Cristiane Conceição Chagas Rudnick

Influence of TNF and IL10 gene polymorphisms in the immunopathogenesis of leprosy in the south of Brazil

OBJECTIVEnTo determine whether cytokine polymorphisms are associated with leprosy and/or their subtypes in a Brazilian population.nnnMETHODSnGenotyping using polymerase chain reaction with sequence-specific primers (PCR-SSP) was performed for: TNF(-308/-238), IL2(-330/+166), IL6(-174), IFNG(+874), TGFB1(+869/+915), and IL10(-592/-819/-1082) in 240 healthy controls and 167 patients with leprosy.nnnRESULTSnFor TNF(-308), a higher frequency of GG genotype (85.5% vs. 74.1% in healthy controls, p = 0.009), along with a decreased frequency of GA/AA genotypes was observed among leprosy patients as compared to the control group (14.5% vs. 25.9%, p = 0.009). The GG genotype was particularly higher in patients with tuberculoid (TT) and borderline (BB) leprosy (90.5% and 89.8%, respectively). Analysis of IL10 genotypes revealed a lower frequency of GCC/GCC haplotype in lepromatous leprosy (LL) patients (6.2%) in comparison to controls (15.4%).nnnCONCLUSIONnIt is suggested that the G-->A substitution at position -308 in the TNF promoter region plays an important role in leprosy patients.

Association between killer‐cell immunoglobulin‐like receptor genotypes and leprosy in Brazil

The aim of this study was to investigate the role of killer cell immunoglobulin-like receptor (KIR) genes in leprosy immunopathogenesis. Genotyping of KIR and human leukocyte antigen (HLA) genes was performed by polymerase chain reaction with sequence-specific oligonucleotide probes in 165 leprosy patients. Both activating KIR2DS2 and KIR2DS3 frequencies were higher in tuberculoid leprosy (TT) patients than in lepromatous leprosy (LL) patients, and the inhibitory KIR with its ligand, KIR2DL1-C2/C2, was elevated in TT patients in comparison to all other leprosy subgroups and controls. However, a negative association between KIR2DL3-C1 and KIR2DL3-C1/C1 and the TT group was identified. Borderline patients exhibited a higher frequency of KIR3DL2-A3/11 than the controls and LL patients, and a lower frequency of KIR2DL1-C2 than the controls and TT subgroup. Some KIR-HLA genotypes could be associated to the development of clinical forms of leprosy and should be investigated further.

Killer cell immunoglobulin-like receptor gene diversity in a Southern Brazilian population from the state of Paraná

Killer cell immunoglobulin-like receptors (KIR) are encoded by polymorphic genes and have as binding human leukocyte antigen (HLA) class I molecules. The aim of this study was to investigate the distribution of KIR genes and inhibitory KIR/HLA pairs in a population from Southern Brazil, in the state of Paraná, and to compare the results with results from other populations. The genotyping of 16 KIR genes and HLA class I alleles of 289 unrelated individuals was accomplished by reverse sequence-specific oligonucleotide Luminex (One Lambda, Inc., Canoga Park, CA). This Brazilian population demonstrated several similarities to Caucasian populations with regard to the frequency of KIR genes. Thirty-eight genotypes were defined in which the most frequent was the homozygous haplotype A (33.2%). Therefore, it was possible to define two new genotypes. Most of the individuals demonstrated at least one inhibitory KIR/HLA pair. Two pairs were the most frequent (40.4%), followed by three pairs (38.2%), one pair (14.6%), and four pairs (6.4%). The KIR2DL2/3 + HLA-C1 pair was the most frequent (79.9%) and the least frequent pair was KIR3DL2 + HLA-A3/11 (25.0%). This study demonstrated the diversity of KIR genes in a population of Paraná, as well as the characteristic pattern of Caucasians with racial admixture, which enabled the definition of two new genotypes and the identification of one individual without the inhibitory KIR/HLA pair.

