Investigation of single-strand conformational polymorphism of the TP53 gene in women with a family history of breast cancer

Breast cancer in families with germ line mutations in the TP53 gene has been described in the medical literature. Mutation screening for susceptibility genes should allow effective prophylactic and preventive measures. Using single-strand conformational polymorphism, we screened for mutations in exons 5, 6, 7 and 8 of gene TP53 in the peripheral blood of 8 young non-affected members (17 to 36 years old) of families with a history of breast cancer. Studies of this type on young patients (mean age, 25 years) are very rare in the literature. The identification of these mutations would contribute to genetic counseling of members of families with predisposition to breast cancer. The results obtained did not show any polymorphism indicating mutation. In our sample, the familial tumorigenesis is probably related to other gene etiologies.

Detection of somatic mutations of the PIG-A gene in Brazilian patients with paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal syndrome characterized by intravascular hemolysis mediated by complement, thrombotic events and alterations in hematopoiesis. Basically, the molecular events which underlie the complexity of the syndrome consist of the absence of the glycosylphosphatidylinositol (GPI) anchor as a consequence of somatic mutations in the PIG-A gene, located on the X chromosome. The GPI group is responsible for the attachment of many proteins to the cytoplasmic membrane. Two of them, CD55 and CD59, have a major role in the inhibition of the action of complement on the cellular membrane of blood cells. The absence of GPI biosynthesis can lead to PNH. Since mutations in the PIG-A gene are always present in patients with PNH, the aim of this study was to characterize the mutations in the PIG-A gene in Brazilian patients. The analysis of the PIG-A gene was performed using DNA samples derived from bone marrow and peripheral blood. Conformation-sensitive gel electrophoresis was used for screening the mutation and sequencing methods were used to identify the mutations. Molecular analysis permitted the identification of three point mutations in three patients: one G->A transition in the 5' portion of the second intron, one T->A substitution in the second base of codon 430 (Leu430->stop), and one deletion deltaA in the third base of codon 63. This study represents the first description of mutations in the PIG-A gene in a Brazilian population.

HHV-8 infection in patients with AIDS-related Kaposi's sarcoma in Brazil

The aims of the present study were to determine the prevalence of human herpesvirus type 8 (HHV-8) in HIV-positive Brazilian patients with (HIV+/KS+) and without Kaposi's sarcoma (HIV+/KS-) using PCR and immunofluorescence assays, to assess its association with KS disease, to evaluate the performance of these tests in detecting HHV-8 infection, and to investigate the association between anti-HHV-8 antibody titers, CD4 counts and staging of KS disease. Blood samples from 66 patients, 39 HIV+/KS+ and 27 HIV+/KS-, were analyzed for HHV-8 viremia in peripheral blood mononuclear cells by PCR and HHV-8 antigenemia for latent and lytic infection by immunofluorescence assay. Positive samples for latent nuclear HHV-8 antigen (LNA) antibodies were titrated out from 1/100 to 1/409,600 dilution. Clinical information was collected from medical records and risk behavior was assessed through an interview. HHV-8 DNA sequences were detected by PCR in 74.3% of KS+ patients and in 3.7% of KS- patients. Serological assays were similar in detecting anti-LNA antibodies and anti-lytic antigens in sera from KS+ patients (79.5%) and KS- patients (18.5%). HHV-8 was associated with KS whatever the method used, i.e., PCR (odds ratio (OR) = 7.4, 95% confidence interval (CI) = 2.16-25.61) or anti-LNA and anti-lytic antibodies (OR = 17.0, 95%CI = 4.91-59.14). Among KS+ patients, HHV-8 titration levels correlated positively with CD4 counts (rho 0.48, P = 0.02), but not with KS staging. HHV-8 is involved in the development of KS in different geographic areas worldwide, as it is in Brazil, where HHV-8 is more frequent among HIV+ patients. KS severity was associated with immunodeficiency, but no correlation was found between HHV-8 antibody titers and KS staging.

