TNF-alpha and IL-17 cytokine levels in Brazilian patients with ankylosing spondylitis after anti-TNF therapy

Abstract The aim of this study was to evaluate tumor necrosis factor alpha (TNF-α), interleukin (IL)- 17A/F levels in the serum of ankylosing spondylitis (AS) patients after anti-TNF therapy, in order to understand how these cytokines are involved in this therapeutic response. Forty-four AS patients were included in the study: thirty using anti-TNF therapy were classified according to their therapy response as responders (15) and non-responders (15) and 14 without anti-TNF therapy were classified as AS control. Fifteen healthy individuals formed the control group. Serum levels of TNF-α were determined using Luminex technology and for IL-17A and IL-17F using ELISA. The non-responder patients presented higher serum levels of TNF-α than the responders and AS control; the same results were found when HLA-B*27 positive or negative patients were separately analyzed. IL-17A and IL17F serum levels were similar for all groups. According to the clinical disease activity, AS patients with BASDAI ≥4 had higher serum levels of TNF-α than AS patients with BASDAI <4. Positive correlation was found between TNF-α levels and BASDAI. In AS patients, TNF-α serum levels were associated with anti-TNF therapy and disease activity independently of HLA-B*27, and IL-17A and IL-17F were not related to anti-TNF treatment.

Genetic polymorphisms of toll-like receptors in leprosy patients from southern Brazil.

Leprosy is a chronic disease and also a global health issue, with a high number of new cases per year. Toll-like receptors can respond to mycobacterial molecules in the early stage of infection. As important components of the innate immune response, alterations in genes coding for these receptors may contribute to susceptibility/protection against diseases. In this context, we used a case-control study model (183 leprosy cases vs. 185 controls) to investigate whether leprosy patients and the control group, in southern Brazil, have different frequencies in TLR1 (TLR1 G>T; rs5743618), TLR2 (TLR2 T>C, rs1816702 and rs4696483), and TLR4 (TLR4 A>G, rs1927911) polymorphisms. Analysis of the TLR1 1805G>T polymorphism presented the G/G genotype more frequently in the control group. TLR2 T>C rs1816702 and TLR2 T>C rs4696483, the T/T and C/T genotype, respectively, were more frequent in the control group than in leprosy patients, suggesting protection from leprosy when the T allele is present (rs4696483). Haplotype analyses between TLR1 (rs5743618) and TLR2 (rs1816702 and rs4696483) polymorphisms suggest risk for the presence of the TCC haplotype and protection in the presence of the TCT haplotype. This study suggests that polymorphisms in TLR1 and TLR2 are factors that may contribute to development/resistance of leprosy.

Influence of IL10 (rs1800896) Polymorphism and TNF-α, IL-10, IL-17A, and IL-17F Serum Levels in Ankylosing Spondylitis.

Ankylosing spondylitis (AS) is a chronic autoimmune inflammatory disease that mainly affects the axial and sacroiliac joints. Single-nucleotide polymorphisms (SNPs) in genes encoding cytokines have been associated with AS, which can interfere with the production of these cytokines and contribute to the development of AS. In order to contribute to a better understanding of the pathology of AS, our objective was to investigate a possible association of the IL10 -1082 A>G SNP (rs1800896) with AS and to evaluate the serum levels of TNF-α, IL-10, IL-17A, and IL-17F in AS patients and controls comparing them with their respective genotypes (TNF rs1800629, IL10 rs1800896, IL17A rs2275913, and IL17F rs763780). Patients and controls were selected from the Maringá University Hospital and the Maringá Rheumatism Clinic, in Paraná State, Southern Brazil, and they were diagnosed by the ASAS Criteria. In total, 149 patients and 169 controls were genotyped for the IL10 -1082 A>G polymorphism using a polymerase chain reaction with sequence specific primers (PCR-SSP); the measurement of TNF-α serum levels was performed through the immunofluorimetric test and IL-10, IL-17A, and IL-17F using an ELISA test. There was a high frequency of the IL10 -1082 G allele in AS patients compared with controls with an odds ratio of 1.83 and 95% confidence interval of 1.32 to 2.54, and a significant difference in the genotype frequencies of the IL10 -1082 A/G+G/G between patients and healthy controls, with an odds ratio of 3.01 and 95% confidence interval of 1.75 to 5.17. In addition, increased serum levels of IL-10 were observed in AS patients: 2.38 (IQR, 0.91) pg/ml compared with controls 1.72 (IQR 0.93) pg/ml (P = 0.01). Our results also showed an association between IL17F rs763780 C/T+T/T genotypes and increased serum levels of IL-17F in patients with AS and also in controls. We can conclude that patients with the A/G and G/G genotypes for -1082 A>G (rs1800896) in the IL10 gene are three times more likely to develop AS, that the serum level of IL-10 was higher in AS patients and that the IL17F rs763780 polymorphism can affect the levels of IL-17F in the serum of patients and controls in the same way.

