Rare splicing defects of FAS underly severe recessive autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is a prototypic disorder of impaired apoptosis characterized by autoimmune features and lymphoproliferation. Heterozygous germline or somatic FAS mutations associated with preserved protein expression have been described. Very rare cases of homozygous germline FAS mutations causing severe autosomal recessive form of ALPS with a complete defect of Fas expression have been reported. We report two unrelated patients from highly inbred North African population showing a severe ALPS phenotype and an undetectable Fas surface expression. Two novel homozygous mutations have been identified underlying rare splicing defects mechanisms. The first mutation breaks a branch point sequence and the second alters a regulatory exonic splicing site. These splicing defects induce the skipping of exon 6 encoding the transmembrane domain of CD95. Our findings highlight the requirement of tight regulation of FAS exon 6 splicing for balanced alternative splicing and illustrate the importance of such studies in highly consanguineous populations.

[Listeriosis in Tunis: seven cases reports]. / Les infections à Listeria monocytogenes à Tunis: à propos de sept cas.

Listeria monocytogenesis a Gram positive facultative intracellular bacterium that can be responsible for severe infections, affecting essentially pregnant women, immunocompromised patients at the early and later stages of life. In Tunisia, invasive L. monocytogenes infections are thought to be exceptional and limited data are available about listeriosis. We reported seven cases (five newborn children and two infants) of human listeriosis that occurred in Tunis from 2000 to 2008. The newborn children were hospitalized for suspicion of maternofoetal infections. The two infants were hospitalized for fever associated with digestive signs in one case and neurological signs in the other. L. monocytogenes-was isolated from culture of cerebrospinal fluid in four cases, peripheral samples in two cases and from blood culture in one case. Isolates identification was based on conventional methods. Antimicrobial susceptibility was realized according to the recommendation of the "Comité de l'antibiogramme de la Société française de microbiologie". All L. monocytogenes isolates were sensitive to amoxicillin and aminoside but resistant to 3rd generation cephalosporins. Investigations of the immune system were realized for the two infants including phenotypic analysis of peripheral blood cells by flow cytometry, lymphocyte proliferation assays, phagocytic cell functions and measurement of immunoglobulins as well as complement. All these explorations were normal for both infants. The outcome was fatal in only one case (a newborn child), and all the other patients recovered after adapted antibiotic treatment. In conclusion, our study shows that listeriosis is not exceptional in Tunis. Thus, it is necessary to know how to evoke this diagnosis, at any age, in order to establish an early and adapted antibiotic treatment and to avoid fatal outcome.

Diagnosis and management of autoimmune hemolytic anemia in children.

BACKGROUND AND AIM: The aim of this study is to evaluate the clinical, biological and hematological profiles of autoimmune hemolytic anemia (AIHA) in children and to specify its etiologies, therapeutic modalities, and treatment responses. METHODS: This is a 14-year retrospective study of AIHA cases collected at the department of pediatric emergency and reanimation of Hedi Chaker University Hospital in Sfax. We included patients under 14 years old with clinical and biological features of hemolysis and a positive direct antiglobulin test (DAT). The selected patients' demographic characteristics, physical signs, laboratory findings, and treatment responses were recorded. RESULTS: Thirteen cases of AIHA were collected, including 8 girls and 5 boys. The median age at diagnosis was 4 years and 6 months (range: 8 months to 13 years). Consanguinity was reported in 6 cases and 4 patients had a previous infection history. The onset of AIHA was progressive in 9 cases, marked by an anemic syndrome and hemolysis symptoms in 6 and 8 cases, respectively. The clinical triad (pallor, jaundice and splenomegaly) was found in only 4 cases. At the time of diagnosis, the median hemoglobin (Hb) level was 6g/dL (range: 4.2 to 9.2g/dL), anemia was non-regenerative in 2 patients. Thrombocytopenia and neutropenia were noted in 5 and 1 patient, respectively. Peripheral smear examination showed spherocytosis in 2 cases. All the patients had a positive DAT. Of these, 10 were positive with IgG and 3 with both IgG and C3d. AIHA was secondary to other conditions in 9 patients: infection (3 cases), autoimmune disease (4 cases), and immunodeficiency (2 cases). All the patients received first-line corticosteroid therapy but only 8 of them required blood transfusions due to severe anemia. Complete remission was obtained in 7 cases. Corticosteroid resistance and dependence were noted in 1 and 2 cases, respectively. During evolution, additional therapy was indicated in 4 patients and it included cyclosporine A, azathioprine, and mycophenolate mofetil (MMF). After a median follow-up of 4.5 years, the cure rate was 80% and only 1 patient (a boy) died due to his underlying pathology. CONCLUSION: Our study highlights the rarity, severity, and heterogeneity of etiological contexts of AIHA in children. The therapeutic difficulties justify specific expertise in pediatric hematology.

