[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.

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.

Agammaglobulinemia ligada al sexo. Reconstitución in vitro / No disponible

No disponible

Agammaglobulinemia ligada al sexo. Reconstitución in vitro / No disponible

No disponible

Major histocompatibility complex haplotypes in Spanish immunoglobulin A deficiency patients: a comparative fine mapping microsatellite study.

The most consistent finding in Immunoglobulin A deficiency (IgAD) genetics is the presence of susceptibility factors located in the major histocompatibility complex (MHC). We have described the existence of at least two distinct susceptibility genes in the MHC present in different haplotypes. The aim of the present study was to locate with precision the susceptibility genes present in DR1- and DR7-positive haplotypes, taking advantage of their structural diversity, as opposed to the conserved nature of the DR3-extended susceptibility haplotype (DR3/B8), that hampers a more exhaustive scrutiny. A detailed analysis with 20 markers along the MHC in the 400 haplotypes present in 100 IgAD families, with special density at Class II locations, was performed to define the minimal shared susceptibility region present in all haplotypes carrying DR1 and, on the other hand, in all DR7-positive haplotypes. A comparison of the fine microsatellite allele structure of DR-extended haplotypes in the Spanish population with those described for Swedish and British families revealed no difference in DRB1*0101 and DRB1*0102 haplotypes between both populations. Our data suggest that the etiologic mutation present in DRB1*0101 and DRB1*0102 in North Europe (Sweden and UK) is missing in the Spanish DRB1*0101 haplotypes but is present in the DQB1/DRB1 region in DRB1*0102 haplotypes. The results obtained also indicated that the most likely susceptibility gene in the DR7 haplotypes is either DQA1 or DRB1.

Three novel mutations reflect the variety of defects causing phenotypically diverse X-linked hyper-IgM syndrome.

X-linked hyper-IgM syndrome (HIGM1) (MIM musical sharp 308230), is a severe primary immunodeficiency caused by mutations in the gene coding for CD40 ligand (CD40L or CD154), a member of the tumour necrosis factor (TNF) superfamily. The interaction of this protein with its ligand, CD40, mediates crucial processes in the immune response. The variety of defects that have been described in HIGM1 patients range from a complete lack of CD40L protein expression to missense mutations that interfere with its interaction with CD40L. In this study we describe three families - a total of seven HIGM1 patients and carriers, presenting a spectrum of severity in clinical evolution. In two of these families, patient DNA samples were available for genetic studies. In the third, carrier detection was performed on female family members. The results of immunological studies - the different patterns of CD40L expression and binding capacity as measured by flow cytometry - and molecular diagnosis are presented. Three novel mutations were identified: an intron mutation that partially interferes with the splicing process (intron 3, position + 5 G/T); a missense mutation (Ser222 Phe) located in the molecular region which interacts with the receptor and which abrogates binding capacity; and a 14 base pair deletion leading to a frameshift and a premature truncated mutation (del I 171 X 195). An attempt to correlate protein expression and function of the CD40L mutants with clinical disease evolution is described.

Epistatic effects occurring among susceptibility and protective MHC genes in IgA deficiency.

Immunoglobulin A deficiency (IgAD), the most prevalent primary immunodeficiency in Caucasian populations, shows strong evidence of polygenic inheritance with several associated genes being located in the major histocompatibility complex (MHC). Our aims were to determine which previously described MHC associations were primary and not secondary to a decrease or an increase in other MHC haplotype frequencies, to study the genetic interactions between all disease-associated MHC haplotypes and, finally, to ascertain the relative importance of protection vs susceptibility. A relative predispositional effect (RPE) study showed that in addition to the primary positive association of IgAD with HLA-DRB1*0102, DR3/TNFa2b3, and DR7 carrying haplotypes, DRB1*1501 was a marker of a primary protective factor in the Spanish population. Our data also indicate that the combined presence in an individual of two MHC susceptibility haplotypes notably increases the predisposition to the disease and that DRB1*1501 positive haplotypes eliminate the susceptibility conferred by any other MHC haplotype.