IL17F: A Possible Risk Marker for Spondyloarthritis in HLA-B*27 Negative Brazilian Patients.

HLA-B*27 is an important marker for spondyloarthritis (SpA), however, many SpA patients are HLA-B*27 negative. Thus, the aim of this study was to investigate the influence of IL17, TNF and VDR gene polymorphisms in SpA patients who were HLA-B*27 negative. This case-control study was conducted in 158 patients [102 patients with ankylosing spondylitis (AS) and 56 with psoriatic arthritis (PsA)] and 184 controls. HLA-B*27 genotyping was performed using PCR-SSP and IL17A (rs2275913), IL17F (rs763780), TNF-308 (rs1800629), TNF-238 (rs361525), FokI C>T (rs2228570), TaqI C>T (rs731236), ApaI A>C (rs7975232), and BsmI C>T (rs1544410) using PCR-RFLP. Statistical analyses were performed by Chi-square and logistic regression using OpenEpi and SNPStats software. The IL17F C allele frequency was higher in patients with SpA, AS and PsA compared to controls. The IL17F T/C genotype frequency was higher in SpA patients in an overdominant inheritance model and when men and women were separately analyzed. IL17A_IL17F AC haplotype was significantly associated to the risk for SpA patients. As for VDR, the ApaI a/a was a potential risk factor for SpA in men. In conclusion, IL17F C variant contributed to the risk of SpA in Brazilian patients who were HLA-B*27 negative and could be a potential marker for SpA.

Toll-like receptor gene polymorphisms in patients with myeloproliferative neoplasms.

Toll-like receptors (TLRs) are a family of transmembrane receptors whose signaling control cellular processes of cell proliferation, survival, apoptosis, angiogenesis, remodeling, and repair of tissues. Polymorphisms in TLR genes can change the balance between pro and anti-inflammatory cytokines, modulating the risk of infection, chronic inflammation, and cancer. Although many studies have demonstrated the direct involvement of TLR signaling in the benefit of tumor cells in certain cancers, little is known about the influence of these gene polymorphisms on myeloproliferative neoplasms (MPNs). In this context, the objective of the study was to investigate a possible association between the TLR polymorphisms and the development of MPNs. 167 patients diagnosed with MPN and 222 healthy controls from the same region were evaluated. Genomic DNA was extracted and the TLR2 (rs5743708), TLR4 (rs4986790, rs4986791), TLR9 (rs5743836, rs187084) and JAK2V617F polymorphisms were genotyped by PCR-RFLP. The statistical analysis was performed by OpenEpi and SNPstat software. The JAK2V617F mutation was found in 68.32% of patients. TLR9-1486C/T CT genotype was less frequent in patients with polycythemia vera (PV) (OR 0.39, 95% CI 0.20-0.78, P = 0.025). When haplotype frequencies were analyzed, -1237T/-1486C (TLR9) was also less frequent in men (OR 0.58, 95% CI 0.36-0.94) and JAK negative men patients (OR 0.43, 95% CI 0.21-0.88). We can infer that the TLR9-1486 CT genotype could be associated with protection for PV and the TLR9-1237T/-1486C haplotype, protection for men, as well as for JAK negative men patients with MPN. There were no associations between TLR2 and TLR4 gene polymorphisms and MPN.

