Association between polymorphisms of TLR2-1-6 and bipolar disorder in a tunisian population.

BACKGROUND: Bipolar disorder (BD) is a complex neuropsychiatric disease that has been strongly linked to immune dysregulation. In particular, an abnormal inflammatory response mediated by toll-like receptor 2 - 1/6 (TLR2-1/6) was described in BD. Nevertheless, genetic factors' contribution is still unknown. Thus, we suggested that functional polymorphisms of TLR2, 1 and 6 could be involved in BD predisposition. METHODS AND RESULTS: TLR2, 1 and 6 polymorphisms were genotyped by PCR-RFLP in 292 controls and 131 patients from a Tunisian population. Polymorphisms and haplotype associations were explored in BD and binary logistic regression analysis was performed for more powerful associations. In dominant model, we found a significantly higher genotype and minor allele frequencies in healthy females compared to patients for TLR2-196-174Ins/Del (p = 0.04; OR = 0.3, p = 0.04; OR = 0.3, respectively) and for TLR6-S249P only with minor allele (p = 0.03; OR = 0.2). In contrast, TLR2-R677W CT + TT and T allele frequencies were significantly higher in BD (padjusted<10- 4; ORadjusted =46.6, p < 10- 4; OR = 6.3, respectively), specifically in females (CT + TT: 100%). Similarly, TLR1-R80T showed significantly increased GC + CC and C allele frequencies in patients compared to controls (padjusted=0.04; ORadjusted=4, p = 0.009; OR = 4.3, respectively). Moreover, haplotype investigation demonstrated that InsGTCGT (p < 10- 4, OR = 275) and delGCCGT (p = 0.03, OR = 18.5) were significantly overrepresented in BD patients compared to controls. CONCLUSIONS: We suggest that TLR2-196-174Ins/Del and TLR6-S249P could be protective factors of females against BD. However, TLR2-R677W and TLR1-R80T could be strongly associated with higher risk of BD. Interestingly, TLR2-R677W could be a genetic marker for BD in females. However, further studies with larger groups are needed to confirm these findings.

TLR4 Polymorphisms (T399I/D299G) Association with Schizophrenia and Bipolar Disorder in a Tunisian Population.

Immune dysregulation has been widely described in the pathophysiology of schizophrenia (SCZ) and bipolar disorder (BD). Particularly, TLR4-altered activation was proposed as one of the underlying processes of psychosis onset. Since TLR4 activation was altered by T399I and D299G polymorphisms, we hypothesized that those variants could present common genetic factors of SCZ and BD. A total of 293 healthy volunteers and 335 psychotic patients were genotyped using PCR-RFLP. Genotype, allele, and haplotype distribution between controls and patients were evaluated according to clinical parameters. Statistical analyses were adjusted by logistic regression. In dominant model, T399I CT + TT and allele frequency were significantly higher in controls compared to psychotic population (p = 0.004, p = 0.002, respectively), SCZ (p = 0.02, p = 0.01, respectively), and BD (p = 0.03, p = 0.02, respectively). Similarly, D299G AG + GG and allele frequency were significantly higher in controls compared to psychotic population (p = 0.04, p = 0.04, respectively) and SCZ (p = 0.04, p = 0.03, respectively). T399I CT + TT and T allele were overrepresented in controls compared to paranoid subgroup (Padjusted = 0.04, p = 0.04, respectively) and type I BD (p = 0.04). Moreover, T399I and D299G were less prevalent in SCZ late-onset age (p = 0.03, p = 0.02, respectively). TA haplotype was associated with protection from psychoses (p = 0.02) and particularly from schizophrenia (p = 0.04). In conclusion, TLR4 polymorphisms could present a preventive genetic background against psychoses onset in a Tunisian population. While T399I could be associated with protection against SCZ and BD, presenting an overlapping genetic factor between those psychoses, D299G was suggested to be associated with protection only from schizophrenia.

Transfusion strategy for weak D Type 4.0 based on RHD alleles and RH haplotypes in Tunisia.

