Bordetella pertussis infection in 2-month-old infants promotes type 1 T cell responses.

Neonatal immaturity of the immune system is currently believed to generally limit the induction of immune responses to vaccine Ags and to skew them toward type 2 responses. We demonstrated here that Bordetella pertussis infection in very young infants (median, 2 mo old) as well as the first administration of whole-cell pertussis vaccine induces B. pertussis Ag-specific IFN-gamma secretion by the PBMC of these infants. IFN-gamma was secreted by both CD4(+) and CD8(+) T lymphocytes, and the levels of Ag-induced IFN-gamma secretion did not correlate with the age of the infants. Appearance of the specific Th-1 cell-mediated immunity was accompanied by a general shift of the cytokine secretion profile of these infants toward a stronger Th1 profile, as evidenced by the response to a polyclonal stimulation. We conclude that the immune system of 2-mo-old infants is developmentally mature enough to develop Th1 responses in vivo upon infection by B. pertussis or vaccination with whole-cell pertussis vaccines.

Diversion of intestinal flow decreases the numbers of interleukin 4 secreting and interferon gamma secreting T lymphocytes in small bowel mucosa.

BACKGROUND/AIMS: The intestinal immune system faces large amounts of antigens, and its regulation is tightly balanced by cytokines. In this study, the effect of intestinal flow diversion on spontaneous secretion of interleukin (IL)-4 and interferon (IFN)- gamma was analysed. METHODS: Eight patients (two with Crohn's disease, four with ulcerative colitis, and two with previous colon cancer) carrying a double lumen small bowel stoma after a total colectomy procedure were included in the study. For each patient, eight biopsy samples were taken endoscopically from both the diverted and non-diverted part of the small bowel. Intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) were isolated separately and assayed for numbers of cells spontaneously secreting IL-4 and/or IFN-gamma by an ELISPOT technique. RESULTS: Compared with the non-diverted mucosa, a significant decrease in the number of spontaneously IFN-gamma secreting CD3 lymphocytes was observed in the diverted small bowel mucosa among both IELs (p = 0.008) and LPLs (p = 0.007). The same results, although less significant, were obtained for IL-4, especially in LPLs (p = 0.01). CONCLUSION: The intestinal content influences the spontaneous secretion of IFN-gamma and IL-4 by intestinal lymphocytes. These results could help to elucidate the anti-inflammatory role of split ileostomy in patients suffering from inflammatory bowel diseases.

A novel syndrome of severe neutrophil dysfunction: unresponsiveness confined to chemotaxin-induced functions.

We have identified a patient with a number of neutrophil dysfunctions. The patient was a female baby who lived for 8 months. During her life, she developed severe bacterial infections and showed omphalitis, impaired wound healing, and a pronounced leukocytosis. She was not a patient with leukocyte adhesion deficiency, because all leukocyte CD18 complex proteins were expressed at normal levels. Yet, neutrophil polarization and chemotaxis to platelet-activating factor, leukotriene B4, or formyl-methionyl-leucyl-phenylalanine (FMLP) were completely absent. We found a strong defect in actin polymerization in response to chemotactic stimuli, but only a retarded or even normal reaction with other stimuli. This indicates that the cellular dysfunctions were not due to an intrinsic defect in actin metabolism. Instead, the regulation of actin polymerization with chemotactic stimuli seemed to be defective. We concentrated on FMLP-induced responses in the patient's neutrophils. Functions dependent on activation of complement receptor type 3, such as aggregation or adherence to endothelial cells, were normally induced. Binding to serum-coated coverslips was normal in cell number; however, spreading was not observed. Exocytosis from the specific granules was readily induced. In contrast, FMLP failed to induce a respiratory burst activity or degranulation of the azurophil granules. FMLP induced a normal increase in free intracellular Ca2+, but a decreased formation of diglycerides (especially the 1-O-alkyl,2-acyl compounds). Thus, we have described a patient whose neutrophils show a severe defect in functional activation via chemotaxin receptors, resulting in a selective absence of NADPH oxidase activity, exocytosis from the azurophil granules, and actin polymerization. Our findings show that actin polymerization for neutrophil spreading and locomotion is regulated differently from that for phagocytosis. Also, the release of azurophil and specific granule contents is clearly shown to be regulated in a different way.

Heparin-binding hemagglutinin, from an extrapulmonary dissemination factor to a powerful diagnostic and protective antigen against tuberculosis.