The Influence of Vitamin D Receptor Gene Polymorphisms in Spondyloarthritis.

Spondyloarthritis (SpA) is an inflammatory rheumatic disease related to low bone mineral density. Because vitamin D plays an important role in bone metabolism and immune system modulation, the aim of this study was to evaluate the influence of polymorphisms in vitamin D receptor genes (VDR) in the development of SpA. In this case-control study, a total of 244 patients with SpA and 197 individuals with no SpA were included. Among the patients, 174 had ankylosing spondylitis (AS) and 66 had psoriatic arthritis (PsA). Genotyping of FokI (rs2228570 C > T), BsmI (rs1544410 C > T), ApaI (rs7975232 A > C), and TaqI (rs731236 T > C) was performed using PCR-RFLP, while genotyping of HLA-B∗27 was performed using PCR-SSP. Serum levels for hydroxy (OH) vitamin D and the clinical activity index of the disease (BASDAI) were also evaluated. SNPStats and OpenEpi software were used for statistical analysis. The ApaI a allele and ApaI a/a genotype were less frequent in PsA compared with controls. The ApaI a/a genotype was associated with a protecting factor for PsA in females, and ApaI A/a was associated with a protecting factor for the disease in HLA-B∗27 positive patients. Notwithstanding, the ApaI a/a genotype was a risk factor for SpA and AS in males. The FokI f/f genotype was associated with a better clinical activity in PsA. When considering the covariates, vitamin D sufficiency, and gender, the FokI F/F genotype was associated with a risk factor in males with SpA and AS compared with females with this same genotype. In conclusion, the ApaI rs7975232 polymorphism was associated with PsA, and the FokI rs2228570 polymorphism was associated with better clinical PsA activity. ApaI and FokI were associated with SpA and AS when considering gender and vitamin D sufficiency.

Optimization of HLA-B*27 ALLELE Genotyping by PCR-SSP.

OBJECTIVES: HLA-B27 is strongly associated with ankylosing spondylitis (AS) and its presence helps to confirm AS diagnosis. Due to the high HLA polymorphism and the differentiated contribution of alleles and molecules encoded by them, HLA-B*27 allele identification is relevant in the clinical follow-up, diagnosis, and treatment of this spondyloarthropathy. Inexpensive genotyping techniques with high specificity and sensitivity are of great interest in histocompatibility laboratories. This work aimed to optimize HLA-B*27 genotyping by Polymerase Chain Reaction Sequence-specific Primer (PCR-SSP), which is an accessible and inexpensive technique. METHODS: The PCR-SSP was standardized using 26 HLA-B*27 positive and 3 HLA-B*27 negative samples previously defined by Polymerase Chain Reaction Sequence-specific Oligonucleotide Probes (PCR-SSOP) (medium resolution, One Lambda®) and primers described by Duangchanchot et al. (2009). For validating the technique, 397 samples were genotyped using PCR-SSP as well as PCR-SSOP. RESULTS: The PCR-SSP technique was standardized for identifying the alleles HLA-B*27:02, HLA-B*27:CAFRW (05/13/16/17/28/37/38/39/42), HLA-B*27:CAFRZ (08/26/40), HLA-B*27:09 and HLA-B*27:12, which were found in 90 positive samples (22.67%). There was 100% agreement between the two techniques for heterozygous samples; however, two homozygous samples could not be detected by PCR-SSP. CONCLUSION: The HLA-B*27 genotyping using PCR-SSP, an easy-to-use, specific, and affordable technique, was optimized for heterozygous samples. This technique may contribute to AS diagnosis.

Association of MBL2 Exon 1 Polymorphisms With Multibacillary Leprosy.

