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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.

BACKGROUND: Most individuals infected with Mycobacterium tuberculosis do not develop tuberculosis (TB) and can be regarded as being protected by an appropriate immune response to the infection. The characterization of the immune responses of individuals with latent TB may thus be helpful in the definition of correlates of protection and the development of new vaccine strategies. The highly protective antigen heparin-binding hemagglutinin (HBHA) induces strong interferon (IFN)- gamma responses during latent, but not active, TB. Because of the recently recognized importance of CD8(+) T lymphocytes in anti-TB immunity, we characterized the CD8(+) T lymphocyte responses to HBHA in subjects with latent TB. RESULTS: HBHA-specific CD8(+) T lymphocytes expressed memory cell markers and synthesized HBHA-specific IFN- gamma . They also restricted mycobacterial growth and expressed cytotoxicity by a granule-dependent mechanism. This activity was associated with the intracellular expression of HBHA-induced perforin. Surprisingly, the perforin-producing CD8(+) T lymphocytes were distinct from the IFN- gamma -producing CD8(+) T lymphocytes. CONCLUSION: During latent TB, the HBHA-specific CD8(+) T lymphocyte population expresses all 3 effector functions associated with CD8(+) T lymphocyte-mediated protective immune mechanisms, which supports the notion that HBHA may be protective in humans and suggests that markers of HBHA-specific CD8(+) T lymphocyte responses may be useful in the monitoring of protection.

Although post-translational modifications of protein antigens may be important componenets of some B cell epitopes, the determinants of T cell immunity are generally nonmodified peptides. Here we show that methylation of the Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA) by the bacterium is essential for effective T cell immunity to this antigen in infected healthy humans and in mice. Methylated HBHA provides high levels of protection against M. tuberculosis challenge in mice, whereas nonmethylated HBHA does not. Protective immunity induced by methylated HBHA is comparable to that afforded by vaccination with bacille Calmette et Guerin, the only available anti-tuberculosis vaccine. Thus, post-translational modifications of proteins may be crucial for their ability to induce protective T cell-mediated immunity against infectious diseases such as tuberculosis.

Whooping cough still represents a major health problem, despite the use of effective vaccines for several decades. Being classically a typical childhood disease, whooping cough in young adults is now more common than it used to be, suggesting that protection after vaccination wanes during adolescence. As an alternative to the current vaccines, we wish to develop live attenuated vaccines to be delivered by the nasal route, such as to mimic the natural route of infection and to induce long lasting immunity. Bordetella pertussis, the etiological agent of whooping cough, produces a number of virulence factors, including toxins. Its recently determined genome sequence makes it now possible to apply functional genomics, such as transcriptomics and systematic knock-out mutagenesis. The expression of most known B. pertussis virulence genes is controlled by the two-component system BvgA/S. DNA microarray analyses have led to the identification of novel genes in the BvgA/S regulon, some of which are activated by BvgA/S and others are repressed by BvgA/S. In addition, some genes appear to be differentially modulated by nicotinic acid and MgSO4, both known to modulate the expression of BvgA/S-regulated genes. Among others, the functional genomics approach has uncovered two strongly BvgA/S-activated genes, named hotA and hotB (for 'homolog of toxin'), the products of which show high sequence similarities to pertussis toxin subunits. The identification of the full array of virulence factors, as well as an integrated understanding of the bacterial physiology should allow us to design attenuated B. pertussis strains useful for intranasal vaccination. A first generation of attenuated strains has already shown full protection in mice after a single intranasal administration. Such strains may also serve as vaccine carriers for heterologous antigens, in order to vaccinate against several different pathogens simultaneously.

