 patients. Cure (normalization of clinical and laboratory parameters and resolution of hepatic lesions) was achieved in 113 patients (98.3%). Two patients with large PLAs required surgery. Patients were hospitalized 7-24 days (mean, 9 days). In the last eight patients, all abscesses were evacuated in one session. Neither complications nor deaths ensued. Recurrence of PLA was not observed in any patient during follow-up (6-36 months). CONCLUSION: US-guided PNA with antibiotic therapy in treatment for PLA is a valid alternative to prolonged catheter drainage.
Abstract: Increased levels of serum IgE and eosinophilia have been described in human immunodeficiency virus (HIV) infection, almost exclusively in patients with CD4+ cell count < 200 cells/microliters. IgE production is regulated by CD4+ T helper type 2 (Th-2) lymphocytes, producing interleukin 4 (IL-4) and expressing a ligand for the B cell-specific CD40 molecule (CD40 ligand [L]). A shift to a Th-2-like pattern of cytokine secretion has been postulated to be associated with progression toward acquired immunodeficiency syndrome (AIDS). We studied three AIDS patients with very high levels of IgE and almost complete depletion of CD4+ lymphocytes, suggesting that IgE synthesis could not be driven by CD4+ cells. IgE in vitro synthesis by cells from such patients was, however, inhibited by anti-IL-4. We show that both CD8+ T cell lines and the majority of CD8+ T cells clones derived from these patients produce IL-4, IL-5, and IL-6 in half of the cases together with interferon gamma (IFN-gamma). 44% of CD8+ T cell clones expressed a CD40L, and the supernatants of the clones were capable of inducing IgE synthesis by normal B cells costimulated with anti-CD40. CD8+ T cells in these patients therefore functionally mimic Th-2 type cells and may account for hyper-IgE and eosinophilia in the absence of CD4+ cells. The presence of such CD8+ cells may also provide a source of IL-4 directing the development of predominant Th-2 responses in HIV infection.
Abstract: In this study we describe a series of nine patients affected by acquired immunodeficiency syndrome (AIDS) or AIDS-related complex who had hypereosinophilia and hyperimmunoglobulinemia E (hyper-IgE) with chronic dermatitis and recurrent staphylococcal infections. These patients had features similar to those present in hyper-IgE syndrome, a primary immunodeficiency disease. In addition, immunologic characterization of these patients with human immunodeficiency virus (HIV) infection, compared with 51 HIV-positive patients without hyper-IgE, both atopic and nonatopic, and three patients affected by the primary hyper-IgE syndrome, also revealed an increase in IgA and a severe decrease in B and CD4+ lymphocytes. Spontaneous in vitro synthesis of IgE by peripheral blood mononuclear cells was confirmed in both hyper-IgE conditions, together with increased levels of circulating eosinophil cationic protein. Serum-soluble CD23, usually increased in atopic conditions and hyper-IgE, was similar to that of normal control subjects in the HIV-positive patients with hyper-IgE. On the basis of our findings, we conclude that a hyper-IgE-like syndrome represents a distinct aspect of the clinical manifestations associated with HIV infection and that the immunologic mechanisms in this condition seem to differ from those known in primary hyper-IgE syndrome, because CD4+ TH2 type cells, which are currently believed to have a role in IgE production, are severely depleted in HIV-positive patients.
Abstract: Intranasal immunotherapy (IT) has been proposed as a means to induce an effective immunity of the nasal mucosa in patients with allergic rhinitis, avoiding systemic side effects. In the present study 20 individuals with chronic allergic rhinitis, and skin prick test reactive to Dermatophagoides pteronyssinus (DP) only, were randomized and subjected to a three months' double-blind placebo-controlled trial of intranasal IT with DP extract. All patients received also sodium cromoglycate as pre-medication. Before and at the end of the treatment the patients performed specific nasal provocation tests, and samples of serum and nasal secretions were collected to measure total and specific IgE, levels of eosinophil cationic protein (ECP), and mast-cell-derived tryptase. A clinical score was computed by the symptoms indicated by the patients. The clinical score did not change in the two groups after the treatment, whereas a decrease in nasal reactivity was observed. Total IgE increased only in secretions from placebo-treated patients, but were not modified in sera. IgE to DP in sera and nasal secretions did not change significantly. Tryptase levels in nasal secretions decreased in both groups, while ECP was unchanged after IT. Serum ECP levels decreased more in actively treated patients than in the placebo group. The data suggest that changes of IgE and inflammatory mediators may be affected by the use of sodium cromoglycate in both groups, but some parameters change early in different directions in IT- and placebo-treated groups.