Mannose-binding lectin (MBL) is a serum protein of innate immunity, with a central role in the activation of the complement system through the lectin pathway. This protein is encoded by MBL2 gene, and single-nucleotide polymorphisms located at exon 1, such as rs5030737 C>T (D variant), rs1800450 G>A (B variant), and rs1800451 G>A (C variant), may change the MBL structure and the serum concentration. MBL2 polymorphisms have been associated with several infectious diseases, including leprosy. Host immune response has a major impact on the clinical manifestation of leprosy since only a few individuals infected with Mycobacterium leprae will develop the disease. Therefore, the aim of this study was to evaluate the influence of MBL2 exon 1 polymorphisms (rs5030737, rs1800450, and rs1800451) on the MBL levels and leprosy immunopathogenesis. This case-control study included 350 leprosy patients from Southern Brazil, with 279 classified as multibacillary (MB) and 71 as paucibacillary (PB). The control group consisted of 350 non-consanguineous individuals, who were not diagnosed with leprosy or other infectious and autoimmune diseases. Genotyping was performed by PCR-sequence specific primers, and the MBL serum concentrations were evaluated by ELISA. MBL2 exon 1 polymorphisms were analyzed individually and grouped as genotypes, considering "A" as the wild allele and "O" as the presence of at least one polymorphism (D, B, or C variants). Differences were not observed in the distribution of genotypic and allelic frequencies between leprosy per se patients and controls. However, in a haplotypic analysis, the TGG haplotype presented a risk for development of leprosy per se in women when compared to the wild haplotype (CGG) (OR = 2.69). Comparing patients with MB and PB, in a multivariate analysis, the B variant was associated with the susceptibility of developing the MB form of leprosy (OR = 2.55). Besides that, the CAG haplotype showed an increased susceptibility to develop MB leprosy in women compared to men. It was observed that the A/O genotype in women was associated with a susceptibility to leprosy development per se (OR = 1.66) and progression to MB leprosy (OR = 3.13). In addition, the MBL serum concentrations were in accordance with the genotyping analysis. In summary, our data suggest that MBL2 exon 1 polymorphisms are associated with an increased risk to leprosy development and progression.

Association of functional IL16 polymorphisms with cancer and cardiovascular disease: a meta-analysis.

INTRODUCTION: Interleukin-16 (IL-16) is a chemotactic cytokine that is found to increase in Cancer and cardiovascular diseases (CVD). Single nucleotide polymorphisms (SNPs) in IL16 were associated with diseases. Thus, we conducted a systematic review and meta-analysis to evaluate possible associations between IL16 rs4778889, rs11556218, rs4072111, and rs1131445 SNPs and the risk for cancer or CVD. MATERIALS AND METHODS: This study was performed according to the PRISMA statement. Medline, Web of Science, and Scopus databases were systematically reviewed, and a meta-analysis was conducted. RESULTS: The analysis comprised 6386 individuals with cancer and 2415 with CVD. The SNP rs11556218 was significantly associated with an increased risk for cancer in Chinese in different genetic inheritance models. Also, to the best of our knowledge, this is the first meta-analysis to show an association of rs4778889 with an increased risk of gastric cancer and rs11556218 with an increased risk of CVD in Chinese. CONCLUSIONS: Our meta-analysis suggested that the SNPs rs11556218 and rs4778889 of IL16 were associated with an increased risk for cancer in Chinese and rs11556218 with increased risk for CVD in Chinese, highlighting the need for further studies on the impact of these polymorphisms on cancer treatment and surveillance.

Association of IL16 polymorphisms with periodontitis in Brazilians: A case- control study.