Strict lifelong compliance to a gluten-free diet (GFD) minimizes the long-term risk of mortality, especially from lymphoma, in adult celiac disease (CD). Although serum IgA antitransglutaminase (IgA-tTG-ab), like antiendomysium (IgA-EMA) antibodies, are sensitive and specific screening tests for untreated CD, their reliability as predictors of strict compliance to and dietary transgressions from a GFD is not precisely known. We aimed to address this question in consecutively treated adult celiacs.In a cross-sectional study, 95 non-IgA deficient adult (median age: 41 yr) celiacs on a GFD for at least 1 yr (median: 6 yr) were subjected to 1) a dietician-administered inquiry to pinpoint and quantify the number and levels of transgressions (classified as moderate or large, using as a cutoff value the median gluten amount ingested in the overall noncompliant patients of the series) over the previous 2 months, 2) a search for IgA-tTG-ab and -EMA, and 3) perendoscopic duodenal biopsies. The ability of both antibodies to discriminate celiacs with and without detected transgressions was described using receiver operating characteristic curves and quantified as to sensitivity and specificity, according to the level of transgressions.Forty (42%) patients strictly adhered to a GFD, 55 (58%) had committed transgressions, classified as moderate (or = 18 g of gluten/2 months; median number 6) in 27 and large (18 g; median number 69) in 28. IgA-tTG-ab and -EMA specificity (proportion of correct recognition of strictly compliant celiacs) was 0.97 and 0.98, respectively, and sensitivity (proportion of correct recognition of overall, moderate, and large levels of transgressions) was 0.52, 0.31, and 0.77, and 0.62, 0.37, and 0.86, respectively. IgA-tTG-ab and -EMA titers were correlated (p0.001) to transgression levels (r = 0.560 and R = 0.631, respectively) and one to another (p0.001) in the whole patient population (r = 0.834, N = 84) as in the noncompliant (r = 0.915, N = 48) group. Specificity and sensitivity of IgA-tTG-ab and IgA-EMA for recognition of total villous atrophy in patients under a GFD were 0.90 and 0.91, and 0.60 and 0.73, respectively.In adult CD patients on a GFD, IgA-tTG-ab are poor predictors of dietary transgressions. Their negativity is a falsely secure marker of strict diet compliance.

Interleukin-10 (IL-10) is currently being extensively studied in clinical trials for the treatment of Crohn's disease (CD). Only marginal effects have, however, been reported, and the dose-response curve was bell-shaped contrasting with the reported data from in vitro experiments.To use another in vitro model to analyze the effect of rhIL-10 and rhIL-4 on the spontaneous mucosal TNF-alpha secretion in patients with CD, and to characterize the phenotype of the cells targeted by rhIL-10.Non-inflamed colon biopsies from CD patients were cultured for 16 hours in presence of different concentrations of rhIL-10 or rhIL-4. The numbers of TNF-alpha-secreting cells among isolated lamina propria mononuclear cells (LPMNC) were estimated by Elispot.Both rhIL-10 and rhIL-4 down-regulate TNF-alpha secretion by LPMNC from CD patients, with a more pronounced effect with rhIL-10. These effects were closely linked to the cytokine concentrations used, with a bell-shaped dose-response curve. Residual TNF-alpha secretion, in the presence of optimal rhIL-10 concentration was mainly attributable to CD3+ T cells. In contrast, at higher rhIL-10 concentrations, CD3- cells contributed significantly to the TNF-alpha secretion.The in vitro model we used, demonstrates that IL-4, but mostly IL-10, efficiently suppresses TNF-alpha secretion in LPMNC from CD patients, with a dose-response curve similar to results obtained in vivo. Resistance at high rhIL-10 concentrations was associated with a change in the phenotype of TNF-alpha-secreting cells.

Background Individuals with genetic defects in CD40 ligand (CD40L) or B-cell antigen receptor coreceptor molecules CD19 and CD81 suffer from an antibody deficiency. Still, these patients carry low levels of memory B cells and serum antibodies. Objective We sought to assess why the remaining memory B cells and antibodies in the blood of these patients do not provide functional immunity. Methods We included CD19-deficient patients (n = 8), CD40L-deficient patients (n = 8), and healthy controls (n = 50) to perform detailed flow cytometry on blood B cells, molecular analysis of IgA and IgG transcripts, as well as functional analysis of B-cell activation. Results CD19-deficient and CD40L-deficient patients carried reduced numbers of all memory B-cell subsets except CD27 − IgA + B cells. Their immunoglobulin heavy chain class-switched transcripts contained less somatic mutations and reduced usage of IgM-distal IgG2 and IgA2 subclasses. The selection strength of mutations for antigen binding was significantly lower than in controls, whereas selection to maintain superantigen binding was normal. Furthermore, the patients showed impaired selection against inherently autoreactive properties of their immunoglobulins. Somatic hypermutation analysis revealed decreased activation-induced cytidine deaminase and uracil-DNA glycosylase 2 activity in CD40L deficiency and increased uracil-DNA glycosylase 2 but decreased mismatch repair in CD19 deficiency. B-cell activation studies revealed that this was at least in part due to transcriptional regulation of DNA repair genes. Conclusions This study on CD19 and CD40L deficiencies illustrates that both the B-cell antigen receptor and CD40 signaling pathways are required for the selection of immunoglobulin reactivity. Still, they differentially mediate DNA repair pathways during somatic hypermutation, thereby together shaping the human in vivo antigen-experienced B-cell repertoire.