Abstract: CD8+CD45RA+ T lymphocytes present two distinct subpopulations expressing the CD11a molecule (LFA-1 alpha chain) with different intensity. CD11adim cells represent the unprimed population within the CD8+CD45RA+ subset, whereas CD11abright cells are activated and may be considered as memory lymphocytes. The aim of this study was to analyze the expression of CD11a and CD18 within the CD8+CD45RA+ population in young HIV-infected individuals at different stages of disease as a marker of activation and of disease progression. Blood cells from 82 HIV-infected individuals and 23 age-matched healthy controls were stained with unconjugated CD11a, CD18, PE-goat F(ab')2 anti-mouse, FITC-CD45RA, and TRI-Color CD8. Quantitative analysis for three-color immunofluorescence was carried out by flow cytometry. HIV+ subjects were subdivided into three groups according to their CD4+ cell number (group A, CD4+ cells > 500/microliters [20 subjects], group B, CD4+ cells between 500 and 200/microliters [36 subjects], and group C, CD4+ lymphocytes < 200/microliters [26 subjects]). We found a significant increase of CD11abright in the CD8+CD45RA+ subpopulation throughout the progression of the disease. The CD11abright percentage of positivity (mean) within the CD8+CD45RA+ subpopulation was 31% in healthy donors, 51% in group A, 52% in group B, and 68% in group C. CD11abright expression was closely related to CD18bright (p < 0.001), but not to CD38. The relative increase of CD11a and CD18 expression in CD8+CD45RA+ T lymphocytes parallels the decrease of CD4+ cells and the progression of disease: an inverse correlation between the percentages of CD4+ cells/microliter and CD8+CD45RA+CD11abright cells (p < 0.001) and a direct correlation between the number of CD4+ lymphocytes per microliter and both the number of CD8+CD45RA+CD11adim cells (p < 0.001) and the number of CD8+CD45RA+CD11abright (p = 0.002) was observed. The relative increase of CD8+CD45RA+CD11abright cells may represent an additional marker for monitoring HIV-induced immunodeficiency.
Abstract: The immunologic defect in X-linked immunodeficiency and hyperimmunoglobulinemia M (HIM) are related to defective expression of the CD40 ligand (CD40L). We have studied two female patients with HIM to evaluate the role of CD40/CD40L in the pathogenesis of impaired immunoglobulin switching. In addition to recurrent infections characteristic of humoral immunodeficiencies, the two patients had chronic hepatitis caused by type C virus. Phenotypic characterization of peripheral blood mononuclear cells showed a similar picture in both patients, with a reduction in the absolute numbers of CD4 cells and increased numbers of CD8 and CD3/DR cells. B cells (CD19+) were reduced in one patient, but CD40 was expressed on all CD19+ cells in both patients. The expression of CD40L was normal on peripheral blood mononuclear cells from the two patients with HIM on both resting and stimulated cells. The combination of anti-CD40 and cytokines (interleukin-2, interleukin-4, and interleukin-10) was able to restore proliferative capacity to anti-IgM. Peripheral blood mononuclear cells from the two patients with HIM showed a high spontaneous production of IgM in vitro and no production of IgG or IgE. Our data suggest that the defect of isotype switching in female patients with HIM is not related to defective expression of the CD40/CD40L receptor system. A possible role for chronic hepatitis C virus infection in the pathogenesis of the disease is suggested by the detection of specific production of anti-hepatitis C virus IgM.
Abstract: We examined the expression of T cell markers in the peripheral blood of five immunologically normal human fetuses at 18-20 weeks of gestational age. The distribution of T cells expressing CD1, CD3, CD4, CD8, CD56, and the alpha/beta and gamma/delta receptors for antigen was comparable to that of newborns and normal adults, except for the absence of gamma/delta cells expressing the delta TCS-1 epitope. The V beta repertoire, as evaluated by two-color flow cytometry using mAbs to specific V beta families, was also comparable to that of adult samples. A significant fraction (8.9 to 16.4%) of fetal CD3+ T cells expressed the alpha chain of IL-2R (CD25) in the absence of HLA-DR; this suggests that antigenic stimuli trigger, during intrauterine life, an unusual pathway of T cell activation. Consistent with this, 7 to 27% of fetal T cells were found to express the CD45R0 marker of "memory" cells.
