Role of polymorphisms in the TNFRSF13B (TACI) gene in Spanish patients with immunoglobulin A deficiency.

Mutations in the TNFRSF13B (TACI) gene have been associated with common variable immunodeficiency, and a role in immunoglobulin A deficiency (IgAD) has also been suggested. We aimed at studying the role of several polymorphisms along this gene in IgAD susceptibility. Three TNFRSF13B mutations (C104R, A181E and R202H) and eight additional single nucleotide polymorphisms in the gene were genotyped in 338 Spanish IgAD patients and 553 ethnically matched healthy controls and tested for association. Data from parents of 114 IgAD patients were also collected and used for additional analysis. No statistically significant differences were observed after comparing patients and controls for any single nucleotide polymorphism analysed. Therefore, our work seems to discard a role of TNFRSF13B mutations in IgAD, concordantly with the most recent published studies.

Naturally occurring Bruton's tyrosine kinase mutations have no dominant negative effect in an X-linked agammaglobulinaemia cellular model.

X-linked agammaglobulinaemia (XLA) is characterized by absence of mature B cells because of mutations in the Bruton's tyrosine kinase (Btk) gene. Btk-deficient early B cell precursors experience a block in their differentiation potentially reversible by the addition of an intact Btk gene. Btk expression was measured in 69 XLA patients with 47 different mutations and normal expression was detected in seven. We characterized these Btk mutant forms functionally by transfection into a lymphoma cell line that lacks endogenous Btk expression (Btk-/- DT40 cells) and analysed the calcium flux in response to B cell receptor stimulation. To test whether co-expression of a mutated form could compromise the function of the intact Btk transfection, studies in wild-type (WT) DT40 cells were also performed. Study reveals that none of the seven Btk mutants analysed was able to revert the absence of calcium mobilization upon IgM engagement in Btk-/- DT40 cells, as does intact Btk. In addition, calcium mobilization by anti-IgM stimulation in DT40 Btk+/+ cells was unaffected by co-expression with Btk mutants. These results suggest that gene addition would be feasible not only for patients with XLA and mutations that prevent Btk expression, but for those with expression of a mutant Btk.

Clinical and molecular analysis of patients with defects in micro heavy chain gene.

Autosomal recessive disorders of B cell development are rare and heterogeneous. To determine the proportion of affected patients who have defects in the micro heavy chain (IGHM) gene, we used single-stranded conformational polymorphism analysis to screen genomic DNA from 40 unrelated patients with early onset infections, profound hypogammaglobulinemia, and absent B cells. All of the patients were genotypically normal in BTK, the gene that underlies X-linked agammaglobulinemia. Eight different mutations in the micro heavy chain were identified in 19 members of 12 unrelated families. Four of the mutations were large deletions that removed more than 40 kb of DNA in the IGHM locus. In six of the 12 families, the affected patients had an identical single base pair substitution, a G-->A, at the -1 position of the alternative splice site. Immunoglobulin haplotype analysis showed that this mutation occurred on at least three different haplotypes, indicating that this is a hot spot for mutations. Compared with patients with mutations in Btk, patients with defects in the micro heavy chain had an earlier onset of disease and more complications. Our study indicates that at least 20-30% of patients with autosomal recessive defects in B cell development have mutations in the micro heavy chain.

Co-receptor usage of HIV-1 primary isolates, viral burden, and CCR5 genotype in mother-to-child HIV-1 transmission.