Interactions of Mycobacterium tuberculosis with macrophages have long been recognized to be crucial to the pathogenesis of tuberculosis. The role of non-phagocytic cells is less well known. We have discovered a M. tuberculosis surface protein that interacts specifically with non-phagocytic cells, expresses hemagglutination activity and binds to sulfated glycoconjugates. It is therefore called heparin-binding hemagglutinin (HBHA). HBHA-deficient M. tuberculosis mutant strains are significantly impaired in their ability to disseminate from the lungs to other tissues, suggesting that the interaction with non-phagocytic cells, such as pulmonary epithelial cells, may play an important role in the extrapulmonary dissemination of the tubercle bacillus, one of the key steps that may lead to latency. Latently infected human individuals mount a strong T cell response to HBHA, whereas patients with active disease do not, suggesting that HBHA is a good marker for the immunodiagnosis of latent tuberculosis, and that HBHA-specific Th1 responses may contribute to protective immunity against active tuberculosis. Strong HBHA-mediated immuno-protection was shown in mouse challenge models. HBHA is a methylated protein and its antigenicity in latently infected subjects, as well as its protective immunogenicity strongly depends on the methylation pattern of HBHA. In both mice and man, the HBHA-specific IFN-gamma was produced by both the CD4(+) and the CD8(+) T cells. Furthermore, the HBHA-specific CD8(+) T cells expressed bactericidal and cytotoxic activities to mycobacteria-infected macrophages. This latter activity is most likely perforin mediated. Together, these observations strongly support the potential of methylated HBHA as an important component in future, acellular vaccines against tuberculosis.

In vitro expansion of CD4+CD25highFOXP3+CD127low/- regulatory T cells from peripheral blood lymphocytes of healthy Mycobacterium tuberculosis-infected humans.

CD4+CD25highFOXP3+ regulatory T (Treg) cells have recently been found at elevated levels in the peripheral blood of tuberculosis patients, compared to Mycobacterium tuberculosis latently infected (LTBI) healthy individuals and non-infected controls. Here, we show that CD4+CD25highFOXP3+ T lymphocytes can be expanded in vitro from peripheral blood mononuclear cells (PBMC) of LTBI individuals, but not of uninfected controls by incubating them with BCG in the presence of TGF-beta. These expanded cells from the PBMC of LTBI subjects expressed CTLA-4, GITR and OX-40, but were CD127low/- and have therefore the phenotype of Treg cells. In addition, they inhibited in a dose-dependant manner the proliferation of freshly isolated mononuclear cells in response to polyclonal stimulation, indicating that they are functional Treg lymphocytes. In contrast, incubation of the PBMC with BCG alone preferentially induced activated CD4+ T cells, expressing CD25 and/or CD69 and secreting IFN-gamma. These results show that CD4+CD25highFOXP3+ Treg cells can be expanded or induced in the peripheral blood of LTBI individuals in conditions known to predispose to progression towards active tuberculosis and may therefore play an important role in the pathogenesis of the disease.

Are the reference values of B cell subpopulations used in adults for classification of common variable immunodeficiencies appropriate for children?

Detailed phenotypic characterization of B cell subpopulations is of utmost importance for the diagnosis and management of humoral immunodeficiencies, as they are used for classification of common variable immunodeficiencies. Since age-specific reference values remain scarce in the literature, we analysed by flow cytometry the proportions and absolute values of total, memory, switched memory and CD21(-/low) B cells in blood samples from 168 healthy children (1 day to 18 years) with special attention to the different subpopulations of CD21(low) B cells. The percentages of total memory B cells and their subsets significantly increased up to 5-10 years. In contrast, the percentages of immature CD21(-) B cells and of immature transitional CD21(low)CD38(hi) B cells decreased progressively with age, whereas the percentage of CD21(low) CD38(low) B cells remained stable during childhood. Our data stress the importance of age-specific reference values for the correct interpretation of B cell subsets in children as a diagnostic tool in immunodeficiencies.

TCR V alpha- and V beta-gene segment use in T-cell subcultures derived from a type-III bare lymphocyte syndrome patient deficient in MHC class-II expression.

Previously, we and others have shown that MHC class-II deficient humans have greatly reduced numbers of CD4+CD8- peripheral T cells. These type-III Bare Lymphocyte Syndrome patients lack MHC class-II and have an impaired MHC class-I antigen expression. In this study, we analyzed the impact of the MHC class-II deficient environment on the TCR V-gene segment usage in this reduced CD4+CD8- T-cell subset. For these studies, we employed TcR V-region-specific monoclonal antibodies (mAbs) and a semiquantitative PCR technique with V alpha and V beta amplimers, specific for each of the most known V alpha- and V beta-gene region families. The results of our studies demonstrate that some of the V alpha-gene segments are used less frequent in the CD4+CD8- T-cell subset of the patient, whereas the majority of the TCR V alpha- and V beta-gene segments investigated were used with similar frequencies in both subsets in the type-III Bare Lymphocyte Syndrome patient compared to healthy control family members. Interestingly, the frequency of TcR V alpha 12 transcripts was greatly diminished in the patient, both in the CD4+CD8- as well as in the CD4-CD8+ compartment, whereas this gene segment could easily be detected in the healthy family controls. On the basis of the results obtained in this study, it is concluded that within the reduced CD4+CD8- T-cell subset of this patient, most of the TCR V-gene segments tested for are employed. However, a skewing in the usage frequency of some of the V alpha-gene segments toward the CD4-CD8+ T-cell subset was noticeable in the MHC class-II deficient patient that differed from those observed in the healthy family controls.