Abstract: The interactions between B and T lymphocytes, leading to the development of humoral responses, are reviewed with references to the changes occurring in aged people. Aging is perceived as a process of impairment of immune functions; it is known that T cells from aged subjects have a reduced ability to produce IL-2. However, other functions seem to be upregulated in elderly subjects; indeed, IL-1, IL-3, IL-4, IL-6 and TNF alpha production are increased both in aged mice and humans. These cytokines are known to control B cell differentiation, through isotype switch and Ig production. A significant increase in IgG subclasses and IgA is observed in sera of aged subjects. This contrasts with the significant decrease in circulating B lymphocytes. The impairment of primary responses to immunization, and other aspects of humoral immunity, including mucosal responses, autoantibody production and correlations with phenotypic markers of T and B cell subsets, are discussed.
Abstract: The production of cytokines during aging, except interleukin (IL)-2, has been neglected in humans. We measured the in vitro production of IL-6, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma and IL-1 beta by peripheral mononuclear cells from selected healthy young (mean age 26.8 years) and aged (mean age 80.2 years) subjects. Significant increases of IL-6, TNF-alpha and IL-1 beta levels were found in mitogen-stimulated cultures from aged donors, occurring at 24 to 72 h after stimulation. No significant differences were observed for IFN-gamma production. Proliferative capability of cells stimulated with PHA was not impaired in aged subjects. Since the amounts of all cytokines studied were similar in unstimulated cultures from young and aged subjects, and also serum levels of TNF-alpha did not differ, these data indicate that the cellular machinery for the production of these cytokines is well preserved in aging, and also that cells from old people are able to up-regulate their production in response to appropriate stimuli. The increases in cytokine synthesis were not dependent on changes in the number of monocytes, nor were they related to the significant rise of CD45RO+, and the concomitant decrease of CD45RA+, occurring in both CD4+ and CD8+ lymphocytes from aged subjects. The increased production of pro-inflammatory cytokines by stimulated mononuclear cells of healthy aged subjects may be relevant to several aspects of age-associated pathological events, including atherosclerosis, osteoporosis, fibrosis and dementia.
Abstract: We have determined the percentages and absolute numbers of lymphocyte subpopulations in 49 patients with common variable immunodeficiency (CVI). In vitro production of IL-6 by PHA-stimulated PBMC of 28 patients was also evaluated. Our data indicate profound alterations in the lymphocyte subpopulations evaluated, including increased CD8 cells (these cells outnumbered CD4 cells in 26 cases), severe defect of CD4 cells (< 400/mm3) in 10 cases and reduction of CD19 cells (< 50/mm3) in 11/41 cases. Increased production in vitro of IL-6 by PBMC of CVI patients does not correlate with the absolute numbers of lymphocytes, lymphocyte subpopulations, or monocytes. In addition, IL-6 was unable to significantly modify the amount of PWM-driven IgM production by CVI cells.
Abstract: Several immunological abnormalities have been observed in ataxia-telangiectasia (AT), the most consistent being defects of immunoglobulin isotypes, decreased T-cell numbers, and reduced proliferative responses to mitogens. We examined the distribution of T lymphocytes expressing distinctive surface Ag characteristic of "naive" (CD45RA+) and "memory" (CD29+, CD45RO+) T cells, in both CD4+ and CD8+ (bright and dim) lymphocytes from 13 AT patients, compared with healthy age-matched controls. We found that, irrespective of age, patients with AT had a severe deficiency of CD4+/CD45RA+ lymphocytes. This decrease accounted for the reduction of total CD4+ cells, since the absolute numbers of memory CD4+ cells were not significantly different in AT and in controls. Functional tests revealed poor proliferative responses to phytohemagglutinin and normal responses to soluble Ag (tetanus toxoid) in AT patients. These data fit with the distribution of naive and memory cells, which are known to respond predominantly to mitogens or to recall Ag, respectively. CD45RA molecules were normally expressed on CD8+ lymphocytes. This rules out a generalized defect of regulation or differential splicing as the cause of defective expression of CD45RA on CD4+ cells. The selective deficiency of CD4+CD45RA+ may provide a cellular basis for some functional T-cell abnormalities of AT patients. Furthermore, it might practically serve for an early, or even prenatal, diagnosis of this disease.
Abstract: We studied the clinical and immunological effects of three months' treatment with intranasal flunisolide (100 micrograms daily) in 18 allergic patients with perennial rhinitis. 17 were hypersensitive to house dust mite and one to Parietaria pollen only. We found no significant changes in white blood cell count, serum levels of IgE and nasal IgA. However the treatment induced a marked improvement of clinical symptoms in all cases, and we observed a significant reduction of total IgE in nasal secretion. Flunisolide seems to exert this effect through its antiinflammatory action on the nasal mucosa.