OBJECTIVE: To investigate the relationship between CC chemokine receptor 5 (CCR5) genotype, viral load and co-receptor usage of maternal HIV-1 isolates in perinatal HIV-1 transmission. PATIENTS AND METHODS: A total of 181 mothers and infants were studied at the time of delivery. Wild-type (wt) and delta32 CCR5 alleles were determined by means of polymerase chain reaction (PCR). The viral load in maternal plasma samples was determined by a quantitative reverse transcriptase-PCR assay; co-receptor usage of maternal isolates was determined by viral infection in cells stably expressing CCR5 or CXC chemokine receptor 4 (CXCR4) co-receptors. RESULTS: HIV-1 transmission rates in wt/wt and wt/delta32 mothers (14.7 versus 15.8%), and in wt/wt and wt/delta32 infants (14.6 versus 14.3%) were similar. Mothers transmitting infection to wt/delta32 infants had significantly higher HIV-1-RNA levels than those who transmitted infection to wt/wt infants (5.4 versus 4.1 log10 copies/ml, P = 0.03). In wt/wt children there was a positive relationship between transmission rate and maternal viral load over the entire range of HIV-1 values, whereas in wt/delta32 children transmission occurred only at viral loads greater than 4.0 log10 copies/ml. Logistic regression analysis confirmed that the relationship between viral load and transmission varied according to the child's CCR5 genotype (P = 0.035; adjusted for zidovudine prophylaxis and mode of delivery, P = 0.090). Moreover, the majority of wt/wt transmitting mothers had R5-type isolates, whereas none of the wt/delta32 mothers with an R5-type virus transmitted HIV-1 to their wt/delta32 infants. CONCLUSION: Taken together, these findings suggest that CCR5 delta32 heterozygosity exerts a protective effect against perinatal transmission in children exposed to a low maternal viral burden of an R5-type isolate.

Platelet-activating factor: the effector of protein-rich plasma extravasation and nitric oxide synthase induction in rat immune complex peritonitis.

1. The involvement of platelet-activating factor (PAF) in immune complex-induced/polymorphonuclear-mediated tissue injury was studied by use of a reverse passive Arthus (RPA) model in the peritoneal cavity of rats. 2. Extravasation of protein-rich plasma, accumulation of polymorphonuclear leukocytes (PMN), and the production of nitric oxide (NO) by resident peritoneal mononuclear phagocytes were assayed. 3. Treatment of rats with either UR-12460 or BB-823, two compounds which possess different chemical structures, but elicit the same antagonistic effect on the PAF receptor, abrogated protein-rich plasma extravasation. In contrast, they did not show any effect on the accumulation of PMN. 4. Inhibition of NO production with both NG-mono methyl-L-arginine and NG-nitro-L-arginine failed to prevent protein-rich plasma extravasation. 5. The production of NO by peritoneal adherent cells following RPA was measured in cells maintained for 2 to 28 h in culture, and it was significantly increased in cells removed as early as 15 min after RPA induction, as compared to controls. 6. Addition of 10 nM PAF to the culture medium reduced the generation of NO by peritoneal cells from RPA rats, whereas this mediator enhanced NO production in cells from naive control animals. 7. Treatment with either UR-12460 or BB-823 prior to the induction of RPA produced an almost complete inhibition of NO production. 8. Assay of nitric oxide synthase activity in cell homogenates from peritoneal cells showed that the activity was due to the inducible form of the enzyme. 9. Study by Northen blotting of mRNA coding for the inducible NO synthase (iNOS) showed transcription at 6 and 18 h after the induction of RPA, which was inhibited in UR-12460-treated rats.10. These data indicate that PAF is the main mediator of the early plasma leakage observed in RPA,and also that PAF is implicated in the triggering of long-term changes via induction of specific genes, as judged from its ability to promote the expression of iNOS.

Autoantibodies in patients with IgA and IgG2 deficiencies.

Patients with primary immunodeficiencies have a high incidence of autoantibodies, mainly of no clinical significance. It has recently been suggested that patients with a combined IgA-IgG2 deficiency have more autoantibodies than those patients with isolated deficiencies. We have studied 42 patients with selective IgA deficiency, nine with isolated IgG2 deficiency and 13 with combined IgA-IgG2 deficiency, and have found that the combined IgA-IgG2 deficiency has no influence on autoantibody prevalence, except for anti-IgA antibodies. The presence of chronic respiratory infections (a clinical feature commonly associated with both selective IgA and IgG2 deficiencies) is unrelated to the prevalence of autoantibodies. The most frequent autoantibodies found are anti-IgA and anti-cardiolipin. Most of the autoantibodies have been found to be devoid of actual clinical significance. Only three patients had overt autoimmune disease.

