Clinical variables and ethnicity may influenced by polymorphism of CAT -262C/T and MnSOD 47C/T antioxidant enzymes in Algerian type1 diabetes without complications.

The latest studies in Algeria show that the frequency of type 1 diabetes (T1D) without complications is lower than that with complications and represents a significant burden in terms of cost and treatment. For this reason, we are interested in uncomplicated type1 diabetes and risk factors that are related to polymorphisms of antioxidant enzymes in order to prevent its complications. A total of 260 blood samples of young Algerian adults were examined. The genotypic analysis of Catalase gene (CAT -262C/T, rs1001179) and the superoxide dismutase gene (MnSOD 47C/T, rs4880) was performed by real-time PCR using TaqMan technology. The genotypic distribution of the CAT -262C/T promoter gene's polymorphism showed a significant difference between control and T1D patients for the CC genotype (p = 0.009; OR = 0.30) and for the T allele (p = 0.002; OR = 2.82). In addition, the genotypic distribution of the MnSOD 47C/T gene showed an association with T1D for the CT genotype (p = 0.040; OR = 2.37). Our results revealed that polymorphisms of CAT and MnSOD may be associated with physiopathology causing the onset of T1D. Our data, suggest that the genotypic frequencies of these SNPs appear to be influenced by clinical variables and by the Arab-Berber ethnic origin of the Algerian population.

NOD2/CARD15 and IL23R genetic variability in 204 Algerian Crohn's disease.

NOD2/CARD15 and IL23R gene variants play an important role in the susceptibility to Crohn's disease (CD). Studies of genotype-phenotype relationship suggest that these variants are associated with the development of the disease and specific phenotype. Preliminary reports analyzing the association between these variants have never been made on Algerian CD's. In a case-control design, 204 Algerian with CD diagnosed for at least 5years and 201 controls were included were genotyped for single nucleotide polymorphisms (SNP) in the NOD2/CARD15 gene R702W (SNP8, rs2066844), G908R (SNP12, rs2066845) and IL23R R381Q (rs11209026) gene variants were determined using the TaqMan SNP genotyping assays. NOD2/CARD15 908R was carried by 3% of the patients and none in control subjects (χ(2)=8.6, Pc=0.003, OR=13.20). NOD2/CARD15 702W was associated to CD outcome (χ(2)=17.2, Pc=0.00003, OR=12.5) and early onset of disease (group A1, χ(2)=19.3, Pc=1.10(-5), OR=14.05, PM-H=2.10(-6)). IL23R 381Q variants was more frequent in CD's patients than controls (χ(2)=8, Pc=0.005, OR=3.48), it was associated to earlier onset (group A1, χ(2)=7.1, Pc=0.007, OR=1.04, PM-H=0.002), extra-intestinal manifestations (EIM) outcome (χ(2)=10.6, Pc=0.001, OR=1.05, PM-H=0.002) and ileocolonic location (χ(2)=6.8, Pc=0.009, OR=1.05, PM-H=0.001). In this Algerian cohort, NOD2/CARD15 and IL23R variants were associated with CD's outcomes and linked to a particular clinical phenotype.

[Susceptibility genes, HLA and diabetic retinopathy in the Algerian population]. / Gènes de susceptibilité HLA et rétinopathie diabétique chez la population algérienne.

BACKGROUND: Diabetic retinopathy (DR) is the most frequent microvascular complication of type I diabetes (T1D). Some well-controlled type I diabetics may develop DR, while other poorly-controlled diabetics do not develop DR. This might be explained by certain susceptibility genes or protective genes. The purpose of our study is to search for any association between the HLA class I and II markers and DR in the Algerian population. PATIENTS AND METHODS: This study was carried out in 52 T1D subjects with and without DR compared to 140 healthy controls. HLA typing was performed using the "microlymphocytotoxicity" technique. RESULTS: The frequency of HLA-A29 and HLA-DR9 antigens is higher in T1D with DR compared to T1D without DR and to controls with frequencies of HLA-A29 (59.26% vs. 0%, OR=∞, pc=4.6×10(-7)), (59.26% vs. 5.66%, OR=24.24, pc=7.6×10-10) and HLA-DR9 (29.63% vs. 0%, OR=∞, pc=1.310(-3)), (29.63% vs. 4.29%, OR=9.40, pc=7.010(-5)) respectively. However, the frequency of HLA-B49 antigen is significantly lower in T1D with DR than in T1D without DR (3.7% vs. 28%, OR=0.10, pc=8.8×10(-3)) and compared to controls (3.7% vs. 22.64%, OR=0.13, pc=0.011). CONCLUSION: HLA-A29 and HLA-DR9 antigens are probably markers of susceptibility for DR while HLA-B49 antigen is probably associated with a protective effect in the Algerian population.