Staphylococcal scalded skin syndrome: An uncommon symptomatology revealing an immune deficiency.

Primary immune deficiencies associated with hyper-IgE syndrome are rare diseases with clinical features dominated by recurring cutaneous and visceral bacterial infections, particularly infections due to Staphylococcus species. Most of these infections are associated with milder inflammation compared to normal. We report a primary immune deficiency associated with a hyper-IgE syndrome revealed by a staphylococcal scalded skin syndrome in a 5-year-old girl. The patient presented with a severe staphylococcal infection with extensive skin lesions and disseminated intravascular coagulation. She received intravenous fluids to compensate for fluid losses and anti-staphylococcal antibiotics. Coagulopathy was also corrected. However, the progression was rapidly fatal.

High level expression of recombinant Mycobacterium tuberculosis culture filtrate protein CFP32 in Pichia pastoris.

Difficulty in obtaining large quantities of Mycobacterium tuberculosis (MTB) proteins remains a major obstacle in the development of subunit vaccines and diagnostic reagents for tuberculosis. A major reason is because Escherichia coli has not proven to be an optimal host for the expression of MTB genes. In this article, we used the yeast Pichia pastoris to express high levels of CFP32, a culture filtrate protein restricted to the MTB complex and a potential target antigen for serodiagnosis of tuberculosis in patients. Using shaker flasks, we generated a P. pastoris clone expressing CFP32 as a secreted protein fused to the myc- (His)6 tag, at a yield of 0.5 g of purified protein per liter of culture. Recombinant CFP32 (rCFP32) produced in P. pastoris has a molecular weight of 35 kDa, which is slightly higher than that of the native protein. We identified putative acylation and glycosylation sites in the CFP32 amino acid sequence that suggested posttranslational modifications may contribute to the size difference. The NH2-terminal peptide sequencing of rCFP32 showed that the signal peptide alpha factor is correctly excised. In addition, rCFP32 reacted with the sera of patients with tuberculosis. These data are the first to show that P. pastoris is a suitable host for high-yield production of good quality mycobacterium antigens, and especially culture filtrate proteins that have vaccine and diagnostic potential.

[Prevalence of Pneumocystis jiroveci pneumonia in Tunisian primary immunodeficient patients]. / Prévalence de la pneumopathie à Pneumocystis Jiroveci au cours des déficits immunitaires héréditaires observés en Tunisie.

UNLABELLED: Pneumocystis Jiroveci pneumonia (PJP) is a rare opportunistic infection in immunodeficient patients in Tunisia, as well as in other Africain countries including those with a high prevalence of AIDS. In the literature, PJP has been reported in primary immunodeficiency diseases (PID) namely SCID T-B- or T-B+ or X-linked hyper-IgM syndrome. OBJECTIVE: To evaluate the prevalence of PJP in the different PID observed in Tunisia. PATIENTS AND METHODS: This retrospective study concerned 290 cases of PID confirmed by immunological investigation including the study of specific and/or non-pecific humoral and cellular immunity. The identification of P. Jiroveci in patients suspected of pneumocystosis was achieved by parasitological investigation in bronchoalveolar lavages. RESULTS: A PID associated to a parasitologically confirmed pneumocystic infection was found in 9 out of 290 patients (3%) among whom the majority (7 patients) had an HLA class II combined immunodeficiency. The latter is an autosomic recessive disease which has been reported mainly in North African families. Indeed, this population is characterized by a high rate of consanguinity. Interestingly, no PJP has been observed neither in SCID T-B- or T-B+ nor in X-linked hyper-IgM syndrome. DISCUSSION AND CONCLUSION: PJP seems to be particularly frequent in HLA class II deficiency patients, since 7 out of 22 patients with this deficiency had a PJP (31%). Due to this defect, antigen presenting cells are unable to present the antigen to T lymphocytes demonstrating the critical role of CD4+ T lymphocytes responses in the immune response to this pathogen.