Because only 10% of individuals infected with Mycobacterium tuberculosis will eventually develop disease, antigens that are recognized differently by the immune systems of infected healthy and diseased subjects may constitute potential vaccine candidates. Here, the heparin-binding hemagglutinin adhesin (HBHA) is identified as such an antigen. Lymphocytes from 60% of healthy infected individuals (n=25) produced interferon (IFN)-gamma after stimulation with HBHA, compared with only 4% of patients with active tuberculosis (n=24). In the responders, both CD4(+) and CD8(+) cells secreted HBHA-specific IFN-gamma, and the antigen was presented by both major histocompatibility complex class I and II molecules. In contrast to the reduced ability of patients with tuberculosis to produce HBHA-specific IFN-gamma, most of them (82%) produced anti-HBHA antibodies, compared with 36% of the infected healthy subjects. These observations indicate that HBHA is recognized differently by the immune systems of patients with tuberculosis and infected healthy individuals and might provide a marker for protection against tuberculosis.

Most individuals infected with Mycobacterium tuberculosis develop latent tuberculosis infection (LTBI). Some may progress to active disease and would benefit from preventive treatment yet no means currently exists to predict who will reactivate. Here, we provide an approach to stratify LTBI based on IFN-γ responses to two antigens, the recombinant Early-Secreted Antigen Target-6 (rESAT-6) and the latency antigen Heparin-Binding Haemagglutinin (HBHA)., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

We have investigated the impact of MHC class II deficiency on the development of the peripheral T-cell repertoire. During T-cell development, interactions between T-cell receptor (TcR) and MHC class I and class II molecules play a pivotal role in the positive and negative selection events that occur within the thymic micro-environment1,2, 3, 4,5. These MHC dependent selection processes influence in greater part the composition of the peripheral T-cell compartment, where only T-cells with moderate affinity for “self” MHC class I or class II molecules can be found6. Various studies in the mouse have shown that interference in these TcR/MHC interactions has a dramatic effect on the composition of the peripheral T-cell compartment with respect to development of T-cells co-expressing the accessory molecules CD4 or CD8 respectively.7,8,9,10

The tetraspan molecule CD81 is widely expressed on immune cells, such as B-, T-, NK-lymphocytes, monocytes and eosinophils, but also on most stromal and epithelial cells and on hepatocytes. In B-cells it is a member of the CD19 complex (CD19, CD21, CD81, CD225), which is required for signaling together with the B-cell antigen receptor upon antigen recognition. Its functions on other cells are unclear, but murine studies show an antiproliferative role for CD81. On hepatocytes, two different epitopes of CD81 act as a co-receptor for Hepatitis C virus and for Plasmodium infection. We evaluated a 4-year-old girl from consanguineous parents of Moroccan decent. She presented with recurrent infections and an acute nephrotic syndrome: >50% of glomeruli were affected due to focal mesangial hypercellularity. She showed poor weight gain (below 3rd percentile), but normal motor development. Her spleen and liver were enlarged, but function normally. Measurements of retina epithelium and CNS showed no signs of hypercellularity. Although serum IgG levels were strongly decreased (2.4 g/L) and IgM and IgA concentrations low within normal range, she tested positive for anti-platelet antibodies. Flow cytometric immunophenotyping of blood showed normal distribution and absolute numbers of granulocyte, monocyte and lymphocyte subsets; however, no CD19 expression was detected on the patient’s B-cells, whereas CD21 expression levels were normal. The patient carried no mutations in the CD19 and IFITM1 (CD225) genes. Additional immunophenotyping showed that all cells lacked CD81 expression. Sequencing of the CD81 gene showed a homozygous G>A substitution immediately downstream of exon 6 (c.561+1G>A). Spectratyping and quantitative PCR analysis showed clearly reduced total CD81 mRNA expression levels. Nearly all CD81 transcripts contained 13 additional nucleotides downstream of exon 6. This insertion results in a frame-shift and a premature stop (p.Glu188MetfsX13). The hypothetical protein lacks the fourth transmembrane domain. Similar to previously described CD19-deficient patients, our patient had reduced numbers CD5+ B-cells and Ig class switched and non-switched CD27+ memory-B cells. Whereas Vh-Cα and Vh-Cγ transcripts from Ig switched cells contained somatic hypermutations, the response of the patient’s B cells to in vitro stimulation through the B-cell receptor was impaired. The antibody response to rabies, tetanus and pneumococcal vaccinations is currently under investigation, as well as the potential impact of CD81 deficiency on the antigen-specific Th1 and Th2 cytokine production. In conclusion, the here presented CD81 deficiency is a new primary immunodeficiency, which leads to disruption of the CD19 complex and consequent hypogammaglobulinemia comparable to the CD19 deficiency. However, due to the broad tissue distribution, the clinical phenotype is not restricted to the B-cell system. Other organs are affected as well, most likely due to excessive proliferation and hypercellularity, with acute nephritic syndrome as dominant clinical problem. Currently, in vitro studies are being performed to identify whether the CD81 defect directly results in impaired B-cell and T-cell functions and abnormal proliferation of kidney and liver cells.