Purification, preliminary characterization and immunohistochemical localization of POSVP21 in the sand rat (Psammomys obesus) seminal vesicles.

The sand rat Psammomys obesus is a mammalian species with male seasonal reproduction. Previously Gernigon et al. (1994) [Gernigon T, Berger M, Lecher P. Seasonal variations in the ultrastructure and production of androgen-dependent proteins in the seminal vesicles of a saharian rodent (Psammomys obesus). J Endocrinol 1994;142:37-46.] reported that the seminal vesicles of the adult sand rat contained a major secretory protein band (M.W. 21000) regulated by testosterone. This protein is synthesized in large amounts when the androgen level increases, and accounts for over 22% of soluble proteins from homogenate of seminal vesicles during the breeding season. When analyzed by NepHGE the protein band of 21kDa appeared to be composed of at least 3 visible spots with pHi values varying from 4 to 7. Its partially internal sequence was identified and exhibited five peptides. Polyclonal antibodies against POSVP21 were obtained in rabbits. They were also used to study immunohistochemical antigen localization by the means of an avidin-biotin peroxidase procedure. Observation showed that it is localized in the cytoplasm of epithelial cells and in secretory products in the lumen. The whole RNA of seminal vesicles was translated in a cell-free system derived from rabbit reticulocyte lysate and [35S]-methionine. Two major bands of 14.4 and 21kDa were visualized by means of denaturing gel electrophoresis. SDS-PAGE from medium incubation of seminal vesicle tissue with [35S]-methionine revealed one band with an apparent molecular weight of 21kDa. The results obtained indicate that seminal vesicle epithelium is the site of POSVP21 synthesis and the comparison of the partial amino acid composition of the internal sequence, indicated that POSVP21 constitute a family of most unusual proteins.

Constitutive nitric oxide synthase gene polymorphisms and house dust mite respiratory allergy in an Algerian patient group.

Genetic polymorphisms in neuronal nitric oxide synthase (NOS1) and calmodulin-dependent endothelial NOS (NOS3) genes are known to influence the course of allergic respiratory disorders. We investigated the role of NOS1 -84 G-->A and NOS3 -786 T-->C, 894 G-->T and 27 base pair (bp) repeat polymorphisms in 125 patients suffering from asthma and/or rhinitis and monosensitized against Dermatophagoides pteronyssinus (Dpter) and 111 controls from Algeria. We found a higher frequency of the -786 C NOS3 allele in patients than in controls [corrected P value (Pc) = 0.04], especially in female cases (Pc = 0.02) and that the 'ab' genotype of the 27-bp polymorphism was significantly associated with specific immunoglobulin E production against Dpter (P = 0.006). This study brings further support for the participation of NOS3 gene polymorphism in the pathogenesis of respiratory allergic disorders.

[Autoimmune chronic active hepatitis: anatomoclinic's study of 50 patients]. / Les Hépatites auto-immunes (HAI) chroniques de l'adulte: étude anatomoclinique d'une série de 50 patients.

PURPOSE: To analyse anatomoclinic and evolutive aspects of autoimmune hepatitis (AIH) through 50 observations collected in two Internal Medicine departments in Algiers from 1998 to 2002 and to make a review of the literature. METHODS: The study is prospective. The diagnosis of autoimmune hepatitis (AIH) is established according to the recommendations of the score of the International Autoimmune Hepatitis Group (1991) or/and hepatitic damage confirmed by histology. RESULTS: Fifty patients were studied: (32 women-18 men) and the mean age was 38 years (17 to 73). Autoimmune extra-hepatitic manifestations were associated in 26%. The AIH type 1 has been noted in 58%. AIH were type 2 in only 6%. In 22% of the cases AIH were sero-negative and the others AIH represented 14% were classed as overlap-syndrome (5 cases of primary biliary cirrhosis and 2 cases of primary sclerosing cholangitis hepatitis overlap syndrome). The first liver biopsy tissue showed strong necrotic-inflammatory activity in 56% and cirrhosis was identified in 19 patients (38%). The treatment (azathioprine and corticosteroid) was prescribed in 37 patients (74%) in active chronic hepatitis or in compensed cirrhosis. FOLLOW-UP: 28% of the patients died (9-36 months) because cirrhosis's complications or because complications of hepatocarcinoma (3 cases). CONCLUSION: The diagnosis of AIH must be established early for each patient with chronic liver disease particularly is those are supposed as a crypto genetic hepatitis. The prognosis is compromised by delayed diagnosis and the mortality in middle following up is high.