BACKGROUND: With more than 460 RHD alleles, this gene is the most complex and polymorphic among all blood group systems. The Tunisian population has the largest known prevalence of weak D Type 4.0 alleles, occurring in one of 105 RH haplotypes. We aimed to establish a rationale for the transfusion strategy of weak D Type 4.0 in Tunisia. STUDY DESIGN AND METHODS: Donors were randomly screened for the serologic weak D phenotype. The RHD coding sequence and parts of the introns were sequenced. To establish the RH haplotype, the RHCE gene was tested for characteristic single-nucleotide positions. RESULTS: We determined all RHD alleles and the RH haplotypes coding for the serologic weak D phenotype among 13,431 Tunisian donations. A serologic weak D phenotype was found in 67 individuals (0.50%). Among them, 60 carried a weak D Type 4 allele: 53 weak D Type 4.0, six weak D Type 4.2.2 (DAR), and one weak D Type 4.1. An additional four donors had one variant allele each: DVII, weak D Type 1, weak D Type 3, and weak D type 100, while three donors showed a normal RHD sequence. The weak D Type 4.0 was most often linked to RHCE*ceVS.04.01, weak D Type 4.2.2 to RHCE*ceAR, and weak D Type 4.1 to RHCE*ceVS.02, while the other RHD alleles were linked to one of the common RHCE alleles. CONCLUSIONS: Among the weak D phenotypes in Tunisia, no novel RHD allele was found and almost 90% were caused by alleles of the weak D Type 4 cluster, of which 88% represented the weak D Type 4.0 allele. Based on established RH haplotypes for variant RHD and RHCE alleles and the lack of adverse clinical reports, we recommend D+ transfusions for patients with weak D Type 4.0 in Tunisia.

Alteration in systemic markers of oxidative and antioxidative status in Tunisian patients with asthma: relationships with clinical severity and airflow limitation.

OBJECTIVE: This study aims to determine the systemic oxidant-antioxidant status in Tunisian patients with asthma. METHODS: We evaluated the levels of malondialdehyde (MDA) as thiobarbituric acid complexes, total protein carbonyls (PCs) and advanced oxidation protein products (AOPP). The levels of total thiols, protein sulfhydryls, glutathione (GSH), together with hydrogen peroxide, ascorbic acid, iron and total antioxidant status (TAS) were colorimetrically estimated. Glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) activities were assessed in plasma and erythrocytes by spectrophotometry. We also determined the levels of nitric oxide (NO) and peroxynitrite in plasma from asthmatic patients and healthy controls. The volume of fractionated exhaled NO (FeNO) was evaluated by the Medisoft HypAir method. Estimation of DNA damage was determined using the comet assay. RESULTS: Asthmatic patients showed increased levels of MDA in comparison to healthy controls (p < 0.001), while no significant difference was found in protein carbonyls (p = 0.79) and AOPP (p = 0.98). Patients with asthma also had significantly lower levels of total thiols (355.9 ± 15.72 versus 667.9 ± 22.65, p < 0.001), protein sulfhydryls (333.99 ± 16.41 versus 591.95 ± 24.28, p < 0.001) and glutathione (p < 0.001). They also showed decreased GSH-Px activity (p < 0.001), whereas no significant differences in measurements of catalase and SOD enzyme activities were observed between the two groups (respectively, p = 0.06 and p = 0.55). In addition, ascorbic acid and nitric oxide levels were decreased in asthmatics in comparison to controls (p < 0.01). CONCLUSIONS: Our findings highlight that oxidative stress and defective anti-oxidative status are major alterations in Tunisian patients with asthma.

Short duplication within the RHCE gene associated with an in cis deleted RHD causing a Rhnull amorph phenotype in an immunized pregnant woman with anti-Rh29.

BACKGROUND: The rare amorph Rhnull phenotype is caused by silent alleles at the RH locus and usually arises in consanguineous families. To date, only five molecular backgrounds have been identified in five unrelated families. Subjects with Rhnull red blood cells (RBCs) readily produce alloantibodies to high-prevalence Rh antigens. STUDY DESIGN AND METHODS: RBCs from a pregnant woman (G5P3) from Libya, with a positive indirect antiglobulin test were phenotyped by hemagglutination. RHD and RHCE genes were analyzed at the genomic level and mutation inheritance pattern was assessed in the patient's family. RESULTS: Hemagglutination testing showed a D-C-E-c-e- phenotype in the proposita associated with the presence of a high titer anti-Rh29 (4096). Molecular analysis revealed a deletion of RHD and presence of a novel RHCE allele with a 7-bp duplication in Exon 7. This duplication is predicted to introduce a frameshift after His350, a new C-terminal sequence, and a premature stop codon resulting in shortened predicted protein with only 402 amino acids. The mutated allele was found at homozygous state in the proposita and heterozygous state in her parents and one brother. CONCLUSION: This report describes a novel RHCE mutation causing the loss of RhCE antigen expression in association with RHD deletion, leading to an amorph Rhnull phenotype.

