Toll-like receptor 9 polymorphisms in brazilian patients with systemic lupus erythematosus: a pilot study / Polimorfismos do receptor toll-like 9 em pacientes brasileiros com lúpus eritematoso sistêmico: um estudo piloto

Abstract Toll-like receptor 9 (TLR9) is an important component of the innate immune system and have been associated with several autoimmune diseases, such as Systemic Lupus Erythematosus (SLE). The aim of this study was to investigate polymorphisms in TLR9 gene in a Brazilian SLE patients group and their association with clinical manifestation, particularly Jaccoud's arthropathy (JA). We analyzed DNA samples from 204 SLE patients, having a subgroup of them presenting JA (n=24). A control group (n=133) from the same city was also included. TLR9 single nucleotide polymorphisms (SNPs) (−1237 C>T and +2848 G>A) were identified by sequencing analysis. The TLR9 gene genotype frequency was similar both in SLE patients and the control group. In the whole SLE population, an association between the homozygosis of allele C at position −1237 with psychosis and anemia (p < 0.01) was found. Likewise, the homozygosis of allele G at position +2848 was associated with a discoid rash (p < 0.05). There was no association between JA and TLR9 polymorphisms. These data show that TLR9 polymorphisms do not seem to be a predisposing factor for SLE in the Brazilian population, and that SNPs are not associated with JA.

Resumo O receptor Toll-like 9 (TLR9) é um componente importante do sistema imunológico inato e tem sido associado a várias doenças autoimunes, como o Lúpus Eritematoso Sistêmico (LES). O objetivo deste estudo foi investigar polimorfismos no gene TLR9 em um grupo de pacientes brasileiros com LES e sua associação com a manifestação clínica, particularmente a artropatia de Jaccoud (JA). Foram analisadas amostras de DNA de 204 pacientes com LES, e um subgrupo com JA (n=24). Um grupo de controle (n=133) da mesma cidade também foi incluído. Os polimorfismos de nucleotídeos únicos TLR9 (SNPs) (−1237 C>T e +2848 G>A) foram identificados pela análise de sequenciamento. A frequência do genótipo genético TLR9 foi semelhante tanto em pacientes com LES quanto no grupo controle. Em toda a população de LES, foi encontrada associação entre a homozigose do alelo C na posição −1237 com psicose e anemia (p < 0,01). Da mesma forma, a homozigose do alelo G na posição +2848 foi associada a uma erupção cutânea discoide (p < 0,05). Não houve associação entre polimorfismos JA e TLR9. Esses dados mostram que os polimorfismos TLR9 não parecem ser um fator predisponível para o LES na população brasileira, e que os SNPs não estão associados ao JA.

Toll-like receptor 9 polymorphisms in brazilian patients with systemic lupus erythematosus: a pilot study / Polimorfismos do receptor toll-like 9 em pacientes brasileiros com lúpus eritematoso sistêmico: um estudo piloto

Toll-like receptor 9 (TLR9) is an important component of the innate immune system and have been associated with several autoimmune diseases, such as Systemic Lupus Erythematosus (SLE). The aim of this study was to investigate polymorphisms in TLR9 gene in a Brazilian SLE patients group and their association with clinical manifestation, particularly Jaccoud’s arthropathy (JA). We analyzed DNA samples from 204 SLE patients, having a subgroup of them presenting JA (n=24). A control group (n=133) from the same city was also included. TLR9 single nucleotide polymorphisms (SNPs) (−1237 C>T and +2848 G>A) were identified by sequencing analysis. The TLR9 gene genotype frequency was similar both in SLE patients and the control group. In the whole SLE population, an association between the homozygosis of allele C at position −1237 with psychosis and anemia (p < 0.01) was found. Likewise, the homozygosis of allele G at position +2848 was associated with a discoid rash (p < 0.05). There was no association between JA and TLR9 polymorphisms. These data show that TLR9 polymorphisms do not seem to be a predisposing factor for SLE in the Brazilian population, and that SNPs are not associated with JA.