Spontaneous secretion of interferon gamma and interleukin 4 by human intraepithelial and lamina propria gut lymphocytes.

BACKGROUND: Cytokines secreted by intestinal T lymphocytes probably play a critical role in regulation of the gut associated immune responses. AIMS: To quantify interferon gamma (IFN-gamma) and interleukin 4 (IL-4) secreting cells (SC) among human intraepithelial (IEL) and lamina propria (LPL) lymphocytes from the duodenum and right colon in non-pathological situations and in the absence of in vitro stimulation. PATIENTS: Duodenal and right colonic biopsy specimens were obtained from patients with no inflammation of the intestinal mucosa. METHODS: Intraepithelial and lamina propria cell suspensions were assayed for numbers of cells spontaneously secreting IFN-gamma and IL-4 by a two site reverse enzyme linked immunospot technique (ELISPOT). RESULTS: The relatively high proportion of duodenal lymphocytes spontaneously secreting IFN-gamma (IEL 3.6%; LPL 1.9%) and IL-4 (IEL 1.3%; LPL 0.7%) contrasted with the very low numbers of spontaneously IFN-gamma SC and the absence of spontaneously IL-4 SC among peripheral blood mononuclear cells. In the basal state, both IFN-gamma and IL-4 were mainly produced by CD4+ cells. Within the colon, only 0.2% of IEL and LPL secreted IFN-gamma in the basal state, and 0.1% secreted IL-4. CONCLUSIONS: Compared with peripheral lymphocytes substantial proportions of intestinal epithelial and lamina propria lymphocytes spontaneously secrete IFN-gamma and/or IL-4. These cytokines are probably involved in the normal homoeostasis of the human intestinal mucosa. Disturbances in their secretion could play a role in the pathogenesis of gastrointestinal diseases.

Differential inducibility of HLA class I and II antigens by r-IFN gamma in type III bare lymphocyte syndrome.

Case Reports

Nutrient- and hypotonicity-induced insulin secretion: effects of cyclic AMP analogs, IBMX, tenidap and 5-nitro-2-(3-phenylpropylamino)benzoic acid

info:eu-repo/semantics/published

Characterization of immunoregulatory T lymphocytes during ageing by monoclonal antibodies.

Monoclonal antibodies of the OKT series were used to identify T lymphocytes (OKT3+) and their inducer (OKT4+) and suppressor-cytotoxic (OKT8+) subsets in the peripheral blood mononuclear cells (PBMC) of 32 healthy old-aged people more than 70 years old (16 men and 16 women) compared to 47 adults (29 men, 18 women) less than 40 years old. The absolute lymphocyte count in the peripheral blood was not significantly influenced by age or sex. Both the proportions and the absolute numbers of T3+ and T4+ cells were significantly lower in aged than in young participants. The proportions but not the absolute counts of OKT8+ cells were higher in the elderly. Most interesting is the influence of sex and these parameters. Old women have normal numbers and proportions of T3+, T4+ and T8+ cells when compared to young women. The latter have a significantly higher proportion of T8+ cells than young adult males. Old men have a striking reduction of both the numbers and proportions of OKT3+ and OKT4+ cells when compared with young men and with women. In addition, old men have an elevated proportion, but a normal absolute number, of OKT8+ cells. The responses of PBMC to phytohaemagglutinin extent (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) are reduced to the same extent in ageing male and female subjects when compared to young adults. In the older group, the magnitude of the lymphocyte response to PHA and Con A but not to PWM is negatively correlated with the proportions of OKT8+ cells. Surprisingly, these correlations are observed only in old women but not in old men. The latter finding excludes the possibility that the age-associated decline of the lymphocyte response to T cell mitogens is secondary to an imbalance between T4+ and T8+ lymphocytes.

Regulatory T cells depress immune responses to protective antigens in active tuberculosis.