[Auto-immune hepatitis in chronic granulomatous disease in a 2-year-old girl]. / Hépatite auto-immune associée à une granulomatose septique chronique chez une fille de 2 ans.

BACKGROUND: Chronic granulomatous disease is a rare inherited primary immune deficiency disease characterized by recurrent infection and an increased susceptibility to autoimmunity disorders. We report on the case of a girl with autoimmune hepatitis in chronic granulomatous disease to describe the clinical and biological features and treatment implications for patients with chronic granulomatous disease associated with autoimmune disorders. CASE REPORT: An 18-month-old girl was referred to our department for investigation of hepatomegaly. She was the third child of non-consanguineous parents. Her two elder sisters had died from infectious diseases at an early age. She had elevated liver transaminase levels with a normal gamma globulin concentration. Negative results were found for all autoimmune markers (antinuclear antibody, anti-smooth muscle, anti-liver-kidney microsomal, anti-liver cytosol and anti-soluble liver antigen). Her liver biopsy showed features of interface hepatitis with portal fibrosis. The diagnosis of seronegative autoimmune hepatitis was established. Treatment with corticosteroids and azathioprine led to clinical improvement with normalization of transaminases. Six months after initial presentation, at the age of 2 years, she was readmitted for fever. Staphylococcus aureus bacteremia was identified with multiple foci of infection (skin infection, arthritis of the right elbow, pneumonia, buttock abscess). The immunological workup revealed chronic granulomatous disease. The course was marked by a fatal outcome despite appropriate antibiotics and intensive care. CONCLUSION: Early diagnosis of the association between chronic granulomatous disease and autoimmune disorders allows for appropriate treatments, improves the quality of life for affected patients, and reduces the risk of mortality.

Two Novel Frame Shift, Recurrent and De Novo Mutations in the ITGB2 (CD18) Gene Causing Leukocyte Adhesion Deficiency in a Highly Inbred North African Population.

We have identified four different mutations causing leukocyte adhesion Deficiency (LAD) in the ITGB2 gene of patients from a highly inbred population. Two were novel single-bp deletions (1497delG and 1920delG) causing frame shift and the two others were the missense mutations G284S and R593C. In our study, the G284S was a recurrent mutation while the R593C occurred de novo. We have also characterized a novel Xba1 polymorphic site located at the 5' end of the ITGB2 locus. Family studies showed that the 1497delG mutation segregated with this marker and the intragenic AvaII polymorphic marker, suggesting the presence of a founder effect. The observation of a heterogeneous spectrum including de novo and recurrent mutations causing LAD in a highly inbred population is rather unexpected. In view of the literature published on the molecular genetics of LAD and considering the ethnic origin of the patients studied, our findings confirm the heterogeneity of the mutations causing LAD and point out potential mutational hot spots in the ITGB2 gene.

[Primary immunologic deficiency by deficiency of HLA class II antigens: nine new Tunisian cases]. / Déficit immunitaire primitif par défaut d'expression des antigènes HLA de classe II: neuf observations tunisiennes.

BACKGROUND: Bare lymphocyte syndrome is a rare inherited primary immunodeficiency. The majority of the patients reported to date are from North Africa. We report nine new Tunisian cases. POPULATION AND METHODS: Over a period of 5 years, we have established the diagnosis of bare lymphocyte syndrome in nine patients who belong to seven different families. Class II HLA antigen expression was studied on resting peripheral mononuclear cells and PHA blasts. RESULTS: The clinical symptoms started at the mean age of 4.5 months (2-10 months) with chronic diarrhea. The evolution was characterized by appearance of other recurrent infections: pneumopathies (seven cases), thrush (seven cases), otitis (five cases) and septicemia (four cases). Allergic manifestations were observed in four cases. Six patients died at the mean age of 30 months from severe denutrition. Class II HLA antigens were not expressed on resting and activated lymphocytes. The absolute count of TCD4+ lymphocytes was decreased in seven patients. Lymphoproliferative response to specific antigens was absent. Four patients had panhypogammaglobulinemia. CONCLUSION: This study confirms the frequency of this disease among the North African population. The severity of the recurrent infection suggests the diagnosis of bare lymphocyte syndrome. This disease is fatal in the absence of bone marrow transplantation.

