Assessment of human sperm DNA integrity using two cytochemical tests: Acridine orange test and toluidine blue assay.

Primary infertility affects approximately 15% of couples, with male factor infertility accounting for 50% of cases. Semen samples from 41 patients with asthenoteratospermia and 28 men with proven fertility were analysed according to World Health Organization guidelines. Abnormal sperm chromatin structure was assessed by toluidine blue assay (TBA), and DNA denaturation (DD) was detected by the acridine orange test (AOT). The mean (±SEM) rates of DD and abnormal chromatin structure were significantly higher in infertile subjects compared to fertile group respectively p = .003 and p < .001. A significant correlation was established between sperm DD and abnormal chromatin structure (R = .431, p < .001). Sperm DNA damage correlated significantly with abnormal morphology, sperm motility and necrozoospermia. Our study shows that men with increased levels of abnormal sperm chromatin structure have a high incidence of DNA denaturation and altered semen parameters. These findings suggest that male infertility has been linked to sperm DNA damage.

HLA-G 14 bp insertion/deletion polymorphism and its association with sHLA-G levels in Behçet's disease Tunisian patients.

The purpose of this study was to investigate the HLA-G 3'UTR 14 bp polymorphism and sHLA-G levels in Tunisian patients with BD. The study included 119 patients with BD and 170 healthy blood donors (HD). HLA-G 14 bp polymorphism was genotyped by polymerase chain reaction. Serum levels of soluble HLA-G (sHLA-G) were measured using a commercial ELISA kit. A significant increased frequency of the -14 bp HLA-G allele was detected in patients with BD compared to HD (0.58 vs 0.49, p=0.023), and a significant increased frequency of HLA-G -14/-14 bp was observed in patients with BD compared to HD [0.37 vs 0.22, p=0.007, OR 2.04 (95% CI 1.21-3.42)]. The mean plasmatic concentration of sHLA-G levels were significantly increased in patients with active disease [231.63±286.4 U/mL] compared to those with inactive disease (103.14±77.8 U/mL, p=0.03) and HD (121.41±24.1 U/mL, p=0.04). Furthermore, our results showed that there is no association between HLA-G 14 bp polymorphism and sHLA-G plasma levels.

Phenotypic abnormalities of peripheral blood mononuclear cells in patients with Behçet's disease and association with HLA-B51 expression.

The aim of this study was to investigate the subclasses and the immunophenotypic profile of peripheral mononuclear cells in patients with Behçet's disease (BD) and to assess associations between the expression of HLA-B51 antigen and that of other cell markers. Thirty healthy volunteer blood donors and forty patients with BD were enrolled into this study. Phenotyping was performed using two color flow cytometry. HLA-B51 typing was performed using the complement dependent microlymphocytotoxicity assay. Unlike controls, patients with BD presented a modified immunophenotypic profile of lymphocytes. Compared to those in the remission phase, patients with active BD showed an increased mean of MFI ratio of CD56 on CD16+CD56+ cells (32.47 ± 14.26 versus 23.87 ± 10.3; p = 0.032), increased absolute numbers of CD4(-)CD8(bright) and CD4(+)CD8(+) cells (657.1 ± 463.6 cells/µL versus 319.24 ± 116.4 cells/µL; p = 0.017 and 40.77 ± 36.41 cells/µL versus 10.77 ± 9.78 cells/µL; p < 0.0001, respectively) and an elevated mean of MFI ratio of CD19 on B cells (252.3 ± 56.7 versus 205.67 ± 32.3; p = 0.021). However, expression of HLA-B51 was not associated with any specific immunophenotypic profile. In conclusion, abnormal immunophenotypic profile of peripheral lymphocytes was found in patients with BD, especially in active phase, reflecting an immune dysregulation. Moreover, HLA-B51 expression was not found to be related to the expression of other cell markers.

Anti-cardiolipin and anti-beta 2-glycoprotein I antibodies in celiac disease.