RHD genotyping and its implication in transfusion practice.

BACKGROUND: The limitations of serology can be overcome by molecular typing. In order to evaluate the contribution of RH systematic genotyping and its implication in transfusion practice, a genotyping of D- blood donors was initiated. METHODS: Blood samples were collected from 400 unrelated D- individuals. All samples were tested by RHD exon 10 PCR. In order to clarify the molecular mechanisms of RHD gene carrier, we applied molecular tools using different techniques: PCR-multiplex, and PCR-SSPs. RESULTS: Among 400 D- subjects tested, 390 had RHD gene deletion; and 10 had RHD exon 10 of which seven were associated with the presence of the C or E antigens. Among D- carriers, we observed in five cases the presence of RHD-CE-Ds hybrid, in four cases the presence of pseudogene RHD ψ and in one case the presence of weak D type 4. CONCLUSION: Since the majority of aberrant alleles were associated with C or E antigens and the preliminary infrastructure for molecular diagnostic were absent in all Tunisia territory, we recommend to reinforce transfusion practice to consider D- donors but C+/E+ antigens as D+ donors and the application of RHD molecular typing only to solve serologic problems.

Human herpesvirus-8 infection in Tunisian adult acute leukemia patients.

Background: Human herpesvirus 8 (HHV-8) has been linked to the development of Kaposi's sarcoma (KS)and multiple other hematologic malignant disorders. However, the role of HHV-8 in acute leukemia patients is unknown. Objectives: The objective of this study was to determine the prevalence of HHV-8 in Tunisian acute leukemia patients and in healthy blood donors. Methods: An indirect immunofluorescence test was used to detect the presence of anti-HHV8 antibodies. Nested PCR was used for the detection of HHV-8 DNAemia in samples of plasma. Results: The seroprevalence of HHV-8 was significantly higher in acute leukemia patients (21,4% ,15/70) than in healthy blood donors (7,1%, 5/70), (p= 0.02). Gender, type of disease, status of disease, prior blood transfusion, and outcome were not associated with HHV-8 seroprevalence. However, among acute leukemia patients, HHV-8 seroprevalence was statistically associated with older age > 40 years of age, (p=0.002). HHV-8 DNAemia was detected (1,4%) in only one patient of acute myeloid leukemia (AML) and none of the healthy blood donors. Conclusions: The seroprevalence of HHV-8 infection in Tunisian adult acute leukemia patients was three times as high compared to healthy blood donors, suggesting that patients with acute leukemia might be at increased risk of HHV-8 infection.

RHD zygosity assignments based on most probable genotype and hybrid Rhesus box detection in Tunisia.

BACKGROUND: Determination of the RHD zygosity is important for genetic counselling and risk evaluation of hemolytic disease of the newborn HDN in women with D iso-immunisation. OBJECTIVES: We proposed to determine the genotype frequencies of the RHD locus using a PCR-SSP method and assignment of the most probable genotype (MPG) and analyse the concordance between the two methods. METHODS: The complete Rh phenotype and the frequencies of RH haplotypes were determined on 506 blood donors. RHD zygosity was determined by both assignment of the MPG and PCR-SSP specific for the hybrid Rhesus box. For RH:-1 samples, analysis of the RHD exon 10 was done to detect eventual RHD aberrant alleles. RESULTS: Among the 466 RH:1 samples, 54.08% were hemizygous and 45.92% homozygous by PCR-SSP, and 64.16% hemizygous and 35.84% homozygous by the MPG. The comparison between the methods showed discordant results in 135 RH:1 samples. For the 40 RH:-1 samples, hybrid Rhesus box was detected in all samples and RHD exon 10 was detected in three samples suggesting unequivocal alleles identified as one RHDψ, one (C)ce(s) and one weak D type 4. CONCLUSION: The PCR-SSP should replace the MPG. However, studying of aberrant RHD alleles and aberrant Rhesus boxes could confirm the accuracy of this method in Tunisian population.