[Primary immunodeficiency in Tunisia: study of 152 cases]. / Les déficits immunitaires primitifs en Tunisie: étude de 152 cas.

BACKGROUND: Primary immunodeficiencies are rare immunopathological disorders. A multidisciplinary study group was set up in Tunis in 1988 and has since identified 152 cases of such diseases. We herein present our series and compare it to the international registries. POPULATION AND METHODS: Over a period of 8 years (April 1988-April 1996), 295 children suffering from recurrent infections were investigated; primary immunodeficiency was confirmed in 152 out of them. The immunological investigation included a study of specific and/or non specific humoral and cellular immunity. RESULTS: These 152 patients belonged to 129 families among which 70 were consanguine (54%). Familial primary immunodeficiency occurred in 23 of them. In 39 families (30%), one or more deaths occurred during early childhood. In more than half of the cases (89 cases), the immunological investigations revealed a cellular or combined immunodeficiency with a majority of ataxia-telangiectasia syndromes (53 cases), T cell activation immunodeficiencies (12 cases) and HLA class II deficiency (nine cases). A predominant antibody defect was observed in 35 patients with a majority of agammaglobulinemia (11 cases) and hyper-IgM syndromes (11 cases). A defect of non specific cellular immunity was found in 18 cases (11.8%) including seven cases of chronic granulomatous disease and five cases of leukocyte adhesion deficiency. Three children (1.9%) were deficient in the complement system. Deaths occurred so far in 37 patients (24.3%). CONCLUSIONS: Primary immunodeficiencies are relatively frequent in Tunisia, probably because of the high rate of consanguinity among the general population. The distribution of the different groups of primary immunodeficiencies is characterized by high frequency of ataxia-telangiectasia and hyper-IgM syndrome and scarcity of severe combined immunodeficiencies and Wiskott-Aldrich syndrome.

[Agammaglobulinemia with the absence of circulating B-lymphocytes. 9 cases]. / Agammaglobulinémie avec absence de lymphocytes B circulants. 9 observations.

OBJECTIVES: Agammaglobulinemia with absence of circulating B lymphocytes is a rare genetically transmitted immunodeficiency that appears in early childhood and affect mainly boys. The clinical manifestations of the disease are rather heterogeneous. PATIENTS AND METHODS: Nine patients (7 boys and 2 girls) were diagnosed as suffering from agammaglobulinemia with absence of circulating B lymphocytes, over a period of 6 years. Quantitation of immunoglobulins and search for circulating B lymphocytes were respectively performed by the Mancini method and immunofluorescence using T specific (anti-CD3, anti-CD4 and anti-CD8) and B (anti-CD19) monoclonal antibody. RESULTS: The disease started to manifest clinically at the mean age of 8.7 months (4-16 months). The mean age at diagnosis is 4 years (1-11 years). The clinical manifestations were essentially recurrent infections of the lung and the gastrointestinal tract. However, bacterial meningitidis was observed in 3 patients. Severe complications such as an echovirus 27 meningoencephalitis and a chronic active hepatitis (1 patient) and a pericarditis (1 patient) were observed. All of our patients lacked circulating B lymphocytes and had low or null immunoglobulin levels. Five patients were treated by intravenous immunoglobulin (Ig) and 3 were treated by intramuscular immunoglobulin with a residual IgG level respectively of 5.5 g/l and 3.3 g/l. CONCLUSION: Recurrent infections are the principal manifestation of the agammaglobulinemia, early Ig treatment is the only therapy allowing improved.

[Chronic septic granulomatous disease. 14 cases]. / Granulomatose septique chronique. 14 observations.