O receptor Toll-like 9 (TLR9) é um componente importante do sistema imunológico inato e tem sido associado a várias doenças autoimunes, como o Lúpus Eritematoso Sistêmico (LES). O objetivo deste estudo foi investigar polimorfismos no gene TLR9 em um grupo de pacientes brasileiros com LES e sua associação com a manifestação clínica, particularmente a artropatia de Jaccoud (JA). Foram analisadas amostras de DNA de 204 pacientes com LES, e um subgrupo com JA (n=24). Um grupo de controle (n=133) da mesma cidade também foi incluído. Os polimorfismos de nucleotídeos únicos TLR9 (SNPs) (−1237 C>T e +2848 G>A) foram identificados pela análise de sequenciamento. A frequência do genótipo genético TLR9 foi semelhante tanto em pacientes com LES quanto no grupo controle. Em toda a população de LES, foi encontrada associação entre a homozigose do alelo C na posição −1237 com psicose e anemia (p < 0,01). Da mesma forma, a homozigose do alelo G na posição +2848 foi associada a uma erupção cutânea discoide (p < 0,05). Não houve associação entre polimorfismos JA e TLR9. Esses dados mostram que os polimorfismos TLR9 não parecem ser um fator predisponível para o LES na população brasileira, e que os SNPs não estão associados ao JA.

Toll-like receptor 9 polymorphisms in brazilian patients with systemic lupus erythematosus: a pilot study

Abstract Toll-like receptor 9 (TLR9) is an important component of the innate immune system and have been associated with several autoimmune diseases, such as Systemic Lupus Erythematosus (SLE). The aim of this study was to investigate polymorphisms in TLR9 gene in a Brazilian SLE patients group and their association with clinical manifestation, particularly Jaccouds arthropathy (JA). We analyzed DNA samples from 204 SLE patients, having a subgroup of them presenting JA (n=24). A control group (n=133) from the same city was also included. TLR9 single nucleotide polymorphisms (SNPs) (1237 C>T and +2848 G>A) were identified by sequencing analysis. The TLR9 gene genotype frequency was similar both in SLE patients and the control group. In the whole SLE population, an association between the homozygosis of allele C at position 1237 with psychosis and anemia (p 0.01) was found. Likewise, the homozygosis of allele G at position +2848 was associated with a discoid rash (p 0.05). There was no association between JA and TLR9 polymorphisms. These data show that TLR9 polymorphisms do not seem to be a predisposing factor for SLE in the Brazilian population, and that SNPs are not associated with JA.

Resumo O receptor Toll-like 9 (TLR9) é um componente importante do sistema imunológico inato e tem sido associado a várias doenças autoimunes, como o Lúpus Eritematoso Sistêmico (LES). O objetivo deste estudo foi investigar polimorfismos no gene TLR9 em um grupo de pacientes brasileiros com LES e sua associação com a manifestação clínica, particularmente a artropatia de Jaccoud (JA). Foram analisadas amostras de DNA de 204 pacientes com LES, e um subgrupo com JA (n=24). Um grupo de controle (n=133) da mesma cidade também foi incluído. Os polimorfismos de nucleotídeos únicos TLR9 (SNPs) (1237 C>T e +2848 G>A) foram identificados pela análise de sequenciamento. A frequência do genótipo genético TLR9 foi semelhante tanto em pacientes com LES quanto no grupo controle. Em toda a população de LES, foi encontrada associação entre a homozigose do alelo C na posição 1237 com psicose e anemia (p 0,01). Da mesma forma, a homozigose do alelo G na posição +2848 foi associada a uma erupção cutânea discoide (p 0,05). Não houve associação entre polimorfismos JA e TLR9. Esses dados mostram que os polimorfismos TLR9 não parecem ser um fator predisponível para o LES na população brasileira, e que os SNPs não estão associados ao JA.

n-3 polyunsaturated fatty acid supplementation reduces insulin resistance in hepatitis C virus infected patients: a randomised controlled trial.