A young girl presenting with recurrent pulmonary infections and atypical lupus erythematosus was totally deficient in C4. In one sister, also deficient in C4, the same symptoms developed. Results of family studies were consistent with an autosomal recessive mode of transmission and with linkage of the genes determining C4 deficiency to those of the major histocompatibility complex. The patient's serum and red cells were Chido- and Rodgers-negative. Humoral and cellular immunity were normal, except for a low lymphocyte response in mixed lymphocyte culture. The cellular function of the patient's polymorphonuclear leukocytes was normal, for both phagocytosis and bactericidal activity using Candida albicans. However, in the presence of C4-deficient serum, opsonin generation and bactericidal indexes were diminished. These defects were completely reversible upon addition of purified C4.

The highly polymorphic fourth component of human complement (C4) is usually encoded by two genes, C4A and C4B, adjacent to the 21-hydroxylase (21-OH) genes and is also remarkable by the high frequency of the null alleles, C4A*Q0 and C4B*Q0. Complete C4 deficiency is exceptional because this condition appears only in homozygotes for the very rare double-null haplotype C4AQ0,BQ0. This condition in most cases gives rise to systemic lupus erythematosus and an increased susceptibility to infections. The molecular basis for complete C4 deficiency has not yet been established. Therefore we studied the DNA of three previously described C4 deficient patients belonging to unrelated families by restriction fragment length polymorphism analysis using C4 and 21-OH probes. These studies revealed a deletion of the C4B and 21-OHA genes in two patients and no deletion at all in the third patient. Therefore, complete C4 deficiency as a result of homozygosity for the C4AQ0, BQ0 haplotype is not a consequence of a deletion of the C4 genes. The molecular basis of this genetic abnormality is certainly very complex and may vary also from one case to another.

Between May 1979 and April 1983, 18 previously healthy African patients were hospitalized in Belgium with opportunistic infections (cryptococcosis, Pneumocystis carinii pneumonia, central-nervous-system toxoplasmosis, progressive cutaneous herpes simplex virus infection, disseminated cytomegalovirus infection, candidiasis, or cryptosporidiosis) or Kaposi's sarcoma, or with both. Ten of them died. During the same period five other patients were hospitalized with an illness consistent with a prodrome of the acquired immunodeficiency syndrome (chronic lymphadenopathy, fever, weight loss, and diarrhea). All patients tested had a marked decrease in helper T cells; an inversion of the normal ratio of helper to suppressor T cells, and a decreased or absent blastogenic response of lymphocytes to mitogens. Twenty patients had anergy. There was no evidence of an underlying immunosuppressive disease and no history of blood-product transfusion, homosexuality, or intravenous-drug abuse. This syndrome in patients originating in Central Africa is similar to the acquired immunodeficiency syndrome reported in American patients.

Echovirus meningoencephalitis and polymyositis are classical complications of X-linked agammaglobulinemia (1). The treatment of meningoencephalitis is troublesome since intravenous (2), intrathecal (3) and intraventricular (4) administration of gammaglobulins have been reported successful, but failure also occurred in some cases (5). We report our experience of high dose intravenous treatment.

Measurement of antigen-specific T cell responses is an adjunctive parameter to evaluate protection induced by a previous Bordetella pertussis infection or vaccination. The assessment of T cell responses is technically complex and usually performed on fresh peripheral blood mononuclear cells (PBMC). The objective of this study was to identify simplified methods to assess pertussis specific T cell responses and verify if these assays could be performed using frozen/thawed (frozen) PBMC. Three read-outs to measure proliferation were compared: the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) dilution test, the number of blast cells defined by physical parameters, and the incorporation of (3)H-thymidine. The results of pertussis-specific assays performed on fresh PBMC were compared to the results on frozen PBMC from the same donor. High concordance was obtained when the results of CFSE and blast read-outs were compared, an encouraging result since blast analysis allows the identification of proliferating cells and does not require any use of radioactive tracer as well as any staining. The results obtained using fresh and frozen PBMC from the same donor in the different T cell assays, including IFNγ and TNFα cytokine production, did not show significant differences, suggesting that a careful cryopreservation process of PBMC would not significantly influence T cell response evaluation. Adopting blast analysis and frozen PBMC, the possibility to test T cell responses is simplified and might be applied in population studies, providing for new instruments to better define correlates of protection still elusive in pertussis.

Peritoneal tuberculosis (TB) remains difficult to diagnose because of its non-specific clinical features and the lack of efficient microbiological tests. As delayed diagnosis is associated with high mortality rates, new diagnostic tools are needed., SCOPUS: ar.j, info:eu-repo/semantics/published