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Natalia Shulzhenko

Publications and source records attributed to Natalia Shulzhenko.

11 recordsLinked to original sources
PubMed2006

A novel strategy for defining haplotypes by selective depletion using restriction enzymes.

Various single nucleotide polymorphisms (SNPs) have been investigated regarding association with gene expression levels or human diseases. Although different SNPs within one gene are frequently analyzed individually, it is highly probable that in the majority of the cases, a precise combination of SNP alleles, i.e., haplotype, determines a functional trait. Methods commonly used for haplotype determination, involving studies in families, cloning, or somatic cell hybrids, are expensive and time-consuming. We herein suggest a novel and simple strategy for haplotype determination, involving selective haplotype depletion with a restriction enzyme, followed by sequencing. We studied 11 LTA gene polymorphisms in 102 Brazilian individuals, and we applied this novel methodology for haplotyping 67 out of 70 LTA heterozygous individuals. We concluded that the method is rapid and efficient, and, as it includes only simple and widespread-used techniques, it could be used in most of the laboratories without further investment in equipments. The wider usage of haplotyping could be important to clarify contradictory results frequently observed among studies that focus on a single SNP.

Alleles↗
PubMed2006

Molecular profiling improves diagnoses of rejection and infection in transplanted organs.

The monitoring of transplanted hearts is currently based on histological evaluation of endomyocardial biopsies, a method that is fairly insensitive and that does not always accurately discriminate between rejection and infection in the heart. Accurate diagnosis of rejection and infection is absolutely crucial, however, as the respective treatments are completely different. Using microarrays, we analyzed gene expression in 76 cardiac biopsies from 40 heart recipients undergoing rejection, no rejection, or Trypanosoma cruzi infection. We found a set of genes whose expression patterns were typical of acute rejection, and another set of genes that discriminated between rejection and T cruzi infection. These sets revealed acute rejection episodes up to 2 weeks earlier, and trypanosome infection up to 2 months earlier than did histological evaluation. When applied to raw data from other institutions, the 2 sets of predictive genes were also able to accurately pinpoint acute rejection of lung and kidney transplants, as well as bacterial infections in kidneys. In addition to their usefulness as diagnostic tools, the data suggest that there are similarities in the biology of the processes involved in rejection of different grafts and also in the tissue responses to pathogens as diverse as bacteria and protozoa.

Bacterial Infections↗
PubMed2005

Selection of control genes for quantitative RT-PCR based on microarray data.

Use of internal reference gene(s) is necessary for adequate quantification of target gene expression by RT-PCR. Herein, we elaborated a strategy of control gene selection based on microarray data and illustrated it by analyzing endomyocardial biopsies with acute cardiac rejection and infection. Using order statistics and binomial distribution we evaluated the probability of finding low-varying genes by chance. For analysis, the microarray data were divided into two sample subsets. Among the first 10% of genes with the lowest standard deviations, we found 14 genes common to both subsets. After normalization using two selected genes, high correlation was observed between expression of target genes evaluated by microarray and RT-PCR, and in independent dataset by RT-PCR (r = 0.9, p < 0.001). In conclusion, we showed a simple and reliable strategy of selection and validation of control genes for RT-PCR from microarray data that can be easily applied for different experimental designs and tissues.

Algorithms↗
PubMed2005

Identification of new alternative splice events in the TCIRG1 gene in different human tissues.

Two transcript variants (TV) of the T cell immune regulator gene 1 (TCIRG1) have already been characterized. TV1 encodes a subunit of the osteoclast vacuolar proton pump and TV2 encodes a T cell inhibitory receptor. Based on the search in dbEST, we validated by RT-PCR six new alternative splice events in TCIRG1 in most of the 28 human tissues studied. In addition, we observed that transcripts using the TV1 transcription start site and two splice forms previously described in a patient with infantile malignant osteopetrosis are also expressed in various tissues of healthy individuals. Studies of these nine splice forms in cytoplasmic RNA of peripheral blood mononuclear cells showed that at least six of them could be efficiently exported from the nucleus. Since various products with nearly ubiquitous tissue distribution are generated from TCIRG1, this gene may be involved in other processes besides immune response and bone resorption.

Alternative Splicing↗
PubMed2005

[HLA polymorphism in a racially admixed sample of the population of Teresina, Piauí].

OBJECTIVE: To establish the frequencies of HLA-A, B, DRB1 and DQB1 specificities in a racially admixed sample of the city of Teresina, Piauí to characterize its genetic composition. METHODS: Polymerase chain reaction-sequence specific primers (PCR-SSP) were used to determine HLA-A, B, DRB1 and DQB1 specificities of 97 unrelated healthy racially admixed people of Teresina. The genotypic frequencies were estimated and compared to those described in samples of Brazilian Caucasian, Portuguese, Black and Amerindian populations using Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA). RESULTS: The frequencies of HLA-A, B, DRB1 and DQB1 specificities observed in the study sample were intermediate between Blacks and Caucasians and the typical elevation of HLA-specificities seen in the Amerindian race was not observed in the study population. The PCA and HCA analysis revealed that Teresina's racially admixed are very close to both Black and Caucasian and do not show similarities with the Amerindians. CONCLUSION: The genetic composition of Teresina's racially admixed is predominantly bi-hybrid of genes originated from Blacks and Caucasians with little contribution from Amerindian genes.

Brazil↗
PubMed2004

T cell receptor excision circles (TRECs) in relation to acute cardiac allograft rejection.