OBJECTIVES: Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency. Affected children are mostly boys. The most common clinical features are recurrent bacterial and fungal infections starting at early childhood. We report 14 cases, including 5 girls, of CGD in Tunisian children. PATIENTS AND METHODS: This retrospective study concerned 14 clinical observations of CGD recorded between April 1988 and December 1998. The diagnosis was established upon determination of a defective respiratory burst in the patients' neutrophils at the tetrazolium nitroblue test (NBT). In 4 cases, the diagnosis was also confirmed by chemiluminescence assay. RESULTS: The patients (9 boys and 5 girls) belonged to 12 families, 75% of which were consanguineous. In 6 families, there had been several deaths in early childhood. The mean age at onset of clinical signs was 6.8 months (7 days to 24 months). Clinical signs included lung (10 cases), nodal (8 cases), skin (7 cases), and intestinal (7 cases) infections. Seven patients developed invasive pulmonary aspergillosis with parietal extension in 4 cases. Salmonella and Staphylococcus infections were rare in our series. Six children (42.8%) including 2 girls, died. Aspergillosis was fatal in 4 cases. CONCLUSION: Recurrent infections are the main clinical fetus of chronic granulomatous disease. Prognosis has been improved by the use of prophylactic antibiotics. Early diagnosis of the disease is crucial.

Genetic and immunological assessment of a bone marrow transplantation in a patient with a primary immune defect: leukocyte adhesion deficiency.

Leukocyte adhesion deficiency (LAD) was suspected in a three weeks old girl from a family with an established history of LAD with a lack (less then 1%) of the beta 2 integrins CD 11a, b/CD 18 expression at the leukocytes surface, was engrafted with her mother HLA identical bone marrow at the age of 14 months. Repeated post transplantation (up to 22 months). Immunological assessments showed a good engraftment with 97% of the lymphocytes expressing CD11a/CD18. Cells proliferated normally in response to PHA and to Tetanus toxoïd after revaccination. The level of serum immunoglobulins was normal. Investigation of the CD18 intragenic polymorphic marker Avall before and after bone marrow transplantation (BMT) showed a transition from the Avall +/+ genotype to the mother's Avall +/- genotype. Similarly DNA fingerprints obtained with the patient genomic DNA, prepared from PBMC, prior and after transplantation, showed that the patient's DNA fingerprints pattern matched the mother's one. These findings are consistent with the good engraftment observed clinically. This study emphasizes the usefulness of the molecular techniques to evaluate the degree of chimerism in monitoring the outcome of bon marrow transplantation.

Cloning of the rat CR3 alphaM (CD11b) subunit, expression and binding assay of recombinant isolated CD11b VA (A-domain) and ICAM-1 Ig modules.

The leukocyte beta2 integrin CR3 (CD11/CD18), is a surface heterodimeric glycoprotein that functions as a divalent cation-dependent adhesive complex. It mediates several important cell-substrate and cell-cell adhesive interactions among which the interaction with vascular endothelial cells that lead to leukocyte transmigration. We have isolated cDNA clones-coding for the rat complement receptor type 3 (CR3) alphaM subunit (CD11b) from a cDNA library. The cDNA sequence showed respectively 89.4% and 74.6% homology with its mouse and human counterpart. We have expressed the sequence coding for the VA module or Von Willebrand type domain (A-domain) and produced it in E. coli as a soluble recombinant fusion protein with GST. Simultaneously, we have cloned DNA fragments specific to the rat ICAM-1 domain 1 and domain 3 and expressed each clone in E. coli as recombinant soluble (rs) fusion proteins with GST. Recombinant CD11b A-domain was released from the fusion protein by thrombin cut. Purified ICAM-1 fusion peptides and CD11b A-domain were used to develop a direct binding assay that showed a specific binding between the rat ICAM-1 Ig like domain 3 and CD11b A-domain. These data demonstrate that the IgSF modules can be produced as a soluble recombinant fusion protein and used to study direct binding to the VA module displayed by members of the integrin superfamily.

Genetic and mutational heterogeneity of autosomal recessive chronic granulomatous disease in Tunisia.