Periodontitis (PD) is a chronic inflammatory process resulting from the relationship of the immune response with the components in dental plaque. Cytokines and their genetic polymorphisms seem to be involved in the immunopathogenesis of this disease. This study aimed to evaluate the correlation of IL16 polymorphism with PD. A case-control study was conducted in a sample of individuals from southern Brazil. The genotyping of IL16, rs11556218 T>G, rs4072111 C>T e rs4778889 T>C, was performed using the PCR-RFLP methodology. The serum level of IL-16 was determined using an IL-16 ELISA kit for humans. SNPStats and OpenEpi software and Wilcoxon's U test were used to perform statistical analysis. IL16 rs11556218 polymorphism was significantly associated to PD in nonsmoking patients: individuals with G/G genotype were less likely to develop PD compared to the T/T genotype (OR = 0.10; Pc = 0.019, codominant model). In addition, the TTT haplotype was associated with a high risk for PD (OR = 2.45; P = 0.01). A low IL-16 serum level was observed among individuals with PD when compared to controls (P = 0.027). Thus, the IL16 rs16556218 polymorphism and the serum levels of IL-16 were associated with periodontitis in a Brazilian sample, and this was influenced by environmental factors such as smoking.

Influence of inflammasome NLRP3, and IL1B and IL2 gene polymorphisms in periodontitis susceptibility.

The pathogenesis of periodontitis (PD) involves several molecules of the immune system that interact in a network to eliminate the periodontopathogens, yet, they contribute to periodontal tissue destruction. The different mechanisms that lead to periodontal tissue damage are not clear. Despite this, immune response genes have been related to the development of PD previously, such as those involved in inflammasomes which are multiprotein complexes and cytokines including Interleukin-1. The aim of the study was to evaluate the polymorphisms in NLRP3 inflammasome, cytokine and receptor of cytokines genes in the development of periodontitis. This case-control study was conducted in 186 patients with PD (stage II and III and grade B) and 208 controls (localized gingivitis and periodontally healthy individuals). Genotyping was performed using PCR-RFLP for the SNP rs4612666 in NLRP3 and using PCR-SSP for IL1A, IL1B, IL1R, IL1RN, IL4RA, INFG, TGFB1, TNF, IL2, IL4, IL6, and IL10. Cytokine serum levels were measured using Luminex technology. SNPStats and OpenEpi software were used to perform statistical analysis. The higher frequencies of NLRP3 T/C and IL1B -511 T/T genotypes and IL2 (+166, -330) GT haplotype were observed in patients with PD compared to controls. The SNPs in NLRP3, IL1R +1970, IL6-174, TNF -308, IL2 +166 and -330, TGFB1 +869 and +915, IL4RA +1902, IL4-1098 and -590 were associated to PD in men. In conclusion, polymorphisms in NLRP3, IL1B and IL2 genes were associated to PD susceptibility. Men carrying the NLRP3, IL1R, IL6, TNF, IL2, TGFB1, IL4RA and IL4 polymorphisms had greater susceptibility than women for developing PD.

HLA-A, -B, -DRB1, -DQA1, and -DQB1 genotyping of 641 individuals from southern Brazil.

Sixty hundred and forty-one Brazilian individuals from the north and northwestern state of Paraná, southern Brazil, were selected for the study. The HLA-A, -B, -DRB1, -DQA1, and -DQB1 genotyping were performed using rSSO and Micro SSP analysis. These genotype data are available in the Allele Frequencies Net Database under the population name "Brazil Paraná Caucasian" number "AFND3618".