Molecular background of D-negative phenotype in the Tunisian population.

BACKGROUND: Most studies of the molecular basis of Rhesus D-negative phenotype have been conducted in Caucasian and African populations. A comprehensive survey of RHD alleles was lacking in people from North Africa (Tunisians, Moroccans and Algerians) which could be very efficient for managing donors and patients carrying an RHD molecular variant. We analyse the molecular background of D-negative population in Tunisia in the present study. MATERIALS AND METHODS: Blood samples were collected from native Tunisians. A total of 448 D-negative donors from different regions of Tunisia were analysed by RHD genotyping according to an adopted strategy using real-time PCR, ASP-PCR and sequencing. RESULTS: Among the 448 D-negative samples, 443 were phenotyped unequivocally as true D-negative including three molecular backgrounds which were RHD gene deletion (n = 437), RHDψ pseudogene (n = 2) and RHD-CE-D hybrid gene (n = 4) with the respective frequencies of 0·9900, 0·0023 and 0·0046. The remaining five samples, in discordance with the serological results, were identified as two weak D type 11, one weak D type 29, one weak D type 4·0 and one DBT-1 partial D. CONCLUSION: This study showed that the Tunisian population gets closer to Caucasians, given that the RHD gene deletion is the most prevalent cause of D-negative phenotype, but it is slightly different by the presence of the RHDψ pseudogene which was found with a very low frequency compared with that described in the African population. Nevertheless, the relative occurrence of weak D variants among studied serologically D-negative samples make necessary the adaptation of RHD genotyping strategy to the spectrum of prevalent alleles.

Leucocyte cytokines dominate platelet cytokines overtime in non-leucoreduced platelet components.

BACKGROUND: Leucoreduction of blood components, including platelet components, is strongly encouraged but not yet universal, especially outside high income countries. As both leucocytes and platelets secrete copious amounts of pro-inflammatory cytokines/chemokines under various conditions and during storage, we investigated the potential of the respective secretory programmes of these cells in order to evaluate their subsequent pathophysiological effects. MATERIAL AND METHODS: A total of 158 individual non-leucoreduced platelet components were obtained from Tunisian donors and tested for characteristic biological response modifiers (BRM) of leukocytes (IL-1ß, IL-8), platelets (sCD62P, sCD40L) and both cell types (TNF-α, RANTES) in the presence or absence of thrombin stimulation and after different periods of storage (up to 5 days). BRM levels were determined using enzyme-linked immunosorbent assays and Luminex technology. Platelet-leucocyte aggregate formation during storage was analysed using flow cytometry. RESULTS: Leucocyte- and platelet-associated BRM had clearly distinct profiles both at the onset (day 0) and termination (day 5) of the observation period but altered during the intermediate period so that their respective importance was inverted; in fact, the profiles were merged and indistinguishable on days 2-3. The leucocyte-derived BRM largely dominated over platelet-derived ones and further altered the BRM platelet secretion programme. DISCUSSION: This study contributes substantial, new information on leucocyte/platelet interactions and their likely role in transfusion when leucodepletion cannot be performed or is only partially achieved.

Molecular genetic diagnosis of Tunisian Glanzmann thrombasthenia patients reveals a common nonsense mutation in the ITGA2B gene that seems to be specific for the studied population.