Lack of evidence for superantigen activity of Toxoplasma gondii towards human T cells

Toxoplasma gondii is an obligatory intracellular parasite whose life cycle may include man as an intermediate host. More than 500 million people are infected with this parasite worldwide. It has been previously reported that T. gondii contains a superantigen activity. The purpose of the present study was to determine if the putative superantigen activity of T. gondii would manifest towards human T cells. Peripheral blood mononuclear cells (PBMC) from individuals with no previous contact with the parasite were evaluated for proliferation as well as specific Vß expansion after exposure to Toxoplasma antigens. Likewise, PBMC from individuals with the congenital infection were evaluated for putative Vß family deletions in their T cell repertoire. We also evaluated, over a period of one year, the PBMC proliferation pattern in response to Toxoplasma antigens in patients with recently acquired infection. Some degree of proliferation in response to T. gondii was observed in the PBMC from individuals never exposed to the parasite, accompanied by specific Vß expansion, suggesting a superantigen effect. However, we found no specific deletion of Vß (or Valpha) families in the blood of congenitally infected individuals. Furthermore, PBMC from recently infected individuals followed up over a period of one year did not present a reduction of the Vß families that were originally expanded in response to the parasite antigens. Taken together, our data suggest that T. gondii does not have a strong superantigen activity on human T cells.

Comparison of the quantitative competitive and semiquantitative RT-PCR methods for the determination of interferon-gamma mRNA levels in AIDS-free HIV-infected individuals

IFN-gamma mRNA expression was evaluated in nonstimulated peripheral blood mononuclear cells (PBMC) of HIV-infected and seronegative individuals using quantitative competitive and semiquantitative RT-PCR and the sensitivity of these methods was compared. A significant correlation was found between quantitative competitive and semiquantitative RT-PCR in samples of both HIV-seronegative (P = 0.004) and HIV-infected individuals (P = 0.0004). PBMC from HIV-infected individuals presented a remarkable increase of IFN-gamma mRNA expression, as determined by both types of RT-PCR methods. Semiquantitative RT-PCR even without an internal standard is also acceptable for measuring cytokine mRNA expression, but less reliable if small amounts are quantified. Moreover, we found that increased IFN-gammamRNA expression is independent of CD4+ cell count in AIDS-free HIV-infected patients.

Correlation of mixed lymphocyte culture with chronic graft-versus-host disease following allogeneic stem cell transplantation

The purpose of the present study was to evaluate the mixed lymphocyte culture as a predictive assay of acute and chronic graft-versus-host disease (GVHD). We studied 153 patients who received a first bone marrow transplantation from human leukocyte antigen-identical siblings. Acute GVHD was observed in 26 of 128 (20.3%) patients evaluated and chronic GVHD occurred in 60 of 114 (52.6%). One-way mixed lymphocyte culture (MLC) assays were performed by the standard method. MLC results are reported as the relative response (RR) from donor against patient cells. The responses ranged from -47.0 to 40.7%, with a median of 0.5%. The Kaplan-Meier probability of developing GVHD was determined for patients with positive and negative MLC. There was no significant difference in incidence of acute GVHD between the groups studied. However, the incidence of chronic GVHD was higher in recipients with RR >4.5% than in those with RR <=4.5%. The Cox Proportional Hazards model was used to examine the effect of MLC levels on incidence of chronic GVHD, while adjusting for the potential confounding effect of others suspected or observed risk factors. The relative risk of chronic GVHD was 2.5 for patients with positive MLC (RR >4.5%), 2.9 for those who received peripheral blood progenitor cells as a graft, and 2.2 for patients who developed previous acute GVHD. MLC was not useful for predicting acute GVHD, but MLC with RR >4.5% associated with other risk factors could predict the development of chronic GVHD, being of help for the prevention and/or treatment of this late complication.

Association of apolipoprotein E polymorphism in late-onset Alzheimer's disease and vascular dementia in Brazilians