Optimization of HLA-B*27 ALLELE Genotyping by PCR-SSP

OBJECTIVES: HLA-B27 is strongly associated with ankylosing spondylitis (AS) and its presence helps to confirm AS diagnosis. Due to the high HLA polymorphism and the differentiated contribution of alleles and molecules encoded by them, HLA-B*27 allele identification is relevant in the clinical follow-up, diagnosis, and treatment of this spondyloarthropathy. Inexpensive genotyping techniques with high specificity and sensitivity are of great interest in histocompatibility laboratories. This work aimed to optimize HLA-B*27 genotyping by Polymerase Chain Reaction Sequence-specific Primer (PCR-SSP), which is an accessible and inexpensive technique. METHODS: The PCR-SSP was standardized using 26 HLA-B*27 positive and 3 HLA-B*27 negative samples previously defined by Polymerase Chain Reaction Sequence-specific Oligonucleotide Probes (PCR-SSOP) (medium resolution, One Lambda®) and primers described by Duangchanchot et al. (2009). For validating the technique, 397 samples were genotyped using PCR-SSP as well as PCR-SSOP. RESULTS: The PCR-SSP technique was standardized for identifying the alleles HLA-B*27:02, HLA-B*27:CAFRW (05/13/16/17/28/37/38/39/42), HLA-B*27:CAFRZ (08/26/40), HLA-B*27:09 and HLA-B*27:12, which were found in 90 positive samples (22.67%). There was 100% agreement between the two techniques for heterozygous samples; however, two homozygous samples could not be detected by PCR-SSP. CONCLUSION: The HLA-B*27 genotyping using PCR-SSP, an easy-to-use, specific, and affordable technique, was optimized for heterozygous samples. This technique may contribute to AS diagnosis.

Impact of SNPs/Haplotypes of IL10 and IFNG on the Development of Diffuse Large B-Cell Lymphoma.

The purpose of this study was to assess the influence of single-nucleotide polymorphisms (SNPs) on cytokine genes in the development of diffuse large B-cell lymphoma (DLBCL). One hundred and twelve patients and 221 controls were investigated. Among them, 97 patients treated with R-CHOP were subdivided into two groups: (i) complete remission of the disease and (ii) patients who progressed to death, relapsed, or had disease progression. The SNPs investigated by PCR-SSP were TNF -308G>A (rs1800629), IFNG +874A>T (rs2430561), IL6 -174G>C (rs1800795), IL10 -1082A>G (rs1800896), IL10 -819C>T (rs1800871), IL10 -592C>A (rs1800872), and TGFB1 codon10T>C (rs1982073) and codon25G>C (rs1800471). In general, the genotypes that have been associated in the literature with lower production or intermediate production of IL-10 and higher production of IFN-γ were associated with the protection of the development of the disease, possibly favoring the Th1 immune response and diminishing the capacity of cell proliferation. However, patients receiving R-CHOP treatment presented unfavorable prognoses in the presence of genotypes related to the intermediate production of IL-10 and high production of TGF-ß1, indicating that cytokines may be related to the response to treatment and action mechanisms of Rituximab.

Vitamin D Receptor Gene Polymorphisms Are Associated With Leprosy in Southern Brazil.

Vitamin D, together with its nuclear receptor (VDR), plays an important role in modulating the immune response, decreasing the inflammatory process. Some polymorphisms of the VDR gene, such as BsmI (G>A rs1544410), ApaI (G>T rs7975232), and TaqI (T>C rs731236) could affect its stability and mRNA transcription activity, while FokI T>C (rs2228570) gives a truncated protein with three fewer amino acids and more efficiency in binding vitamin D. This study evaluated these four polymorphisms in the immunopathogenesis of leprosy in 404 patients and 432 control individuals without chronic or infectious disease in southern Brazil. When analyzing differences in the allele and genotype frequency of polymorphisms between patients (leprosy per se, multibacillary, and paucibacillary clinical forms) and controls, we found no statistically significant association. Regarding haplotype analysis, the bAt haplotype was associated with protection from leprosy per se (P = 0.004, OR = 0.34, CI = 0.16-0.71) and from the multibacillary clinical form (P = 0.005, OR = 0.30, CI = 0.13-0.70). In individuals aged 40 or more years, this haplotype has also showed protection against leprosy per se and multibacillary (OR = 0.26, CI = 0.09-0.76; OR = 0.26, CI = 0.07-0.78, respectively), while the BAt haplotype was a risk factor for leprosy per se in the same age group (OR = 1.34, CI = 1.04-1.73). In conclusion, despite having found no associations between the VDR gene polymorphisms with the development of leprosy, the haplotypes formed by the BsmI, ApaI, and TaqI polymorphisms were associated with leprosy per se and the multibacillary clinical form.

The impact of KIR/HLA genes on the risk of developing multibacillary leprosy.