RATIONALE: Tuberculosis (TB) remains a leading cause of death, and the role of T-cell responses to control Mycobacterium tuberculosis infections is well recognized. Patients with latent TB infection develop strong IFN-gamma responses to the protective antigen heparin-binding hemagglutinin (HBHA), whereas patients with active TB do not. OBJECTIVES: We investigated the mechanism of this difference and evaluated the possible involvement of regulatory T (Treg) cells and/or cytokines in the low HBHA T-cell responses of patients with active TB. METHODS: The impact of anti-transforming growth factor (TGF)-beta and anti-IL-10 antibodies and of Treg cell depletion on the HBHA-induced IFN-gamma secretion was analyzed, and the Treg cell phenotype was characterized by flow cytometry. MEASUREMENTS AND MAIN RESULTS: Although the addition of anti-TGF-beta or anti-IL-10 antibodies had no effect on the HBHA-induced IFN-gamma secretion in patients with active TB, depletion of CD4(+)CD25(high)FOXP3(+) T lymphocytes resulted in the induction by HBHA of IFN-gamma concentrations that reached levels similar to those obtained for latent TB infection. No effect was noted on the early-secreted antigen target-6 or candidin T-cell responses. CONCLUSIONS: Specific CD4(+)CD25(high)FOXP3(+) T cells depress the T-cell-mediated immune responses to the protective mycobacterial antigen HBHA during active TB in humans.

Molecular dissociation of FHA-induced IL-10 and IL-12 secretion by human dendritic cells.

info:eu-repo/semantics/nonPublished

Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin.

In addition to antibodies, Th1-type T cell responses are also important for long-lasting protection against pertussis. However, upon immunization with the current acellular vaccines, many children fail to induce Th1-type responses, potentially due to immunomodulatory effects of some vaccine antigens, such as filamentous haemagglutinin (FHA). We therefore analysed the ability of FHA to modulate immune functions of human monocyte-derived dendritic cells (MDDC). FHA was purified from pertussis toxin (PTX)-deficient or from PTX- and adenylate cyclase-deficient Bordetella pertussis strains, and residual endotoxin was neutralized with polymyxin B. FHA from both strains induced phenotypic maturation of human MDDC and cytokine secretion (IL-10, IL-12p40, IL-12p70, IL-23 and IL-6). To identify the FHA domains responsible for MDDC immunomodulation, MDDC were stimulated with FHA containing a Gly→Ala substitution at its RGD site (FHA-RAD) or with an 80-kDa N-terminal moiety of FHA (Fha44), containing its heparin-binding site. Whereas FHA-RAD induced maturation and cytokine production comparable to those of FHA, Fha44 did not induce IL-10 production, but maturated MDDC at least partially. Nevertheless, Fha44 induced the secretion of IL-12p40, IL-12p70, IL-23 and IL-6 by MDDC, albeit at lower levels than FHA. Thus, FHA can modulate MDDC responses in multiple ways, and IL-10 induction can be dissociated from the induction of other cytokines.

Anoctamin 1 (Ano1) is required for glucose-induced membrane potential oscillations and insulin secretion by murine β-cells.

Anions such as Cl(-) and HCO3 (-) are well known to play an important role in glucose-stimulated insulin secretion (GSIS). In this study, we demonstrate that glucose-induced Cl(-) efflux from β-cells is mediated by the Ca(2+)-activated Cl(-) channel anoctamin 1 (Ano1). Ano1 expression in rat β-cells is demonstrated by reverse transcriptase-polymerase chain reaction, western blotting, and immunohistochemistry. Typical Ano1 currents are observed in whole-cell and inside-out patches in the presence of intracellular Ca(++): at 1 μM, the Cl(-) current is outwardly rectifying, and at 2 μM, it becomes almost linear. The relative permeabilities of monovalent anions are NO3 (-) (1.83 ± 0.10) > Br(-) (1.42 ± 0.07) > Cl(-) (1.0). A linear single-channel current-voltage relationship shows a conductance of 8.37 pS. These currents are nearly abolished by blocking Ano1 antibodies or by the inhibitors 2-(5-ethyl-4-hydroxy-6-methylpyrimidin-2-ylthio)-N-(4-(4-methoxyphenyl)thiazol-2-yl)acetamide (T-AO1) and tannic acid (TA). These inhibitors induce a strong decrease of 16.7-mM glucose-stimulated action potential rate (at least 87 % on dispersed cells) and a partial membrane repolarization with T-AO1. They abolish or strongly inhibit the GSIS increment at 8.3 mM and at 16.7 mM glucose. Blocking Ano1 antibodies also abolish the 16.7-mM GSIS increment. Combined treatment with bumetanide and acetazolamide in low Cl(-) and HCO3 (-) media provokes a 65 % reduction in action potential (AP) amplitude and a 15-mV AP peak repolarization. Although the mechanism triggering Ano1 opening remains to be established, the present data demonstrate that Ano1 is required to sustain glucose-stimulated membrane potential oscillations and insulin secretion.