AIMS: To determine the frequency of anti-cardiolipin (aCL) and anti-ß2-glycoprotein I antibodies (aß2GPI) in celiac disease (CD) patients. PATIENTS AND METHODS: Sixty-three untreated CD patients and 40 healthy blood donors (HBD) were studied. IgG, IgA and IgM aCL and aß2GPI were detected by Elisa. RESULTS: The frequency of antiphospholipid antibodies (aPL) (aCL and/or aß2GPI) was significantly higher in CD patients (12 out of 63) than in HBD (two out of 40) (19% vs 5%, P=0.04). Six CD patients out of 63 (9.5%) and one HBD out of 40 (2.5%) had aCL. Ten CD patients (15.9%) and two HBD (5%) had aß2GPI. Only aß2GPI-IgA was significantly more frequent in CD patients than in HBD (14.3% vs 2.5%, P=0.048). In CD patients, aß2GPI-IgA (nine out of 63) was significantly more frequent (14.3%) than aß2GPI-IgG (1.6%) and IgM (1.6%) (P=0.008). In CD patients, the frequency of aCL-IgA and IgM was 6.3% (four out of 63) and aCL-IgG were not detected. Simultaneous presence of positive antibodies was found in four CD patients: one patient had four aPL, one had three aPL and two had two aPL. The four patients who had aCL-IgA had also aß2GPI-IgA and three of them had a titer higher than 50 units. Among nine patients with aß2GPI-IgA, four had a titer higher than 100 units. The highest titers were found in adults. CONCLUSIONS: aPL and particularly aß2GPI-IgA are frequent in CD. The significance of these antibodies has to be determined.

IgA anti-actin antibodies in celiac disease.

AIMS: The purpose of this study was to determine the sensitivity and specificity of IgA anti-actin antibodies (IgA-AAA) for celiac disease (CD), to investigate their usefulness as a marker of compliance in CD patients to the gluten-free diet (GFD), and to assess the relationship between their presence in the sera of CD patients and severity of intestinal mucosal damage. PATIENTS AND METHODS: A total of 182 patients with CD were studied: 63 patients were untreated; 50 patients were following a strict GFD; and 69 patients were non-compliant with a GFD. IgA-AAA was detected using a homemade enzyme-linked immunosorbent assay (ELISA). RESULTS: IgA-AAA showed a sensitivity of 41.3% and a specificity of 71.4% for a diagnosis of CD. In children, the frequency of IgA-AAA detection was lower in those following a strict GFD (23.1%) compared with untreated patients (39.4%) and those not complying with a GFD (32.5%). In patients following a strict GFD, IgA-AAA detection was significantly less frequent in children than in adults (23.1% vs. 58.3%, respectively; P<0.001). IgA-AAA was found in 17 out of 52 CD patients with total villous atrophy (32.7%), and in one out of 11 patients with subtotal villous atrophy (9%). CONCLUSION: IgA-AAA cannot replace anti-endomysium and anti-tissue transglutaminase antibodies in the diagnosis algorithm of CD, but it can serve as a reliable marker of severe intestinal mucosal damage in CD patients.

Uveitis responding on gluten free diet in a girl with celiac disease and diabetes mellitus type 1.

A 9-year old girl with a history of diabetes mellitus type 1, presented with visual loss of the left eye. The right eye examination was unremarkable. Slit-lamp examination revealed few small and fine keratic precipitates. We noted 2+ flare in the vitreous. There was no choroiditis, papillitis or retinal vasculitis. No aetiology was found. The patient was treated by topical and systemic corticosteroids without any improvement. Celiac disease was discovered by the presence of celiac antibodies in the work-up of joint pain and diabetes mellitus type 1. Antiendomysium antibodies and anti-transglutaminase antibodies were both positive. A small bowel biopsy confirmed celiac disease. A gluten free diet was set up and corticosteroids were tapered off. Recovery of the uveitis was obvious during gluten free diet and normalized within two months.

A role for tissue transglutaminase in alpha-gliadin peptide cytotoxicity.

In coeliac disease, gliadin peptides p56-88, p57-68 and p31-49 have been demonstrated to be involved in the pathogenic damage of the small intestine via their immunogenicity or toxicity to epithelial cells. To try to understand the mechanism of their toxicity, we investigated the effect of synthetic peptides (p31-49, p56-88, p57-68, p69-82) and of their deamidated analogues on Caco2 and FHs 74 Int cell toxicity and tissue tranglutaminase activity. Apoptosis, necrosis and cell viability were assessed by flow cytometry, and peptide deamidation was determined indirectly by measuring its capacity to inhibit tTG activity. The results showed that p56-88 and p57-68 reduced cell growth and concomitantly inhibited tTG activity in both cell types. This effect was abolished when Caco2 cells were treated with antibodies to tTG. Deamidated peptide p57-68 (E(65)) lost practically all of its inhibitory effect on cell growth and on tTG activity. Cellular toxicity was also observed with p31-49, which was not a substrate for tTG. p69-82 was not cytotoxic but became so when glutamine 72 was substituted by glutamic acid. These findings provide evidence for the existence of three types of toxicity among gliadin peptides: (i) peptides that are intrinsically toxic and are not substrates of tTG; (ii) peptides that are non-toxic but become so when they act as substrates of tTG; and (iii) peptides that are non-toxic and are not substrates of tTG but become so when deamidated. A mechanism other than that involving tTG could be responsible for the deamidation of glutamine residues of gliadin in the intestinal tract.