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.

Characterization of Herpesvirus saimiri-transformed T lymphocytes from common variable immunodeficiency patients.

Common variable immunodeficiency (CVID) is a very frequent but heterogeneous syndrome of antibody formation. The primary defect remains unknown, but many reports describe peripheral blood T lymphocyte dysfunctions in a substantial proportion of CVID patients, which may impair T--B cell collaboration. In order to investigate whether such putative defects were intrinsic to T cells or, rather, secondary to quantitative differences in T cell subset distribution, or to other described disorders, we have used Herpesvirus saimiri (HVS) for the targeted transformation of CVID CD4+ and CD8+ T cells and subsequent functional evaluation by flow cytometry of their capacity to generate cell surface (CD154, CD69) or soluble (IL-2, TNF-alpha, IFN-gamma) help after CD3 engagement. Unexpectedly, the results showed that 40 different CVID blood samples exposed to HVS gave rise with a significantly increased frequency to transformed CD4+ T cell lines, compared to 40 age-matched controls (27% versus 3%, P < or = 0.00002) suggesting the existence of a CVID-specific signalling difference which affects CD4+ cell transformation efficiency. The functional analysis of 10 CD4+ and 15 CD8+ pure transformed T cell lines from CVID patients did not reveal any statistically significant difference as compared to controls. However, half of the CD4+ transformed cell lines showed CD154 (but not CD69) induction (mean value of 46.8%) under the lower limit of the normal controls (mean value of 82.4%, P < or = 0.0001). Exactly the same five cell lines showed, in addition, a significantly low induction of IL-2 (P < or = 0.04), but not of TNF-alpha or IFN-gamma. None of these differences were observed in the remaining CD4+ cell lines or in any of the transformed CD8+ cell lines. We conclude that certain CVID patients show selective and intrinsic impairments for the generation of cell surface and soluble help by CD4+ T cells, which may be relevant for B lymphocyte function. The transformed T cell lines will be useful to establish the biochemical mechanisms responsible for the described impairments.

Soluble HLA class I antigens in serum and synovial fluid from patients with rheumatoid arthritis and other arthropathies.

OBJECTIVES: To investigate the presence of soluble HLA class I (s-HLA) antigens in serum and synovial fluid (SF) from a large cohort of rheumatic patients. METHODS: We studied clinical and analytical data and serum samples from 300 patients [122 patients with rheumatoid arthritis (RA), 38 with osteoarthritis or osteoporosis, 29 with seronegative spondyloarthropathies, 45 patients with other rheumatic diseases] and 66 healthy controls. In addition, we studied 25 paired samples of serum and SF from these groups of subjects. In RA patients, we examined whether the levels of s-HLA in serum and SF were related to the activity of the disease. RESULTS: The mean concentrations of s-HLA molecules in serum were slightly higher in RA patients (1.2 microg/ml) than in the other four groups (1.08, 1.01, 1.09 and 0.94 microg/ml respectively). We found no correlation between serum s-HLA levels and any variable of inflammatory disease activity in RA patients. s-HLA molecules were found in SF and at levels that correlated with those found in serum (P=0.04; r=0.4). Furthermore, s-HLA levels were higher in SF from patients with RA (1.3 microg/ml) or crystal-induced arthritis (0.98 microg/ml) than in SF from those with osteoarthritis (0.38 microg/ml) (P<0.05 and P<0.005 respectively), and these levels were correlated inversely and significantly with the score on the visual analogue scale of pain (P=0.02), the number of painful joints (P=0.05) and the level of C-reactive protein (P=0.03) in RA patients. CONCLUSIONS: This is the first report to demonstrate the presence of s-HLA molecules in SF at levels that correlate with serum levels. The mean levels of s-HLA molecules were significantly higher in SF from patients with RA and crystal-induced arthritis than in SF from cases of osteoarthritis, and correlated inversely with certain variables of disease activity in RA patients.