Quantification of soluble serum HLA class I antigens in healthy volunteers and AIDS patients.

A solid-phase enzyme immunoassay (ELISA) has been used to quantify human soluble Class I histocompatibility antigens in serum samples from voluntary blood donors and AIDS patients. Statistical analysis of the results showed significantly raised levels (p less than 0.01) of free HLA Class I in sera from AIDS patients (2.95 +/- 1.80 micrograms/ml) when compared with the blood donors (1.06 +/- 0.6 micrograms/ml). The assay is specific, reproducible and easy to perform. Potential uses of this determination are discussed.

In vivo effect of chlorophyllin on gamma-ray-induced sister chromatid exchange in murine bone marrow cells.

The aim of the present work is to determine the radioprotective capacity of chlorophyllin, by measuring the reduction of gamma-ray-induced sister-chromatid exchange (SCE) in murine bone marrow cells in vivo. The results obtained in two separate experiments, using 10, 50 and 100 micrograms of chlorophyllin per g of body weight (bw), indicate that chlorophyllin per se did not have any effect on the SCE frequency and that the dose of 100 micrograms/g bw protects 100% against the induction of SCE by 1.0 Gy of gamma-rays; 50 micrograms/g bw protects less than 50% and 10 micrograms/g bw affords no protection.

Effect of chlorophyllin on chromium trioxide-induced micronuclei in polychromatic erythrocytes in mouse peripheral blood.

The effect of chlorophyllin on micronucleated polychromatic erythrocytes (MN-PCE) induction by chromium trioxide (CrO(3)) exposure in peripheral blood of mice was studied. Animals were treated with a single intraperitoneal dose of chlorophyllin (CHL) (20mg/kg), CrO(3) (20mg/kg), and CHL (20mg/kg) 4h before (CHL-CrO(3)) or 4h before and 20h after chromium treatments (20mg/kg; CHL-CrO(3)-CHL). Peripheral blood samples were drawn from the caudal vein at 0, 12 and 48h, and analyzed by the acridine orange (AO) technique. The results obtained in present study shown that CHL injection did not modify the number of MN-PCE. CrO(3) treatment resulted in a significantly increases 12 and 48h after the injection, reaching a four-fold increase 48h after CrO(3) administration. Whereas treatment with 20mg/kg of CHL prior to chromium, decreased the MN frequency induced by chromium in the 12h samples. When the samples were analyzed 48h after CrO(3) injection, no significant differences between CHL-CrO(3) and CHL-CrO(3)-CHL in comparison with CrO(3) treatment, were observed. These results indicate that increase of MN-PCE by CrO(3) is CHL-blocked in both protocols used (CHL-CrO(3) and CHL-CrO(3)-CHL) at 12h after treatment, but it was unable to modify the frequency of MN-PCE measured 48h after CrO(3) injection. The absence of a protective effect by CHL in the MN-PCE induction by CrO(3) at 48h, show that CHL has action only on one of the times of MN induction and suggests the possible action of CrO(3) by two different mechanisms, and not by CHL time-limited in vivo.

[Azidothymidine in the treatment of patients with human immunodeficiency virus infection and persistent generalized adenopathies]. / La azidotimidina en el tratamiento de los pacientes con infección por el virus de la inmunodeficiencia humana y adenopatías generalizadas persistentes.