The genetic basis for dementias is complex. A common polymorphism in the apolipoprotein E (APOE) gene is considered to be the major risk factor in families with sporadic and late-onset Alzheimer's disease as well as in the general population. The distribution of alleles and genotypes of the APOE gene in late-onset Alzheimer's disease (N = 68), other late-life dementias (N = 39), and in cognitively normal controls (N = 58) was determined, as also was the risk for Alzheimer's disease associated with the epsilon4 allele. Peripheral blood samples were obtained from a total of 165 individuals living in Brazil aged 65-82 years. Genomic DNA was amplified by the polymerase chain reaction and the products were digested with HhaI restriction enzyme. APOE epsilon2 frequency was considerably lower in the Alzheimer's disease group (1%), and the epsilon3 allele and epsilon3/epsilon3 genotype frequencies were higher in the controls (84 and 72%, respectively) as were the epsilon4 allele and epsilon3/epsilon4 genotype frequencies in Alzheimer's disease (25 and 41%, respectively). The higher frequency of the epsilon4 allele in Alzheimer's disease confirmed its role as a risk factor, while epsilon2 provided a weak protection against development of the disease. However, in view of the unexpectedly low frequency of the epsilon4 allele, additional analyses in a more varied Brazilian sample are needed to clarify the real contribution of apolipoprotein E to the development of Alzheimer's disease in this population.

Identification of Mycobacterium bovis antigens by analysis of bovine T-cell responses after infection with a virulent strain

Purification and characterization of individual antigenic proteins are essential for the understanding of the pathogenic mechanisms of mycobacteria and the immune response against them. In the present study, we used anion-exchange chromatography to fractionate cell extracts and culture supernatant proteins from Mycobacterium bovis to identify T-cell-stimulating antigens. These fractions were incubated with peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle in lymphoproliferation assays. This procedure does not denature proteins and permits the testing of mixtures of potential antigens that could be later identified. We characterized protein fractions with high stimulation indices from both culture supernatants and cell extracts. Proteins were identified by two-dimensional gel electrophoresis followed by N-terminal sequencing or MALDI-TOF. Culture supernatant fractions containing low molecular weight proteins such as ESAT6 and CFP10 and other proteins (85B, MPB70), and the novel antigens TPX and TRB-B were associated with a high stimulation index. These results reinforce the concept that some low molecular weight proteins such as ESAT6 and CFP10 play an important role in immune responses. Also, Rv3747 and L7/L12 were identified in high stimulation index cell extract fractions. These data show that protein fractions with high lymphoproliferative activity for bovine PBMC can be characterized and antigens which have been already described and new protein antigens can also be identified in these fractions.

Patterns of intracellular cytokines in CD4 and CD8 T cells from patients with mycobacterial infections

Using a short-term bulk culture protocol designed for an intracellular-staining method based on a flow cytometry approach to the frequencies of cytokine-producing cells from tuberculosis and leprosy patients, we found distinct patterns of T cell subset expression. The method also reveals the profile of peak cytokine production and can provide simultaneous information about the phenotype of cytokine-producing cells, providing a reliable assay for monitoring the immunity of these patients. The immune response of Mycobacterium leprae and purified protein derivative (PPD) in vitro to a panel of mycobacteria-infected patients from an endemic area was assessed in primary mononuclear cell cultures. The kinetics and source of the cytokine pattern were measured at the single-cell level. IFN-gamma-, TNF-alpha-, IL-4- and IL-10-secreting T cells were intracytoplasmic evaluated in an attempt to identify M. leprae- and PPD-specific cells directly from the peripheral blood. The analysis by this approach indicated that TNF-alpha was the first (8 h) to be produced, followed by IFN-gamma (16 h), IL-10 (20 h) and IL-4 (24 h), and double-staining experiments confirmed that CD4+ were a greater source of TNF-alpha than of CD8+ T cells (P < 0.05). Both T cell subsets secreted similar amounts of IFN-gamma. We conclude that the protocol permits rapid evaluation of cytokine production by different T cell populations. The method can also be used to define immune status in non-infected and contact individuals.

Superoxide release and cellular gluthatione peroxidase activity in leukocytes from children with persistent asthma