BACKGROUND: Killer-cell immunoglobulin-like receptors (KIRs) are a group of regulatory molecules able to activate or inhibit natural killer cells upon interaction with human leukocyte antigen (HLA) class I molecules. Combinations of KIR and HLA may contribute to the occurrence of different immunological and clinical responses to infectious diseases. Leprosy is a chronic neglected disease, both disabling and disfiguring, caused mainly by Mycobacterium leprae. In this case-control study, we examined the influence of KIRs and HLA ligands on the development of multibacillary leprosy. METHODOLOGY/PRINCIPAL FINDINGS: Genotyping of KIR and HLA genes was performed in 264 multibacillary leprosy patients and 518 healthy unrelated controls (238 healthy household contacts and 280 healthy subjects). These are unprecedented results in which KIR2DL2/KIR2DL2/C1/C2 and KIR2DL3/2DL3/C1/C1 indicated a risk for developing lepromatous and borderline leprosy, respectively. Concerning to 3DL2/A3/A11+, our study demonstrated that independent of control group (contacts or healthy subjects), this KIR receptor and its ligand act as a risk factor for the borderline clinical form. CONCLUSIONS/SIGNIFICANCE: Our finding suggests that synergetic associations of activating and inhibitory KIR genes may alter the balance between these receptors and thus interfere in the progression of multibacillary leprosy.

The Influence of TLR4, CD14, OPG, and RANKL Polymorphisms in Periodontitis: A Case-Control Study.

The pathogenesis of periodontitis involves a complex interaction between the microbial challenge and the host immune response. The individual immunoinflammatory response has a great contribution in the pathogenesis of the disease and becomes a trigger in the process of bone remodeling which is a characteristic of the disease. Thus, the aim of this study was to evaluate the influence of the TLR4 A896G (rs4986790), TLR4 C1196T (rs4986791), CD14 C-260T (rs2569190), RANKL (TNFSF11, rs2277438), and OPG (TNFSF11B C163T, rs3102735) polymorphisms in periodontitis. A case-control study was conducted on patients with periodontitis (N = 203) and controls (N = 213) over 30 years of age, without diabetes mellitus, acute infections, and osteoarthritis, and patients without aggressive periodontitis, i.e., stage IV and C degree of periodontitis, and any periodontal treatment performed in the last 6 months. Genotypes were determined by the PCR-RFLP and sequencing method. The frequency comparisons between case and controls were performed using the chi-square test and logistic regression (OpenEpi and SNPStats software). The risk (OR) was evaluated for values of P < 0.05. Differences in TLR4, CD14, RANKL, and OPG genotype and allele frequency distributions were not observed between patients and controls. However, some variants were a risk factor for the development of periodontitis when considering gender and smoking habits. The TLR4 896 A/G genotype was a risk factor for periodontitis in males (OR = 2.86), and the TLR4 1196C/C genotype was a risk factor for nonsmoking males (OR = 1.85) when compared to women. The RANKL A/A and the OPG T/C genotype was associated with the risk of the disease in nonsmoking men compared to nonsmoking women with the same genotype (OR = 1.96 and OR = 2.9, respectively). In conclusion, TLR4, CD14, RANKL, and OPG variants were not associated with periodontitis. However, TLR4, RANKL, and OPG polymorphisms could be a risk for periodontitis in males regardless of smoking habits.

IL18 Polymorphism and Periodontitis Susceptibility, Regardless of IL12B, MMP9, and Smoking Habits.