The effectiveness and security of azidothymidine (AZT) in the treatment of patients with infection by the human immunodeficiency virus (HIV) and persistent generalized adenopathies (PGA), were assessed. Thirty six patients with HIV infection and PGA participate in the study. Eighteen were treated with AZT and the other 18 were included in the control group, since they did not accept the treatment. Both groups were homogeneous with respect to their clinical, immunological and virological characteristics. A common study protocol was used and the clinical, immunological and virological effectiveness was assessed. Lymphocyte subpopulations were quantified by flow cytometry, viral antigens were determined by sandwich-type ELISA and antibodies against viral proteins (anti-gp120, anti-gp160, anti-gp41, anti-gp24 and anti-p18) were detected by Western blot. Naranjo and Busto's algorithm was used for the causality of adverse effects. We did not observe any significant differences regarding the presence of infection and the evolution of AIDS in both groups. A positive response to thrombocytopenia was observed, more evident in patients under low doses of AZT. The small initial transitory improvement of the immunological parameters was not statistically significant. The viral antigen was not modified by the treatment. With respect to the behaviour of the several antibodies studied, no differences were observed. The initial doses of AZT had to be modified in 44% of patients due to their hematological toxicity, more frequent in the first stages of the treatment. In two patients, the treatment had to be finally discontinued due to severe neutropenia. 25% of patients showed mild to moderate gastrointestinal manifestations.(ABSTRACT TRUNCATED AT 250 WORDS)

[Green tea and its role on chemoprevention in vivo of genotoxic damage induced by carcinogenic metals (chromium [VI])]. / El té verde en la quimioprevención in vivo del daño genotóxico inducido por metales cancerígenos (cromo [VI]).

BACKGROUND: Consumption of green tea, by its antioxidant properties, has been associated with beneficial health effects, because antioxidant may play a role in the risk and pathogenesis of several chronic diseases, especially cardiovascular disease and cancer. On the other hand, it has been reported that metal compounds such as chromium [VI] are carcinogenic and can induce genotoxic damage through the Oxidative Stress. Therefore, it is possible that green tea has a protective effect against the genotoxic damage induced by this compounds. OBJECTIVE: To evaluate the effect of oral administration of green tea over the genotoxic damage induced by Cr [VI] by quantification of micronucleus (MN) in polychromatic erythrocytes (EPC). MATERIALS AND METHODS: We use mice of CD-1 strain that were randomly divided into the following groups: (i) control, (ii) treatment with green tea, (iii) treatment with chromium trioxide, (iv) treatment with green tea and chromium trioxide. The green tea was administrated via intragastric tube every 12 hours over two days (4 doses of 0.25 ml infusions 1.6 g/7.5 ml) and ad libitum (5.6 ml/day for 10 days infusions of 3.2 g/100 ml), while chromium trioxide was administrated via intraperitoneal (20 mg/kg). Blood samples were obtained from the caudal vein, the number of MN in EPC was assessed at 0, 24, 48 and 72 hours after the treatments. RESULTS: The group treated with green tea showed no significant statistical changes in the average of MN. On the other hand, the group that was dosed with the chromium trioxide showed an increase between 4 and 8 MN, which was statistically significant when compared with control group, which confirmed the genotoxic damage. When the green tea treatment was administered before the application of chromium trioxide, there was a decrease in MN frequencies of 31 and 62% at 72 hours, 20 and 35% at 48 hours and 18 and 31% at 24 hours with intragastric and ad libitum respectively, compared with the group treated only with chromium trioxide. Hence, green tea reduced the genotoxic damage induced by chromium trioxide, and the highest protection was presented at 72 hours. CONCLUSIONS: Our findings support the protective effects of green tea against the damage of genetic material, induced by metal compounds such as chromium [VI], suggesting that its antioxidant compounds are those that have a chemopreventive effect on the EOX generated by the Cr [VI] during its reduction to Cr (III). The fact that the largest decrease in the frequency of MN was observed at 72 hours and ad libitum treatment, suggests that, the protective effect depends on the bioavailability, pharmacodynamics and pharmacokinetics of the active ingredient in green tea, so the administration of green tea for a long period of time before the exposure to Cr [VI] could have a more consistent preventive effect.

[Immunological changes in the child infected by vertical HIV transmission]. / Alteraciones inmunológicas en el niño infectado por transmisión vertical.