Asthma is an inflammatory condition characterized by the involvement of several mediators, including reactive oxygen species. The aim of the present study was to investigate the superoxide release and cellular glutathione peroxidase (cGPx) activity in peripheral blood granulocytes and monocytes from children and adolescents with atopic asthma. Forty-four patients were selected and classified as having intermittent or persistent asthma (mild, moderate or severe). The spontaneous or phorbol myristate acetate (PMA, 30 nM)-induced superoxide release by granulocytes and monocytes was determined at 0, 5, 15, and 25 min. cGPx activity was assayed spectrophotometrically. The spontaneous superoxide release by granulocytes from patients with mild (N = 15), moderate (N = 12) or severe (N = 6) asthma was higher at 25 min compared to healthy individuals (N = 28, P < 0.05, Duncan test). The PMA-induced superoxide release by granulocytes from patients with moderate (N = 12) or severe (N = 6) asthma was higher at 15 and 25 min compared to healthy individuals (N = 28, P < 0.05 in both times of incubation, Duncan test). The spontaneous or PMA-induced superoxide release by monocytes from asthmatic patients was similar to healthy individuals (P > 0.05 in all times of incubation, Duncan test). cGPx activity of granulocytes and monocytes from patients with persistent asthma (N = 20) was also similar to healthy individuals (N = 10, P > 0.05, Kruskal-Wallis test). We conclude that, under specific circumstances, granulocytes from children with persistent asthma present a higher respiratory burst activity compared to healthy individuals. These findings indicate a risk of oxidative stress, phagocyte auto-oxidation, and the subsequent release of intracellular toxic oxidants and enzymes, leading to additional inflammation and lung damage in asthmatic children.

Human T-cell lymphotropic virus type I (HTLV-I) proviral DNA viral load among asymptomatic patients and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis

To evaluate the human T-cell lymphotropic virus type I (HTLV-I) proviral DNA load among asymptomatic HTLV-I-infected carriers and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), real time PCR using TaqMan probes for the pol gene was performed in two million peripheral blood mononuclear cells (PBMC). The albumin gene was the internal genomic control and MT2 cells were used as positive control. The results are reported as copies/10,000 PBMC, and the detection limit was 10 copies. A total of 89 subjects (44 HAM/TSP and 45 healthy HTLV-I-infected carriers) followed up at the Institute of Infectious Diseases "Emilio Ribas" and in the Neurology Division of Hospital of Clínicas were studied. The asymptomatic HTLV-I-infected carriers had a median number of 271 copies (ranging from 5 to 4756 copies), whereas the HAM/TSP cases presented a median of 679 copies (5-5360 copies) in 10,000 PBMC. Thus, HAM/TSP patients presented a significantly higher HTLV-I proviral DNA load than healthy HTLV-I carriers (P = 0.005, one-way Mann-Whitney test). As observed in other persistent infections, proviral DNA load quantification may be an important tool for monotoring HTLV-I-infected subjects. However, long-term follow-up is necessary to validate this assay in the clinical setting.

Asynchronous expression of myeloid antigens in leukemic cells in a PML/RARalpha transgenic mouse model

Acute promyelocytic leukemia (APL) is characterized by the expansion of blasts that resemble morphologically promyelocytes and harbor a chromosomal translocation involving the retinoic acid receptor a (RARa) and the promyelocytic leukemia (PML) genes on chromosomes 17 and 15, respectively. The expression of the PML/RARa fusion gene is essential for APL genesis. In fact, transgenic mice (TM) expressing PML/RARa develop a form of leukemia that mimics the hematological findings of human APL. Leukemia is diagnosed after a long latency (approximately 12 months) during which no hematological abnormality is detected in peripheral blood (pre-leukemic phase). In humans, immunophenotypic analysis of APL blasts revealed distinct features; however, the precise immunophenotype of leukemic cells in the TM model has not been established. Our aim was to characterize the expression of myeloid antigens by leukemic cells from hCG-PML/RARa TM. In this study, TM (N = 12) developed leukemia at the mean age of 13.1 months. Morphological analysis of bone marrow revealed an increase of the percentage of immature myeloid cells in leukemic TM compared to pre-leukemic TM and wild-type controls (48.63 ± 16.68, 10.83 ± 8.11, 7.4 ± 5.46%, respectively; P < 0.05). Flow cytometry analysis of bone marrow and spleen from leukemic TM identified the asynchronous co-expression of CD34, CD117, and CD11b. This abnormal phenotype was rarely detected prior to the diagnosis of leukemia and was present at similar frequencies in hematologically normal TM and wild-type controls of different ages. The present results demonstrate that, similarly to human APL, leukemic cells from hCG-PML/RARa TM present a specific immunophenotype.