Genetic variations contribute to the susceptibility in the development of periodontitis. The aim of this study was to investigate the influence of IL18, IL12, and MMP9 polymorphisms in the chronic periodontitis. This case-control study involved 381 individuals matched by gender and age. Genotyping of IL18 (rs187238 and rs1946518) and IL12B (rs3212227) was performed by PCR-SSP and PCR-RFLP was used for MMP9 (rs3918242). IL-18 and MMP-9 were quantified in the serum by ELISA. SNPStats and OpenEpi software were used for statistical analysis and, in order to eliminate smoking as a confounding factor, the analyses were also performed in nonsmoking subjects. The IL18-137G/C genotype was associated with the risk of chronic periodontitis in nonsmokers (P c = 0.03; OR = 1.99; overdominant inherence model). In the multivariate analyses, homozygous IL18-137G/G and IL18-607C/C were more frequent in males compared to women with these same genotypes (OR = 2.51 and OR = 3.30, respectively). The serum levels of the IL-18 in patients were higher than those in healthy controls (P = 0.005). IL12B and MMP9 polymorphisms and MMP-9 serum concentration were similar in patients and controls. In this study, IL18 was associated with chronic periodontitis susceptibility. Men had greater risk than women for developing the disease when IL18 polymorphism was considered and the susceptibility was independent of the smoking status.

Genotyping of Dombrock and Lutheran blood group systems in blood donors from the southwestern region of the state of Paraná, Southern Brazil

Abstract Background Lutheran and Dombrock are two blood group systems with low immunogenic antigens; they can cause mild-to-moderate transfusion reactions. For both, immunophenotyping is not performed in the pretransfusion routine in Brazil. In addition, the distribution of their antigenic frequencies is an important marker of ethnicity. Thus, the goal of this study was to carry out the genotyping of the LU*01, LU*02, DO*01 and DO*02 alleles of the Lutheran and Dombrock blood group systems in blood donors from the southwestern region of the state of Paraná, Southern Brazil. Method Genotyping was performed for 251 blood donors by specific allele-polymerase chain reaction. The genotype and allele frequencies were obtained through direct counting and compared with other Brazilian populations using the chi-square test with Yates correction. Results The distribution of genotype frequencies for LU were 0.4% for LU*01/LU*01, 6.8% for LU*01/LU*02 and 92.8% for LU*02/LU*02 and for DO, they were 19.9% for DO*01/DO*01, 44.6% for DO*01/DO*02 and 35.5% for DO*02/DO*02. The allele and genotype frequencies of LU and DO were similar to those expected for Caucasians, but the DO*01/DO*01 genotype frequency was different to other Brazilian populations. The rare LU*01/LU*01 genotype was found in a loyal blood donor. Conclusion The genotyping techniques allowed the evaluation of the LU*01, LU*02, DO*01 and DO*02 alleles in blood donors registered in the Hemotherapy Center of the southwestern region of Paraná, Southern Brazil, and contributed to a genotyped blood donor database.

Methods for blood group antigens detection: cost-effectiveness analysis of phenotyping and genotyping

Abstract Background Alloimmunization is a major problem in transfusion practice due to the clinical complications of the patients and the difficulty of choosing a unit of compatible blood product. Serological methods are widely used in blood banks, but they not always determine the phenotype. Thus, genotyping is an important complement to the serology tool as it allows one to predict the phenotype from deoxyribonucleic acid (DNA) with high accuracy. Objective To compare the centrifugation gel, microarray, Restriction Fragment Length Polymorphismone PCR (PCR-RFLP) and Sequence-Specific Primer PCR (PCR-SSP) techniques, in terms of cost, reaction time and reliability of the results. Methods The RHCE, Kidd, Kell and Duffy blood group systems were chosen to determine the approximate cost of each technique, considering the reagents used in both methods and considering only one sample. The time required for the development of each reaction was obtained at the Maringa Regional Blood Center and Immunogenetics Laboratory at the State University of Maringa. Data from Microarray reactions were obtained at the Campinas Blood Center. The results of phenotyping and genotyping of the 16 samples were compiled in a spreadsheet and compared. Results The PCR-SSP was more economical compared to other methods, and the serological method was faster than the molecular methods. However, all methods proved to be effective and safe in the detection of erythrocyte antigens. Conclusion Analyzing the advantages and limitations of the molecular and serological methods tested in this study, we note that both are important and complementary. However, the choice of a methodology depends on the reality and needs of each health service.