The vertical transmission of HIV infection depends on factors in the mother and child, as well as characteristics of the virus itself. The body defends itself through the immune response. Today it is known that the presence of the CD4 molecule on the cells does not suffice for them to be able to be infected, being necessary the presence of co-receptors such as CCR5 and CXCR4. The immunological disorders that most frequently appear in infected children are hypergammaglobulinemia and reduction in the CD4+ T lymphocytes and CD4+/CD8+ ratio. In vitro production of immunoglobulins is increased and the proliferative response to mitogens and antigens is reduced. Also, the production of cytokines like IL-2 and IFN(is lower than in controls as a consequence of the reduction in memory CD4+ T cells in these children. The presence of a mutation in the CCR5 gene in some of them seems to contribute to the evolution of the disease because heterozygotes for this gene have less risk of developing severe immunodeficiency and a slower disease progression.

Follow-up of anti-IgA antibodies in primary immunodeficient patients treated with gamma-globulin.

The levels of anti-IgA antibodies and the appearance of adverse reactions following gamma-globulin administration in 41 patients affected by primary antibody defects treated with intramuscular (IMGG) or intravenous gamma-globulin (IVGG), and 3 patients with the Wiskott-Aldrich syndrome (WAS) have been studied during a 31-month period. Anti-IgA antibodies were restricted to patients with circulating B lymphocytes and measurable amounts of IgA. The incidence of anti-IgA antibodies in the immunodeficient patients studied was 22.7%, and 2 of the 3 WAS patients also had high levels of anti-IgA antibodies. The presence of moderate levels of anti-IgA antibodies (up to 1/1,600) was not associated with adverse reactions. Our results indicate a significant relationship (p less than 0.02) between persistence of anti-IgA antibodies and IMGG administration.

Microtubule function in leukocytes from umbilical cord blood.

Polymorphonuclear neutrophils and lymphocytes from umbilical cord blood had a normal distribution of concanavalin A receptors on their surfaces and do not show an abnormal sensitivity to the microtubule disrupting drug colchicine. These experiments suggest that leukocytes from úmbilical cord blood do not show any developmental deficiency of the microtubule system.

Anticardiolipin antibodies in patients with primary immunodeficiency diseases.

The presence of antibodies to cardiolipin was determined (by an ELISA) in 143 patients with primary immunodeficiency diseases. Thirty (21%) had raised anticardiolipin antibody levels compared with only three in 98 age matched controls. The highest prevalence of this autoantibody was found in the Wiskott-Aldrich syndrome. Patients with selective IgA deficiency also showed a high prevalence of this autoantibody (32%), while patients with severe defects in antibody production showed a low prevalence or did not have such autoantibodies. This study provides further evidence of the association between autoimmune phenomena and primary immunodeficiency diseases.

Anti-IgA antibodies in selective IgA deficiency and in primary immunodeficient patients treated with gamma-globulin.

Sera from 106 blood donors, 40 patients with primary immunodeficiencies (ID) treated with gamma-globulin, and 46 patients with selective IgA deficiency were analyzed by an enzyme-linked immunosorbent assay for anti-IgA antibodies. Increased levels of antibodies to IgA were found in 5.6% of the blood donors, 17.5% of the ID patients, and 36.8% of the isolated IgA deficiencies. The percentage was higher in patients with IgA and IgG2 deficiencies (50%). The percentage of patients having increased levels of anti-IgA antibodies was similar to the total prevalence of the 10 other autoantibodies studied. These anti-IgA antibodies were mainly of the IgG class, except from one blood donor with IgM antibodies, and two patients, one with isolated IgA deficiency and the other with common variable immunodeficiency who had anti-IgA antibodies of the IgE class. The latter patient developed a near fatal anaphylactic reaction when intravenous gamma-globulin was administered. Most of the patients with severe adverse reactions to gamma-globulin did not present anti-IgA antibodies. Our data suggest that at least in some immunodeficient patients the elevated amounts of anti-IgA antibodies are not related to the administration of exogenous IgA. The importance of measuring anti-IgA antibodies of the IgG and IgE isotypes in IgA-deficient patients as well as in patients in treatment with gamma-globulin is emphasized.