NADPH oxidase, a multi-subunit protein consisting of cytosolic components and the membrane-bound heterodimer, plays an instrumental role in host defence mechanisms of phagocytes. Genetic deficiency of the enzymatic complex results in an inherited disorder, chronic granulomatous disease (CGD), which is characterized by an impaired phagocyte microbicidal activity. X-Linked (XL) CGD results from a mutation in the CYBB gene encoding the gp91phox subunit, while autosomal recessive (AR) CGD is associated with mutations in one of the NCF1, NCF2 and CYBA genes that encode the p47phox, p67phox and p22phox subunits, respectively. In the study reported here, we investigated genetic defects underlying CGD in 15 Tunisian patients from 14 unrelated families. Haplotype analyses and homozygosity mapping with microsatellite markers around known CGD genes assigned the genetic defect to NCF1 in four patients, to NCF2 in four patients and to CYBA in two patients. However, one family with two CGD patients seemed not to link the genetic defect to any known AR-CGD genes. Mutation screening identified two novel mutations in NCF2 and CYBA in addition to the recurrent mutation, DeltaGT, in NCF1 and a splice site mutation previously reported in a North African patient. Our results revealed the genetic and mutational heterogeneity of the AR recessive form of CGD in Tunisia.

Gamma interferon is dispensable for neopterin production in vivo.

Previous studies have indicated that neopterin is synthesized in vitro by human monocyte-derived macrophages and dendritic cells upon stimulation with gamma interferon (IFN-gamma). Neopterin production under specific conditions in vitro has also been obtained upon stimulation with IFN-alpha and/or IFN-beta. However, it is unknown if any IFN-gamma-independent neopterin synthesis is possible in vivo. In the present study we investigated the serum neopterin concentrations in patients affected by the syndrome of Mendelian susceptibility to mycobacterial disease (MSMD). Indeed, this syndrome is characterized by deeply impaired or absent IFN-gamma production or function due to severe mutations in molecules involved in IFN-gamma/interleukin-12 (IL-12)/IL-23-dependent pathway. Serum neopterin levels were measured by an enzyme-linked immunosorbent assay in 27 patients with MSMD. We found that serum neopterin levels are elevated in the complete absence of IFN-gamma activity due either to a complete deficiency of its receptor or to deleterious mutations of IL-12 or its receptor. These data clearly indicate that, as reported from in vitro studies, other stimuli are able to induce neopterin synthesis in vivo. Consequently, neopterin cannot be used as means of diagnosis of MSMD due to IFN-gamma-, IL-12-, and IL-23-dependent pathway defects.

Common idiotypes expressed on human, monoclonal, abnormal immunoglobulins and cryoglobulins with polyreactive autoantibody activities.

Several human monoclonal immunoglobulins with the same autoantibody activity have been shown to have cross-reactive idiotypes (CRI). In this study, using polyclonal anti-idiotypic antibodies, we found that 28% of human monoclonal immunoglobulins with polyreactive autoantibody activity from myeloma, Waldenström's macroglobulinaemia and cryoglobulinaemia patients shared common idiotype(s). Furthermore, the latter were expressed on human and murine natural MoAbs (respectively in 12% and 22% of the clones tested) and on human IgG preparations used for therapeutic intravenous injections (IVIg) and which contain natural antibodies. These findings suggest that monoclonal immunoglobulins could arise from the proliferation of a clone that normally produces a natural antibody. The existence of common idiotype(s) between monoclonal immunoglobulins and IVIg could be relevant to the improvement noted after treatment with IVIg in patients suffering from peripheral neuropathies associated with monoclonal gammopathy.

Prognostic significance of autoantibodies to laminin in the sera of breast cancer patients: a preliminary report.

Interactions between cancer cells and laminin play an important role in the metastatic cascade. The aim of this retrospective study was to evaluate the presence and the prognostic significance of autoantibodies to laminin in sera of patients with breast cancer. Our study population consisted of 71 high-risk primary breast cancer patients (median follow-up, 6 years). IgG, IgA and IgM were analysed at the time of surgery and before any treatment was given. Concentrations were measured by enzyme immunoassay with a highly purified antigen. IgG, IgA and IgM anti-laminin median values were, respectively 117%, 130% and 97% of normal absorbance. Higher concentrations of IgM to laminin were found in pre-menopausal patients as compared with post-menopausal patients (p < 0.001). Patients with high concentrations of IgG to laminin had lower disease-free (p = 0.003) and overall survival rates (p = 0.003). In breast cancer, assay of IgG to laminin could thus provide a cheap and easy prognostic index available for all patients.