BACKGROUND: Insulin resistance promotes liver disease progression and may be associated with a lower response rate in treated hepatitis C virus (HCV) infected patients. n-3 polyunsaturated fatty acid (PUFA) supplementation may reduce insulin resistance. The present study aimed to evaluate the effect of n-3 PUFA supplementation on insulin resistance in these patients. METHODS: In a randomised, double-blind clinical trial, 154 patients were screened. After applying inclusion criteria, 52 patients [homeostasis model assessment index of insulin resistance (HOMA-IR ≥2.5)] were randomly divided into two groups: n-3 PUFA (n = 25/6000 mg day(-1) of fish oil) or control (n = 27/6000 mg day(-1) of soybean oil). Both groups were supplemented for 12 weeks and underwent monthly nutritional consultation. Biochemical tests were performed at baseline and after intervention. Statistical analysis was performed using the Wilcoxon Mann-Whitney test for comparisons and the Wilcoxon test for paired data. Statistical package r, version 3.02 (The R Project for Statistical Computing) was used and P < 0.05 (two-tailed) was considered statistically significant. RESULTS: Comparisons between groups showed that n-3 PUFA supplementation was more effective than the control for reducing HOMA-IR (P = 0.015) and serum insulin (P = 0.016). The n-3 PUFA group not only showed a significant reduction in HOMA-IR 3.8 (3.2-5.0) versus 2.4 (1.8-3.3) (P = 0.002); serum insulin 17.1 (13.8-20.6) µIU mL(-1) versus 10.9 (8.6-14.6) µIU mL(-1) (P = 0.001); and glycated haemoglobin 5.4% (5.0-5.7%) versus 5.1% (4.8-5.6%) (P = 0.011), but also presented an increase in interleukin-1 97.5 (0.0-199.8) pg mL(-1) versus 192.4 (102.2-266.8) pg mL(-1) (P = 0.003) and tumour necrosis factor 121.2 (0.0-171.3) pg mL(-1) versus 185.7 (98.0-246.9) pg mL(-1) (P = 0.003). CONCLUSIONS: n-3 PUFA supplementation reduces insulin resistance in genotype 1 HCV infected patients.

Coping with genetic diversity: the contribution of pathogen and human genomics to modern vaccinology

Vaccine development faces major difficulties partly because of genetic variation in both infectious organisms and humans. This causes antigenic variation in infectious agents and a high interindividual variability in the human response to the vaccine. The exponential growth of genome sequence information has induced a shift from conventional culture-based to genome-based vaccinology, and allows the tackling of challenges in vaccine development due to pathogen genetic variability. Additionally, recent advances in immunogenetics and genomics should help in the understanding of the influence of genetic factors on the interindividual and interpopulation variations in immune responses to vaccines, and could be useful for developing new vaccine strategies. Accumulating results provide evidence for the existence of a number of genes involved in protective immune responses that are induced either by natural infections or vaccines. Variation in immune responses could be viewed as the result of a perturbation of gene networks; this should help in understanding how a particular polymorphism or a combination thereof could affect protective immune responses. Here we will present: i) the first genome-based vaccines that served as proof of concept, and that provided new critical insights into vaccine development strategies; ii) an overview of genetic predisposition in infectious diseases and genetic control in responses to vaccines; iii) population genetic differences that are a rationale behind group-targeted vaccines; iv) an outlook for genetic control in infectious diseases, with special emphasis on the concept of molecular networks that will provide a structure to the huge amount of genomic data.

Vascular endothelial growth factor (VEGF) levels as a tool to discriminate between malignant and nonmalignant ascites.

Ascites formation is commonly associated with cancer, although it is also present in other diseases. The aim of this study was to evaluate the usefulness of vascular endothelial growth factor as a malignancy marker in ascites of different etiologies. The levels of VEGF in 32 malignant and in 31 non-malignant ascites were determined by enzyme immunoassay (ELISA). VEGF levels were significantly higher in malignant than in non-malignant ascites (median=1265.9 pg/ml x 114.6 pg/ml; p<0.0001). We observed 72% sensitivity and 90% specificity, using 662 pg/ml as a cut-off value. Therefore, this approach can be used as a marker for a first screening to discriminate between malignant and nonmalignant ascites.

Rheumatoid arthritis induced by alpha-interferon therapy.

Interferon (IFN) therapy has been used for the treatment of common diseases such as hepatitis C, myeloproliferative disorders, autoimmune diseases and various types of cancer. Given the biological properties of interferon, it is not surprising that there are a larger number of side effects due to its use. Although rheumatoid arthritis (RA) is one of the most common autoimmune diseases found in clinical practice, it does not seem to be frequently related to IFN therapy. We report a 40-year-old female patient who, after high doses of IFN-alpha therapy for malignant melanoma, developed symmetrical polyarthritis, with pain and oedema in small and large joints, associated with prolonged morning stiffness. She had positive rheumatoid factor and DR4 HLA phenotype. She was treated with deflazacort (6 mg/day), chloroquine and NSAIDs, with a partial response. In conclusion, although the development of RA after IFN therapy is a rare event, IFN may work as a 'trigger' for such complication, leading to deregulation in the immune cascade in a person genetically predisposed.