The Association of the Immune Response Genes to Human Papillomavirus-Related Cervical Disease in a Brazilian Population

The genetic variability of the host contributes to the risk of human papillomavirus (HPV)-related cervical disease. Immune response genes to HPV must be investigated to define patients with the highest risk of developing malignant disease. The aim of this study was to investigate the association of polymorphic immune response genes, namely KIR, HLA class I and II, and single-nucleotide polymorphisms (SNPs) of cytokines with HPV-related cervical disease. We selected 79 non-related, admixed Brazilian women from the state of Paraná, southern region of Brazil, who were infected with high carcinogenic risk HPV and present cervical intraepithelial neoplasia grade 3 (CIN3), and 150 HPV-negative women from the same region matched for ethnicity. KIR genes were genotyped using an in-house PCR-SSP. HLA alleles were typed using a reverse sequence-specific oligonucleotide technique. SNPs of TNF −308G>A, IL6 −174G>C, IFNG +874T>A, TGFB1 +869T>C +915G>C, and IL10 −592C>A −819C>T −1082G>A were evaluated using PCR-SSP. The KIR genes were not associated with HPV, although some pairs of i(inhibitory)KIR-ligands occurred more frequently in patients, supporting a role for NK in detrimental chronic inflammatory and carcinogenesis. Some HLA haplotypes were associated with HPV. The associations of INFG and IL10 SNPs potentially reflect impaired or invalid responses in advanced lesions.

Class-I human leukocyte alleles in leprosy patients from Southern Brazil

INTRODUCTIONnThe present study was designed to investigate a possible role of HLA (histocompatibility leucocyte antigen) class-I alleles (HLA-A, -B, and -C) in leprosy patients from Southern Brazil.nnnMETHODSnTwo hundred and twenty-five patients with leprosy and 450 individuals for the control group were involved in this research. HLA genotyping was performed through PCR-SSO protocols (One Lambda, USA); the frequency of these alleles was calculated in each group by direct counting, and the frequencies were then compared.nnnRESULTSnThere was an association between HLA-A*11 (6.9% vs 4.1%, p=0.0345, OR=1.72, 95% CI=1.05-2.81), HLA-B*38 (2.7% vs. 1.1%, p=0.0402, OR=2.44, 95% CI=1.05-5.69), HLA-C*12 (9.4% vs. 5.4%, p=0.01, OR=1.82, 95% CI=1.17-2.82), and HLA-C*16 (3.1% vs. 6.5%, p=0.0124, OR=0.47, 95% CI=0.26-0.85) and leprosy per se. In addition, HLA-B*35, HLA-C*04, and HLA-C*07 frequencies were different between lepromatous (LL) and tuberculoid (TT) patients. However, after adjusting for the number of alleles compared, Pc values became nonsignificant.nnnCONCLUSIONSnAlthough our results do not support the previous findings that HLA class-I alleles play a role in leprosy pathogenesis, we suggest new studies because of the importance of the association between the HLA and KIR in the innate immune response to leprosy.

Otimização de metodologia para o estudo de genes KIR

The killer cell immunoglobulin-like receptors (KIRs) are molecules expressed on natural killer (NK) cells surface and in T-cell subsets encoded by genes located in chromosome 19q13.4. The interaction between KIR receptors and HLA class I molecules determines if the NK cells will fulfill their cytotoxic function and/or cytokine secretion or if this function will be inhibited. The objective of this work was to optimize KIR genotyping method described by Martin (2004). It was used PCR-SSP (polymerase chain reaction-sequence-specific primers) with primers synthesized by Invitrogen® and visualization of the amplified products on 2% agarose gel electrophoresis, containing ethidium bromide. Some adaptations were made and the reagents had their concentrations increased: the internal control from 100 nM to 150 nM, forward and reverse specific primers KIR12.5/12.3, KIR13.5/13.3, KIR14.5/14.3, KIR22.5/22.3 and KIR36.5/36.3 from 500 nM to 750 nM, and MgCl2 solution from 1.5 mM to 2 mM. Other reagent concentrations and amplification temperatures were maintained. Satisfactory results were obtained with Taq DNA Polymerase Recombinant (Invitrogen®). The results of seventy samples were confirmed by rSSO-Luminex® (One Lambda, Canoga Park, CA, USA). This KIR typing method proved to be accurate, specific, reproducible and cost effective.

Investigation of deletion of 22pb in KIR2DS4 gene in a population of southern Brazil.

The aim of this study was to investigate the distribution of full‐length and deleted variants of KIR2DS4 in a population of southern Brazil and compare the results with other populations, as well as comparing two techniques, PCR‐SSP and PCR‐SSO, for typing of variants.