[An immunoenzymatic technique for the detection of antinuclear antibodies]. / Mise au point d'une technique immunoenzymatique pour la recherche d'anticorps antinucléaires.

In the present work, we propose, for the detection of the anti-nuclear antibodies (ANA) an immunoenzymatic test in simple sandwich in heterogeneous phase (ANA-EIA) allowing the screening of the antinuclear antibodies, and its assessment in comparison with the indirect immunofluorescent assay (IFA) for 292 sera of patients affected by rheumatic diseases and 40 sera of individuals presenting no pathology. The ANA-EIA test proved to have a sensitivity of 97% and a specificity of 96%. Moreover, it gives a global concordance of 96.6% and a discordance of 3.4% compared to IFA. Also, the analysis of correlation between this test and IFA for the positive individual gives a very good concordance according to homogeneous, speckled and nucleolar aspects. On the other hand, the results obtained for cytoplasmic aspect (74%), although acceptable, needs to be confirmed a second time with a more important sampling. According to the results obtained, this test could have an interesting routine application in laboratory of immunopathology given its analytic characteristics similar to those of IFA, and its great easiness of realisation.

[Production of monoclonal antibodies specific for the ABO blood group and rhesus D antigens]. / Production d'anticorps monoclonaux spécifiques des antigènes des groupes sanguins ABO et Rhesus D.

We report, in this work, the techniques to obtain four monoclonal antibodies specific of erythrocytes antigens. Three of this antibodies, react with the ABO Blood Groupe System (A,B and AB), are produced by three mouse hybridomas (M18-2F11, M18-4F6 et M19-45D6), obtained by fusion of Sp2/0 mouse myeloma cell with spleen cell of balb/c mice immunized with human red blood cells and selected on selectif medium (Hypoxanthin, Aminoptérin, Thymidin) (HAT) and cloned by four limiting dilutions. Where as the fourth, it is an human monoclonal antibodies against Rhesus (D) produced by heterohybridomas, realized by fusion of X63 mouse myeloma cell with human B lymphocytes, from actively immunized persons by D antigen, that are purified and transformed by Epstein-Barr virus (EBV), and selected on selectif medium (HAT), presence of ouabain and then cloned by four limiting dilutions. The specificity of the antibodies produced, has been determined by direct hemagglutinin for the mouse monoclonal antibodies and artificial for the human anti-D. The determination of isotype the heavy and light chains is released by the technique immunoenzymatique (ELISA) and immunofixation has shown that mouse monoclonal antibodies belong to class IgM kappa, and human monoclonal antibodies anti-D belong to class IgG lambda.

[Contribution of immunofixation and laser nephelometry in the diagnosis of monoclonal immunoglobulinopathy]. / Apport de l'immunofixation et de la néphélémétrie-laser dans le diagnostic des immunoglobulinopathies monoclonales.

Immunological diagnosis of principal's monoclonal gammapathies [Waldenström's macroglobulinemia and multiple myeloma], used classically electrophoresis and immunoelectrophoresis analysis (AIE) for detection of monoclonal components. Although its specificity and sensibility, the last method stays a long analysis with critical interpreting from time to time. The present study reports results obtained with 100 sera from subjects with these diseases when using immunofixation and nephelometry-laser witch tests amounts of IgG, IgA and IgM and kappa/lambda--ratio both methods together, which are simpler and more faster than AIE.

HLA-B27 polymorphism and worldwide susceptibility to ankylosing spondylitis.

HLA-B27 is strongly associated to ankylosing spondylitis (AS) and represents a family of eleven B27 alleles (B*2701-11). Our aim was to analyze the distribution of B27 subtypes by PCR/SSOP and genomic sequencing in a large group of populations (n = 17). 711 B27-positive samples from Caucasoid, Asian, African, Amerindian and Polynesian populations were selected to ascertain transracial gene mapping of the B27 subtypes. 476 of these were AS patients, chosen to investigate the contribution of B27 alleles to AS susceptibility. Some significant new findings have arisen from this study: 1) B*2705 was the predominant subtype in circumpolar and subarctic areas. B*2702 was found to be practically restricted to Caucasian populations, showing a higher frequency in Middle-East (Jews) and North Africa (Arabs/Berbers) groups. 2) B*2703 appears associated with AS in Western Africans. This is of remarkable interest since it was suggested that B*2703 would be negatively disease-associated. 3) Although B*2706 appears negatively associated with AS in Thais, we identified two patients from northern China carrying it. This may be a reflection of a disease heterogeneity and could indicate that more than one pathogenic agent can be involved in AS. B*2709 has been recently described as negatively associated with AS in Sardinians. The molecular changes His114Asp (B*2706) and Asp116His (B*2709) could modify the genetic susceptibility to AS.