Tissue transglutaminase antibodies in celiac disease, comparison of an enzyme linked immunosorbent assay and a dot blot assay.

AIMS: The purpose of our study is to determine the sensitivity, specificity and predictive values of an enzyme linked immunosorbent assay (ELISA) and a dot blot assay for the detection of IgA class anti-tissue transglutaminase antibodies (IgA-AtTGA) and to compare these results with those of IgA class anti-endomysium antibodies (IgA-AEA), IgA class anti-reticulin antibodies (IgA-ARA) and IgA class anti-gliadin antibodies (IgA-AGA). PATIENTS: Serum samples from 143 patients (97 children, 46 adults) with untreated celiac disease (CD) confirmed by intestinal biopsy and 74 disease controls (64 children, 10 adults) were studied. Methods. - The anti-tissue transglutaminase antibodies were detected by dot blot assay and an ELISA using guinea pig tissue transglutaminase (gp-tTG) as antigen. The anti-endomysium antibodies were detected by an indirect immunofluorescence technique on cryostat sections of human umbilical cord. The anti-reticulin antibodies were also investigated by indirect immunofluorescence on cryostat sections of kidney, liver and stomach of rat. The anti-gliadin antibodies were determined by an ELISA. RESULTS: The sensitivity of an ELISA for the detection of anti-tissue transglutaminase antibodies was 86% in children and 87% in adults and the sensitivity of dot blot assay was 57% in children and 54% in adults. The specificity of an ELISA and dot blot for the detection for anti-tissue transglutaminase antibodies was, respectively, 96% and 88% lower than that of anti-endomysium antibodies (100%). The sensitivity of anti-gliadin antibodies was 97% in children and 91% in adults and their specificity was 85%. The sensitivity of anti-reticulin antibodies was 94% in children and 87% in adults. Their specificity was 100%. CONCLUSIONS: The sensitivity and specificity of an ELISA for the detection of anti-tissue transglutaminase antibodies were better than that of dot blot assay. However, this dot blot assay could screen four celiac patients who have not had anti-tissue transglutaminase antibodies by an ELISA. The sensitivity of anti-endomysium antibodies was better than that of anti-tissue transglutaminase antibodies, anti-reticulin antibodies and anti-gliadin antibodies but in children aged less than 2 years, the sensitivity of anti-gliadin antibodies was better than that of anti-tissue transglutaminase antibodies.

[Clinical and serological characteristics of systemic lupus erythematosus: 128 cases]. / Caractéristiques cliniques et sérologiques du lupus érythémateux systémique: à propos de 128 cas.

OBJECTIVE: To analyse the clinical and serological characteristics of systemic lupus erythematosus (SLE) in the center of Tunisia. METHODS: We studied 128 patients with SLE aged one to 73 years. Antinuclear antibodies (ANA) were detected by an immunofluorescence method. Anti-double-stranded DNA (anti-dsDNA) antibodies, anti-extractable nuclear antigen antibodies (anti-Sm, anti-SS-A, anti-SS-B and anti-RNP) and anti-cardiolipin (aCL of IgG, IgA and IgM isotypes) antibodies were detected by ELISA. RESULTS: Malar rash (71%) and anemia (71%) were the most common clinical manifestations. Arthritis was seen in 62.5%. Severe kidney damage was observed in 39%. Pericarditis and pleuritis were observed in only 23%. Neurological manifestations (16%) were uncommon. Clinical manifestations of anti-phospholipid syndrome (SAPL) were observed in 15%. ANA were detected in 100%, anti-dsDNA in 76%, anti-Sm in 55.5%, anti-SS-A in 64%, anti-SS-B in 33.6%, anti-RNP in 49%. aCL of IgG, IgA and IgM isotypes were detected in 63.5%, 49% and 40.6% of the patients respectively. The only significant positive clinical associations were those of arthritis with anti-dsDNA antibodies (p = 0.022) and malar rash with anti-SS-A antibodies (p = 0.002). CONCLUSIONS: This study suggests that tunisians with SLE present, in general, a mild form of disease predominantly manifested by cutaneous, musculoskeletal and hematologic involvement but low prevalence of major organ damage.