: Glanzmann thrombasthenia is an inherited severe bleeding disease. Mutations associated with Glanzmann thrombasthenia are highly heterogeneous and occur across the two genes coding for the platelet αIIbß3 integrin. This study was aimed at identifying Glanzmann thrombasthenia-associated novel mutations in Tunisian patients. Seven unrelated Glanzmann thrombasthenia patients issued from high consanguineous families (86%; 6/7 of the patients) were studied. Glanzmann thrombasthenia diagnoses were based on patients' bleeding histories and platelet aggregation tests. Screening of ITGA2B and ITGB3 genes was performed by denaturing high-performance liquid chromatography (DHPLC) analysis. Amplicons with abnormal elution profiles were subjected to direct sequencing. DHPLC/sequencing analysis identified a pathogenic homozygous mutation in exon 26 at position c.2702C>A, inducing a substitution of a serine to a stop codon (p.S901*) in the ITGA2B gene, in three patients. This mutation was only previously reported in a Glanzmann thrombasthenia patient of a Tunisian origin and not in other populations. We diagnosed a pathogenic Glanzmann thrombasthenia mutation in ITGA2B screened by DHPLC that appears to be specific to individuals of Tunisian heritage and that deserves to be investigated in first intention. As a result, we determined that performing prenatal diagnosis and setting a prevention strategy via counselling for affected heterozygote individuals will be helpful for Tunisian Glanzmann thrombasthenia families where there is still a high rate of consanguinity.

Chrysin, a natural and biologically active flavonoid suppresses tumor growth of mouse B16F10 melanoma cells: In vitro and In vivo study.

Chrysin (5,7-dihydroxyflavone) is a natural and biologically active compound which has many biological activities as an anticancer agent. The current report is aimed at finding out whether the antitumor potential of chrysin, evidenced in vitro and in vivo, is linked or not to its effect on immunological mechanisms of melanoma-bearing mice. Chrysin-treated B16F10 cells were analyzed for their metabolic rate and apoptotic potentials. In vivo, BALB/c mice received a subcutaneous injection of B16F10 melanoma cells prior to antitumor treatments with chrysin (50 mg/kg b.w) for 14 days and 21 days. The results showed that chrysin inhibited cancer cell growth at a dose-dependent manner by inducing apoptosis and cell cycle arrest at G2/M phase. Moreover, chrysin suppressed melanoma tumor growth at an average of 60% (after 14 days of treatment) and 71% (after 21 days of treatment) compared to the tumor-bearing group. Furthermore, chrysin treatment increased the cytotoxic activity of NK, CTL and macrophages. The findings showed that chrysin antitumor action on the murine melanoma model was very promising, suggesting that chrysin could be a potentially good candidate for future use in alternative anti-melanoma treatments.

Weak D in the Tunisian population.

BACKGROUND: More than 90 weak D types have been discovered to date. As there are no published data on the frequencies of weak D types in the Tunisian population, the aim of this study was to determine the composition of weak D alleles in our population. MATERIAL AND METHODS: Blood samples from 1777 D+ and 223 D- blood donors were tested for markers 809G, 1154C, 8G, 602G, 667G, 446A, and 885T relative to translation start codon by polymerase chain reaction with sequence-specific primers to estimate the frequencies of weak D type 1, weak D type 2, weak D type 3, weak D type 4, weak D type 5 and weak D type 11 in our population. Twenty-three samples with positive reactions were re-evaluated by DNA sequencing of RHD exons 1-10 and adjacent intronic sequences. RESULTS: Among the D+ donor cohort, weak D type 4 was the most prevalent allele (n=33, 1.2%) followed by weak D type 2 (n=6, 0.17%), weak D type 1 (n=4, 0.11%), and weak D type 5 (n=1, 0.28%) and weak D type 11 (n=1, 0.28%). RHD sequencing identified a weak D type 4.0 allele in all 19 samples tested. Among the D- pool, comprising 223 samples, we detected one sample with weak D type 4.0 associated with a C+c+E-e+ phenotype which had been missed by routine serological methods. DISCUSSION: Weak D type 4.0 appears to be the most prevalent weak D in our population. However, all samples must be sequenced in order to determine the exact subtype of weak D type 4, since weak D type 4.2 has considerable clinical importance, being associated with anti-D alloimmunisation. One case of weak D type 4 associated with dCe in trans had been missed by serology, so quality control of serological tests should be developed in our country.

Paternal RHD zygosity determination in Tunisians: evaluation of three molecular tests.