Secondary prevention of type 1 diabetes mellitus: stopping immune destruction and promoting ß-cell regeneration

Type 1 diabetes mellitus results from a cell-mediated autoimmune attack against pancreatic ß-cells. Traditional treatments involve numerous daily insulin dosages/injections and rigorous glucose control. Many efforts toward the identification of ß-cell precursors have been made not only with the aim of understanding the physiology of islet regeneration, but also as an alternative way to produce ß-cells to be used in protocols of islet transplantation. In this review, we summarize the most recent studies related to precursor cells implicated in the regeneration process. These include embryonic stem cells, pancreas-derived multipotent precursors, pancreatic ductal cells, hematopoietic stem cells, mesenchymal stem cells, hepatic oval cells, and mature ß-cells. There is controversial evidence of the potential of these cell sources to regenerate ß-cell mass in diabetic patients. However, clinical trials using embryonic stem cells, umbilical cord blood or adult bone marrow stem cells are under way. The results of various immunosuppressive regimens aiming at blocking autoimmunity against pancreatic ß-cells and promoting ß-cell preservation are also analyzed. Most of these regimens provide transient and partial effect on insulin requirements, but new regimens are beginning to be tested. Our own clinical trial combines a high dose immunosuppression with mobilized peripheral blood hematopoietic stem cell transplantation in early-onset type 1 diabetes mellitus.

Adhesion molecule profiles of B-cell non-Hodgkin's lymphomas in the leukemic phase

We evaluated the expression of 10 adhesion molecules on peripheral blood tumor cells of 17 patients with chronic lymphocytic leukemia, 17 with mantle-cell lymphoma, and 13 with nodal or splenic marginal B-cell lymphoma, all in the leukemic phase and before the beginning of any therapy. The diagnosis of B-cell non-Hodgkin's lymphomas was based on cytological, histological, immunophenotypic, and molecular biology methods. The mean fluorescence intensity of the adhesion molecules in tumor cells was measured by flow cytometry of CD19-positive cells and differed amongst the types of lymphomas. Comparison of chronic lymphocytic leukemia and mantle-cell lymphoma showed that the former presented a higher expression of CD11c and CD49c, and a lower expression of CD11b and CD49d adhesion molecules. Comparison of chronic lymphocytic leukemia and marginal B-cell lymphoma showed that the former presented a higher expression of CD49c and a lower expression of CD11a, CD11b, CD18, CD49d, CD29, and CD54. Finally, comparison of mantle-cell lymphoma and marginal B-cell lymphoma showed that marginal B-cell lymphoma had a higher expression of CD11a, CD11c, CD18, CD29, and CD54. Thus, the CD49c/CD49d pair consistently demonstrated a distinct pattern of expression in chronic lymphocytic leukemia compared with mantle-cell lymphoma and marginal B-cell lymphoma, which could be helpful for the differential diagnosis. Moreover, the distinct profiles of adhesion molecules in these diseases may be responsible for their different capacities to invade the blood stream.

Techniques used to identify the Brazilian variant of HIV-1 subtype B

The purpose of the present study was to compare the sensitivity and specificity of V3 enzyme immunoassay (solid phase EIA and EIA inhibition) and restriction fragment length polymorphism (RFLP) with the DNA sequencing "gold standard" to identify the Brazilian HIV-1 variants of subtype B and B"-GWGR. Peripheral blood mononuclear cells were collected from 61 HIV-1-infected individuals attending a clinic in São Paulo. Proviral DNA was amplified and sequentially cleaved with the Fok I restriction enzyme. Plasma samples were submitted to a V3-loop biotinylated synthetic peptide EIA. Direct partial DNA sequencing of the env gene was performed on all samples. Based on EIA results, the sensitivity for detecting B-GPGR was 70%, compared to 64% for the Brazilian variant B"-GWGR while, the specificity of B-GPGR detection was 85%, compared to 88% for GWGR. The assessment of RFLP revealed 68% sensitivity and 94% specificity for the B-GPGR strain compared to 84 and 90% for the B"-GWGR variant. Moreover, direct DNA sequencing was able to detect different base sequences corresponding to amino acid sequences at the tip of the V3 loop in 22 patients. These results show a similar performance of V3 serology and RLFP in identifying the Brazilian variant GWGR. However, V3 peptide serology may give indeterminate results. Therefore, we suggest that V3 serology be used instead of DNA sequencing where resources are limited. Samples giving indeterminate results by V3 peptide serology should be analyzed by direct DNA sequencing to distinguish between B-GPGR and the Brazilian variant B"-GWGR.