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.

[Molecular diagnosis of primary immunodeficiencies]. / Diagnóstico molecular de inmunodeficiencias primarias.

Knowledge of the molecular defects responsible for some primary immunodeficiency diseases (PIDs) offers undoubted advantages in establishing a reliable diagnosis. Such knowledge would allow us not only to establish a prognosis but also to instigate the most appropriate therapy. After molecular diagnosis, some patients could benefit from gene therapy. However, apart from the diagnosis of the disease, molecular biological techniques also enable more reliable identification of carriers and, when suggested by the family history and when the familial defect is already known, prenatal diagnosis will also be possible, thus establishing the earliest possible treatment. Using the single-stranded conformational polymorphism technique followed by direct sequencing, we found 22 different mutations in 22 patients from unrelated families and with a phenotype compatible with x-linked agammaglobulinemia. Fourteen of these are new, previously undescribed mutations and the remaining eight are already included in the data base (http://www.uta.fi/imt/bioinfo/Btkbase). Analysis of the female carrier was performed in all the mothers and the mutation was de novo in only one patient. Study of the BtK gene enabled differential diagnosis with common variable immunodeficiency disease in some patients who showed absent or very low lymphocyte B counts as well as forms of autosomal recessive agammaglobulinemia. Using the same techniques, we were able to identify mutations in the CD40 ligand gene in three families in which one of the members had clinical and biological phenotype compatible with X-linked hyper-IgM. Molecular diagnosis was very useful in identifying carriers in these families as well as in making the differential diagnosis among patients with common variable immunodeficiency disease. Purely on this were we able to provide appropriate genetic counseling.

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.

Tumor necrosis factor genomic polymorphism in Spanish IGA deficiency patients.

Selective IgA deficiency (IgAD) is the most common form of primary immunodeficiency. Its association with genes within the major histocompatibility complex (MHC) has been repeatedly reported. Recently the susceptibility gene has been located in the class III region, around the tumor necrosis factor (TNF) cluster. In this study we have examined IgAD association with TNF-alpha gene promoter polymorphisms and TNFa and b microsatellites. No significant association was found with the former polymorphisms and the observed associations with TNFa2 allele and haplotypes TNFa2b1 and TNFa2b3 were proven to be secondary to their occurrence on the B14-DR1 and B8-DR3 haplotypes, previously reported to be associated with susceptibility to IgAD. However, a primary negative (protective) association was found between the TNFa10 allele and IgAD.

[Neutropenia as early manifestation of X-linked agammaglobulinemia. Report on 4 patients]. / Neutropenia como manifestación precoz de una agammaglobulinemia ligada al cromosoma X. Descripción de cuatro pacientes.

OBJECTIVE: The aim of this study was to determine the frequency of neutropenia associated to X-linked agammaglobulinemia (XLA) and to describe the clinical characteristics of the children diagnosed in our unit. PATIENTS AND METHODS: A revision of the medical records registered in our unit during a 28 year period (1970-1998) according to the diagnostic criteria of XLA was performed. We included in the study group those patients that expressed a neutropenia. Immunological studies by standard techniques were performed. RESULTS: Of the 37 patients fulfilling the diagnostic criteria of XLA, 4 cases had experienced episodes of neutropenia (10.81%). The frequency of neutropenia within the group without familiar antecedents was 15% and within the group with familiar antecedents 5.88%. In all cases, the neutropenia was present during a serious acute infectious disease. The neutropenia was transient and resolved promptly after the onset of antibiotic therapy in all patients. None of the patients experienced neutropenia while under therapy with intravenous gammaglobulin. CONCLUSIONS: The association of XLA and neutropenia seems to be sufficiently frequent as to include it in the differential diagnosis of neutropenia during infancy. It is important to consider a primary immunodeficiency diagnosis when a child presents neutropenia and a serious acute infectious disease. Quantification of serum immunoglobulin levels and enumeration of lymphocyte subpopulations can lead to an early diagnosis.