BACKGROUND: The choice of a molecular test for first intention determination of paternal RHD zygosity, before entering into invasive diagnostics, is important for the management of pregnancies at risk of haemolytic disease of the foetus and newborn related to anti-RhD. MATERIALS AND METHODS: RHD zygosity was evaluated in 370 RH:1 Tunisian donors by polymerase chain reaction - sequence-specific polymorphism (PCR-SSP) analysis and polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) amplification of hybrid Rhesus box and by real time quantitative polymerase chain reaction (RQ-PCR) specific for RHD exon 5. To evaluate the accuracy of molecular tests in the cases of discordant results, the ten exons of RHD and Rhesus boxes were amplified by PCR and sequenced. RESULTS: Molecular investigations revealed that our 370 donors comprise 193 dizygous and 145 hemizygous individuals and 32 subjects whose zygosity remains unknown. Positive predictive values were higher than 99% for all the methods, reaching 100% for RQ-PCR. Negative predictive values were 83.24%, 87.27% and 98% for PCR-SSP, PCR-RFLP and RQ-PCR respectively. This study also revealed 19 novel Rhesus box polymorphisms and three novel RHD alleles: RHD(Trp185Stop), RHD(Ala176Thr) and RHD(Ile342Ile). DISCUSSION: RQ-PCR is the most convenient method for first intention determination of paternal RHD zygosity in Tunisians. However, taking into account positive and negative predictive values, PCR-RFLP could be an alternative despite the heterogeneity of Rhesus boxes and the complexity of RHD.

Matrix Metalloproteinase-9 (279R/Q) Polymorphism is Associated with Clinical Severity and Airflow Limitation in Tunisian Patients with Chronic Obstructive Pulmonary Disease.

BACKGROUND: The aim of this study was to investigate the role of matrix metalloproteinase-9 (MMP-9) C-1562T and 279R/Q (836G>A) polymorphisms in the development of chronic obstructive pulmonary disease (COPD) in Tunisians and to determine their impact on disease progression and airflow obstruction. METHODS: Pulmonary functional tests were evaluated by body plethysmography. MMP-9 genotypes were determined in patients with COPD (n = 138) and healthy controls (n = 216) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Serum MMP-9 and interleukin-6 (IL-6) levels were determined by enzyme-linked immunosorbent assays (ELISA) and activity of MMP-9 was evaluated by gelatin zymography. RESULTS: No significant association was found between genetic variations in MMP-9 C-1562T and 279R/Q polymorphisms and the risk of development of COPD. However, a significant correlation was retrieved between the 279 R/Q polymorphism and disease severity (P = 0.02). In addition, homozygous Q (A) genotype was associated with a poorer lung function with a fall in forced expiratory volume in 1 s (FEV1) (%) and forced vital capacity (FVC%) among COPD patients compared with both AG and GG individuals (52.06 ± 19.6 vs. 59.08 ± 17.19, P = 0.03 and 72.41 ± 21.42 vs. 82.98 ± 16.48, P = 0.002, respectively). Using ELISA, a higher level of MMP-9 was found in patients with the CT genotype (P = 0.03), while no significant impact of the 279R/Q polymorphism was observed (P = 0.48). In contrast, by using zymography gel analysis, MMP-9 activity was enhanced in individuals carrying the R(G) allele in comparison with those homozygous for the Q(A) variant (P = 0.02). CONCLUSION: Our results support a role for the 279R/Q polymorphism in physiological alterations that may affect progression and severity of COPD. These findings could be related to the decreased activity of MMP-9 among COPD patients carrying the 279Q variant.

Frequency of HNF4A-P.I463V Variant in the Tunisian North-African Population and Its Relation with Diabetes Mellitus.

BACKGROUND: HNF4A-p.I463Vvariant, reported previously in two distinct families suspected of MODY-1, is assessed in this report to determine whether it is a mutation or a polymorphism (frequency >1%). METHODS: 200 Tunisian healthy people were screened for the presence of HNF4A-p.I463V variant, using RFLP-PCR technique and sequencing. Then, the frequency of this variant was estimated in the Tunisian population and compared to other populations registered in genetic databases. We also performed in-silico analysis using PolyPhen2 and Mutation T@sting softwares to assess the probable effect of HNF4A-p.I463V variant. RESULTS: HNF4A-p.I463V had a rare frequency in different populations and was found in 3 control subjects (1.5%) of the studied population. PolyPhen2 predicted that it is a polymorphism, whereas mutation T@sting suggested a probably affected mutant protein. CONCLUSION: HNF4A-p.I463V has a relatively high frequency (>1%) in our control cohort. It is also present in different ethnicities and in- silico analysis showed conflicting results. For these reasons, HNF4A-p.I463V should not be considered as a mutation responsible for MODY-1.