[Rapid micromethod for CH50 determination using an algorithm]. / Détermination rapide par logiciel de la CH50 en microméthode.

The titration of haemolytic complement in biological liquid cause inconvenient in calculating concentration, it was long and fastidious. We report a fast technic based on measure of hemolysis in microplate++ method, which exploitation of results, it does with an appropriate algorithm.

[The association of diabetes and celiac disease: the value of assaying antireticulin antibodies]. / Association diabète et maladie coeliaque: intérêt du dosage des anticorps antiréticuline.

The authors studied the specificity and the sensitivity of serum anti-reticulin IgA type antibody determination for the diagnosis of coeliac disease in diabetic patients. They observed a frequency of 11.1% coeliac disease among a group of 54 type I juvenile diabetic patients. The search for serum antireticulin IgA type antibodies appears to be an efficient method for identifying patients with coeliac disease; it has a specificity of 98% and a sensitivity of 95%. This marker appears to be particularly well suited for the identification of asymptomatic forms, and also for surveillance of diet during the course of coeliac disease.

[Immunoenzymatic determination of free immunoglobulin light chains in the cerebrospinal fluid: its value in the biological diagnosis of multiple sclerosis]. / Dosage immunoenzymatique des chaînes légères libres d'immunoglobulines dans le liquide céphalorachidien: intérêt dans le diagnostic biologique de la sclérose en plaques.

Based on previous data on free immunoglobulin light chains in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS), we evaluated free kappa and lambda light chains in the CSF from 3 patients groups: (a) 42 with MS, (b) 16 with other neurological inflammatory diseases (ONID) and (c) 42 with non inflammatory neurological diseases (NIND) used as control. The kappa and lambda light chains contents were evaluated using a specific and sensitive (0.5 mcg/ml) enzyme linked immunosorbent assay (ELISA). Whole CSF immunoglobulins G (CSF-IgG) concentrations were determined by an immuno-nephelometric method and CSF oligoclonal banding was assessed by agarose gel electrophoresis. Elevated free kappa light chains levels were found in 36 of the 42 (85.7%) MS-CSF, and only in 2 of the 16 (12.5%) ONID-CSF. Moreover, all MS-CSF with oligoclonal banding (21/42) exhibited detectable free kappa light chains. In contrast, elevated free lambda light chains or whole CSF-IgG concentrations were found not so specific. These results suggest that free kappa light chains measurement in MS-CSF may have an important diagnostic usefulness. This assay offers a reliable alternative to other available procedures and may constitute complementary technique in routine investigation of MS-CSF.

Cells expressing intracytoplasmic immunoglobulins in secreting and hyposecreting cases of alpha chain disease.

Alpha chain disease proteins (ACDP) originated probably from secreting plasma-cells predominantly present in diffuse and massive enteromesenteric lymphoid infiltration. Very decreased levels of abnormal alpha chain molecules were detected in sera of patients with immunoblastic lymphoma occurring in the late course of the disease. A direct correlation might exist between the proportion of cells bearing intracytoplasmic IgA determinants and the serum amounts of alpha chain disease protein. Relevant evidence raised from study of proliferating lymphoid cells using the unlabeled peroxidase anti-peroxidase method of immunocytochemistry. The percentage of cells expressing intracytoplasmic alpha chains was found to be greater readily secreting case than in hyposecreting case of alpha chain disease. Furthermore, the cells from secreting situation exhibited much more pronounced specific staining, indicative of probably more active synthesis state. Taken together with histological data, these results suggested a possible late evolutionary pathway without detectable intracytoplasmic and serum alpha chain disease protein. They might also support the hypothesis that alpha chain disease and mediterranean lymphoma were different evolutionary phases of the same entity.

Spontaneous enzymatic cleavage of IgD myeloma protein giving a pattern of delta heavy chain disease.