Abstract: The study of 87 adults of different ages, including 15 centenarians, selected for their healthy status, showed that profound changes of humoral immunity occur throughout life. In particular, a statistically significant age-related increase of the serum level of immunoglobulin classes (IgG and IgA but not IgM) and IgG subclasses (IgG1, 2 and 3, but not IgG4) was detected. A parallel age-related decrease of circulating B cells was also observed. The hypothesis of a complex derangement of B cell function and/or compartmentalization with age is put forward, together with the proposal that healthy centenarians (as representative of successful ageing) may be helpful in identifying the physiological age-related modifications of the immune system.
Abstract: We describe a new simplified solid phase immunoassay for the detection of anti-HIV antibodies. The results obtained analyzing a panel of sera from subjects showing a different pattern of reactivity on enzyme immunoassays and on Western blot demonstrated high sensitivity and specificity. The test does not require experienced laboratory staff nor the use of costly laboratory instruments.
Abstract: Atopic dermatitis (AD) is a chronic inflammatory disease of the skin, frequently associated with a family history of atopy, raised serum IgE levels and other immunological abnormalities. Both eosinophils and their basic proteins have been detected in the skin lesions of AD patients. We measured the levels of eosinophil cationic protein (ECP) in sera of 24 children with AD and found them to be increased, compared to nonatopic controls, both children and adults. High ECP values were also obtained in 3 patients with the hyper-IgE syndrome. However, no direct relationship between IgE and ECP serum levels could be established. We found no correlation between serum ECP and the number of circulating eosinophils, suggesting that part of ECP was produced by cells infiltrating the tissues. Measurement of ECP might represent a noninvasive tool to assess the activity of AD in relation to eosinophil involvement in this disease.
Abstract: We evaluated the clinical response to oligoallergenic dietary treatment and the intestinal absorption of a protein antigen, cow milk beta-lactoglobulin (BLG) in 24 patients with chronic urticaria/angioedema syndrome 13 of whom also suffered from joint symptoms. Sixteen patients (77% of those with arthralgia) responded to diet (RD) with marked reduction of symptoms; the others did not respond (NR). Ten (all but one RD with arthralgia) had increased permeability to BLG after oral administration of cow milk. Four with high titers of IgG to BLG showed the highest absorption of BLG and the groups with arthralgia showed higher BLG levels than those without arthralgia. In all cases, specific IgE to cow milk was absent. These data suggest that the symptoms of a subgroup of patients with chronic urticaria, and especially patients with joint complaints that subside with diet, are related to excess intestinal permeability. The measurement of gut permeability to food proteins may be useful to define those who may benefit from dietary restriction.
Abstract: We measured the in vitro production of interferon-gamma (IFN-gamma) in five cases of hyper-IgE syndrome (HIgE), induced by mitogens, calcium ionophores and phorbol ester. The biosynthesis of IFN-gamma was severely reduced or undetectable in HIgE, while it was near normal in most atopic patients. The in vitro spontaneous production of IgE was increased overall in HIgE patients, although no correlation was found with serum IgE levels. Recombinant interleukin-4 (IL-4) induced a further increase in IgE synthesis, and its effect was totally antagonized by recombinant IFN-gamma; the same pattern of response was also observed in atopic subjects with high production of IgE. IFN-alpha synergized with IL-4 on IgE synthesis, whereas recombinant IL-6 gave opposite changes in individual cases tested. We propose that IFN-gamma deficiency may be responsible for some of the features of HIgE patients, including IgE levels and infections.
Abstract: We studied the clinical and immunological effects of three months' treatment with intranasal flunisolide (100 micrograms daily) in 18 allergic patients with perennial rhinitis. 17 were hypersensitive to house dust mite and one to Parietaria pollen only. We found no significant changes in white blood cell count, serum levels of IgE and nasal IgA. However the treatment induced a marked improvement of clinical symptoms in all cases, and we observed a significant reduction of total IgE in nasal secretion. Flunisolide seems to exert this effect through its antiinflammatory action on the nasal mucosa.
Abstract: We studied 14 patients with irritable bowel syndrome for the presence of increased intestinal permeability to food antigens and their responses to diet with and without disodium cromoglycate. After a standardized oral challenge with cow milk, serum beta-lactoglobulin was increased above control values in three patients. This finding did not correlate with response to hypoallergenic diet or treatment with disodium cromoglycate for 3 weeks. However over 50% of patients improved after diet with and without DSCG (2/5 on diet only and 5/7 with disodium cromoglycate of 12 evaluable cases). Since only two patients had elevated serum IgE levels, our results suggest that intolerance rather than hypersensitivity to foods may play a role in the disease. The tests we used to identify immunologic mechanisms could not predict which patients would do better on the diet and/or the drug.