The purpose of this study was to quantify T cell receptor excision circles (TRECs) in blood mononuclear cells of cardiac transplant recipients and to investigate a possible relationship between TREC levels and rejection episodes. In addition, we investigated the correlation of TREC levels with age and also compared the levels between transplant recipients and healthy individuals. TREC levels were assessed by quantitative competitive PCR in 70 blood samples from 27 graft recipients and in 66 blood samples from 66 healthy individuals. The results showed: (1) higher TREC levels during rejection than during rejection-free periods (medians 9.0 vs. 0.3; p<0.001); (2) no suggestion of correlation with doses of prednisone or time after transplantation; (3) a negative correlation between TREC levels and age; and (4) lower TREC levels in cardiac recipients than in age-matched healthy controls. The value of blood TREC level measurements as an approach to rejection monitoring warrants future investigation.

Acute Disease↗
PubMed2004

Pre- and post-transplant anti-myosin and anti-heat shock protein antibodies and cardiac transplant outcome.

BACKGROUND: The purpose this study was to investigate the relationship of anti-myosin and anti-heat shock protein immunoglobulin G (IgG) serum antibodies to the original heart disease of cardiac transplant recipients, and also to rejection and patient survival after cardiac transplantation. METHODS: Anti-myosin and anti-heat shock protein (anti-hsp) IgG antibodies were evaluated in pre-transplant sera from 41 adult cardiac allograft recipients and in sequential post-transplant serum samples from 11 recipients, collected at the time of routine endomyocardial biopsies during the first 6 months after transplantation. In addition, the levels of these antibodies were determined from the sera of 28 healthy blood donors. RESULTS: Higher anti-myosin antibody levels were observed in pre-transplant sera than in sera from normal controls. Moreover, patients with chronic Chagas heart disease showed higher anti-myosin levels than patients with ischemic heart disease, and also higher levels, although not statistically significant, than patients with dilated cardiomyopathy. Higher anti-hsp levels were also observed in patients compared with healthy controls, but no significant differences were detected among the different types of heart diseases. Higher pre-transplant anti-myosin, but not anti-hsp, levels were associated with lower 2-year post-transplant survival. In the post-transplant period, higher anti-myosin IgG levels were detected in sera collected during acute rejection than in sera collected during the rejection-free period, whereas anti-hsp IgG levels showed no difference between these periods. CONCLUSIONS: The present findings are of interest for post-transplant management and, in addition, suggest a pathogenic role for anti-myosin antibodies in cardiac transplant rejection, as has been proposed in experimental models of cardiac transplantation.

Adult↗
PubMed2003

Associations between cytokine gene polymorphisms and recurrent pregnancy loss.

Since certain cytokines may play a role in unexplained recurrent pregnancy loss (RPL) and also some cytokine gene polymorphisms may affect the level of cytokine production, the aim of the present study was to investigate the relationship between RPL and polymorphisms of the genes coding for TNF-alpha (-308 G-->A), IL-10 (-1082 G-->A), IL-6 (-174 G-->C), and IFN-gamma (+874 A-->T). Genotyping was performed in 48 RPL women and 108 ethnically matched healthy individuals. In addition, we performed a meta-analysis encompassing the present results and those from studies on the association of TNF-alpha, IL-10 and IFN-gamma polymorphisms with RPL published in the literature until December 2001. The results showed: (1) no evidence of association with IL-6 gene polymorphisms; (2) significant associations, revealed by the meta-analysis, with the high cytokine production genotypes of IFN-gamma (+874 T/T: odds ratio (OR)=1.92, P=0.04) and IL-10 (-1082 G/G: OR=1.75, P=0.03), and a trend for association with the high TNF-alpha production genotypes -308 A/A and A/G (OR=1.61; P=0.18). We believe that the associations of these genotypes with RPL are interesting not only as risk factors but also because they represent another piece of evidence that these cytokines might be important in the pathogenesis of RPL.

Abortion, Habitual↗
PubMed2003

Blood and intragraft CD27 gene expression in cardiac transplant recipients.

The present study investigated gene expression of costimulatory molecule CD27 in relation to the occurrence of acute cardiac rejection. CD27 transcripts were measured by means of quantitative competitive reverse transcriptase-polymerase chain reaction in 120 endomyocardial biopsies and in 89 samples of blood mononuclear cells from 31 recipients. Higher levels of CD27 transcripts were observed in biopsies with rejection than in samples without rejection (medians, 7.1 and 1.9; P = 0.06). In contrast, blood mononuclear cells collected during rejection showed lower levels than blood mononuclear cells from rejection-free periods (medians, 3.3 vs. 7.9; P = 0.03). Considering only endomyocardial biopsies without rejection, the values were lower in samples from recipients who did not present any rejection during the first 6 months after transplantation than in those from recipients who had at least one rejection during the same period (medians, 0 vs. 3.5, P < 0.001; percentage of biopsies expressing CD27, 44% vs. 77%). In conclusion, the presence of intragraft CD27 mRNA may identify recipients at risk for developing acute rejection.

Biopsy↗
PubMed2003

Specificity of alternative splice form detection using RT-PCR with a primer spanning the exon junction.

A recently described strategy for splice variant specific detection by RT-PCR is based on the use of a primer spanning the junction between exons of the alternative splice form. However, this reaction may generate false-positive results in the presence of excess principal transcript. In this report, transcript variant 3 of T cell immune regulator gene 1 was used as a model to demonstrate a new method to ensure PCR specificity. Our approach permits the determination of detection specificity considering the full-length transcript amount. Furthermore, we demonstrated that the addition of a few molecules of a specific template dramatically increases the specificity of the reaction and allows for the detection of the alternative form, even in the presence of large amounts of the principal transcript. Competitor DNA for the alternative splice form is suggested as the specific template to achieve the detection specificity. Thus, we describe a simple strategy to avoid nonspecific amplifications for RT-PCR using a primer spanning the exon junction.

Alternative Splicing↗