Increased oxidative stress and altered levels of nitric oxide and peroxynitrite in Tunisian patients with chronic obstructive pulmonary disease: correlation with disease severity and airflow obstruction.

This study was aimed to evaluate the oxidant-antioxidant imbalance in the pathogenesis of chronic obstructive pulmonary disease (COPD) in Tunisians. We assessed 16 parameters related to the oxidative status that include malondialdehyde (MDA), total protein carbonyls (PCs), and advanced oxidation protein products (AOPP). We also examined the activity of glutathione peroxydase (GSH-Px), catalase, and superoxide dismutase (SOD) in the plasma and erythrocytes. Levels of total thiols, reduced glutathione (GSH), total antioxidant status (TAS), hydrogen peroxide, ascorbic acid, iron, and protein sulfhydryls were determined using spectrophotometry. We also evaluated the level of nitric oxide (NO) and peroxynitrite in plasma from COPD patients and healthy controls. Estimation of DNA damage was determined using the comet assay. Pulmonary functional tests were performed by body plethysmography. Levels of MDA, PC, DNA damage, and AOPP were significantly increased while total thiols, GSH, and TAS were decreased in COPD patients. GSH-Px activity was higher in COPD patients while no difference was found for catalase and SOD. We also observed a lower level of NO and peroxynitrite in COPD patients. Decreased levels of peroxynitrite were found to correlate with disease progression, as well as with forced expiratory volume in 1 s/forced vital capacity among COPD patients. Multivariate analysis revealed that NO is associated with pathological pathways that help to predict patient outcome independently of the degree of airflow obstruction. These results indicate the presence of a systemic oxidative stress and highlight the importance of NO and peroxynitrite as major effectors in COPD development and airflow obstruction.

Are polymorphisms of the immunoregulatory factor CD40LG implicated in acute transfusion reactions?

The CD40 ligand (CD40L/CD154), a member of TNF superfamily, is notably expressed on activated CD4+ T-cells and stimulated platelets. CD40L is linked to a variety of pathologies and to acute transfusion reactions (ATR). Mutations in this gene (CD40LG) lead to X-linked hyper-IgM syndrome. Some CD40LG polymorphisms are associated with variable protein expression. The rationale behind this study is that CD40L protein has been observed to be involved in ATR. We wondered whether genetic polymorphisms are implicated. We investigated genetic diversity in the CD40LG using DHPLC and capillary electrophoresis for screening and genotyping (n = 485 French and Tunisian blood donors). We identified significant difference in the CD40LG linkage pattern between the two populations. Variant minor alleles were significantly over-represented in Tunisian donors (P<0.0001 to 0.0270). We found higher heterogeneity in the Tunisian, including three novel low frequency variants. As there was not a particular pattern of CD40LG in single apheresis donors whose platelet components induced an ATR, we discuss how this information may be useful for future disease association studies on CD40LG.

Fetal RhD genotyping by real time quantitative PCR in maternal plasma of RhD-negative pregnant women from the Sahel of Tunisia.

OBJECTIVE: The aim of this study was to evaluate the diagnostic value of RhD fetal genotyping from the plasma of RhD-negative pregnant women. METHODS: We analysed the plasma samples of 65 pregnant women. DNA quantification was done using real time quantitative PCR (RQ-PCR) in multiplex targeting multiple RhD exons 5, 7 and 10, with a standardized pool of plasmid calibrators. Results were compared with serological analysis of cord blood after delivery. RESULTS: Fetal RhD status was predicted with 95.38% accuracy from maternal plasma of pregnant women in the 11(th) to 40(th) weeks of gestation. One false positive but no false negative results were found. Thus the sensitivity of the assay was 100% and the specificity was 94.44 %. CONCLUSION: The present data demonstrates that the fetal RhD genotyping approach could be achieved efficiently with RQ-PCR for RhD-negative tunisian pregnant women.