The monoclonality of myeloma proteins is usually demonstrated by their electrophoretic homogeneity and their reactivity with monovalent antisera directed against isotypic determinants of a single heavy chain and a single type of light chain. The absence of precipitation with anti-sera to immunoglobulin kappa and Lambda light chains is a constant character of heavy Chain Disease Proteins (HCDP). However, homogeneous M-components present in the sera of some patients and reacting only with anti-heavy-chain antisera were identified as IgA and IgD myeloma proteins bearing unreactive Lambda chains. In this study, the electrophoretic pattern of a patient serum showed a paraprotein with heterogeneous electrophoretic mobility and precipitation reaction limited to anti-IgD antiserum. The failure to react with anti-light chain antisera was observed by immuno-electrophoresis, immunofixation and rocket-immunoselection. Further analysis by crossed-immunoelectrophoresis revealed that IgD paraprotein contained two separate populations of molecules, one of them being retained when anti-Kappa and Lambda light chains anti-bodies were incorporated in the first dimension gel. It soon became obvious that the observed pattern was generated by enzymatic cleavage of native IgD myeloma protein.

Biclonal gammopathy of kappa light chain type in a case of multiple myeloma.

This immunoglobulin abnormality was found in the serum of a 33 years old man presenting with anemia, weight loss, bone pain and a single bone lesion. The cellulose acetate electrophoresis of his serum showed 2 homogeneous bands migrating in the gamma and alpha-2 regions. Agar gel immunoelectrophoresis using anti-whole normal serum revealed 2 abnormal precipitin arcs of gamma-2 and alpha-2 respective mobility. These 2 M-components precipitated with anti-human kappa light chain antiserum but failed to react with lambda-light chain specific and H-chains specific anti-sera. Furthermore, the 2 monoclonal kappa chains were still demonstrated in the supernatant of the se-rum treated with 50% saturated ammonium sulphate. The 2 kappa chains were isolated from the supernatant using goat anti-kappa antibodies bound to Sepharose-4B. Their SDS-polyacrylamide gel electrophoresis pattern showed a single protein band of 25 Kd molecular weight. This suggest the presence of 2 monomeric kappa chains of different clonal origin. Subsequently, the difference in their electrophoretic mobility was probably due to different V-kappa sequences. However, it is also possible that the unusual alpha-2 mobility is a consequence of a moderate glycosylation without increase in molecular weight.

[Rapid and sensitive micromethod for protein determination by the Coomassie-blue technique]. / Micro-methode rapide et sensible de dosage des proteines par la technique au bleu de Coomassie.

The Coomassie blue method of proteins quantitation initially reported by Bradford and modified by Macart et al. appears to be simple, fast, sensitive and of low cost. In this study, we report an improved micromethod derived from the technic described by Macart. Applied to the protein quantitation of 100 cerebrospinal fluid (CSF) specimens and 50 serum specimens our method showed a very good correlation with the macromethod of Macart (r = 0.985 for CSF proteins, r = 0.944 for serum proteins) and also with the Lowry technic (r = 0.986 for CSF proteins, r = 0.898 for serum proteins). Our technic may be used with various biological fluids (urines, tears, saliva...) and seems to be of particular interest for low protein level fluids and for little volume samples (it needs only 20 mcl).

[Detection of oligoclonal immunoglobulins in the cerebrospinal fluid using agar gel immunoelectrophoresis with silver salt staining]. / Détection des immunoglobulines oligoclonales dans le l. c. r. par IEF sur gel d'agarose et coloration par sel d'argent.

Detection of oligoclonal immunoglobulins in multiple sclerosis cerebrospinal fluid (MS-CSF) seems to be an important test in the biological diagnosis of the disease. In our laboratory, the classical agarose gel electrophoretic technic allowed the detection of CSF oligoclonal bands in only 40% of the MS patients. This relatively low percentage in comparison with those obtained by other investigators led us to develop a much more resolving electrophoretic technic: an agarose gel isoelectric focusing (IEF) with silver staining. By this method, we detected oligoclonal immunoglobulins in 43 (43%) of 100 MS-CSF exhibiting only polyclonal patterns on classical electrophoresis. Oligoclonal banding appeared particularly in patients with inflammatory type profile (36/60) according to Schuller's classification, but also in those with normal (5/26) or inflammatory transudative (2/8) type profiles. MS-CSF with oligoclonal immunoglobulins on IEF had a higher IgG/Albumin ratio (p = 0.01) than those with polyclonal immunoglobulins, while the mean IgG levels were not significantly different (p = 0.07). These results support the diagnostic usefulness of the IgG/Albumin ratio. Agarose IEF appears to be a useful technic in the detection of oligoclonal immunoglobulins and may be applied at least to CSF of patients with clinical signs of multiple sclerosis.