Stabilization of gas-phase noncovalent macromolecular complexes in electrospray mass spectrometry using aqueous triethylammonium bicarbonate buffer.

The use of triethylammonium bicarbonate (TEAB) solution in electrospray mass spectrometry proved to be a very efficient way for studying proteins or noncovalent protein complexes under "nondenaturing" conditions. The low charge states observed in the mass spectra improve the separation of ions arising from macromolecular species of close masses. Moreover, the multiply charged ions generated in a TEAB solution are significantly more stable than those formed under more conventional conditions (for example, with ammonium bicarbonate or acetate solution). The analytical interest of TEAB for the analysis of macromolecular species that can easily dissociate in the gas phase, such as hemoglobin or other macromolecular noncovalent complexes, is demonstrated.

An NMR and conformational investigation of the trans-syn cyclobutane photodimers of dUpdT.

Both trans-syn cyclobutane-type photodimers of 2'-deoxyuridylyl (3'-5') thymidine (dUpdT) were formed by deamination of the corresponding trans-syn cyclobutane photodimers of 2'-deoxycytidylyl (3'-5') thymidine (dCpdT) and were examined by 1H-, 13C-, and 31P-nmr spectroscopy. One- and two-dimensional nmr experiments provided a nearly complete assignment of the 1H, 13C, and 31P resonances. Scalar and nuclear Overhauser effect contacts were used to determine the conformation of the deoxyribose rings, exocyclic bonds, cyclobutane rings, and glycosidic linkages. Isomer I (S-type class; CB-; SYN-ANTI) and isomer II (N-type class; CB+; ANTI-SYN) exhibit markedly different conformational features. 31P chemical shifts show that the relative flexibility is dUpdT > isomer II > isomer I. The conformations of these species are very similar to those of other previously examined trans-syn photodimers. Among bipyrimidine photodimers of a given diastereomeric form (i.e., trans-syn I or II), the nmr-derived conformational parameters are nearly invariant, regardless of base substitution pattern. This contrasts with the substituent-dependent variation of cyclobutane ring conformation observed by Kim et al. (Biopolymers, 1993, Vol. 33, pp. 713-721) for an analogous series of cis-syn photodimers. Steric crowding of cyclobutane ring substituents is offered as an explanation for the difference in substituent effects between the families of cis-syn and trans-syn photodimers.

An NMR and conformational investigation of the trans-syn cyclobutane photodimers of dTpdU.

Two trans-syn cyclobutane photodimers of thymidylyl (3'-5') deoxyuridine were formed by deamination of the corresponding trans-syn cyclobutane photodimers of thymidylyl (3'-5') deoxycytidine and were examined by 1H-, 13C-, and 31P-nmr spectroscopy. Correlation spectroscopy, nuclear Overhauser enhancement spectroscopy, and one-dimensional heterodecoupling experiments allowed a more complete assignment of the 1H spectra, compared with previous reports by Koning et al. [(1991) European Journal of Biochemistry, Vol. 195, pp. 29-40] and Liu and Yang [(1978) Biochemistry, Vol. 17, pp. 4865-4876]. Deoxyribose ring conformations were calculated from 1H coupling constants by pseudorotational analysis, and rotamer distributions of exocyclic bonds were calculated from the observed homonuclear and heteronuclear coupling constants. The cyclobutane ring configuration (CB) of each isomer was identified, using arguments based upon observed scalar and dipolar couplings. Glycosidic bond conformation was ascertained from nuclear Overhauser enhancements observed between base and deoxyribose protons. Isomer I (S-type class; CB-; SYN-ANTI) and isomer II (N-type class; CB+; ANTI-SYN) exhibit markedly different conformational features. 31P chemical shifts and exocyclic bond rotamer distributions indicate diminished backbone flexibility for both photoproducts relative to parent thymidylyl (3'-5') deoxyuridine. Isomer I (SYN-ANTI) is particularly rigid, while isomer II (ANTI-SYN) maintains some flexibility. Also, 13C spectra were acquired and assigned unequivocally with the aid of short- and long-range two-dimensional heteronuclear shift correlation experiments.

Quantum yields and secondary photoreactions of the photoproducts of dTpdT, dTpdC and dTpdU.

The photoproducts of the dinucleoside monophosphates, dTpdT, dTpdC and dTpdU, have been purified by high performance liquid chromatography and characterized by UV absorption spectroscopy, fast atom bombardment mass spectrometry and by secondary thermal and photoreactions. Four types of photoproducts were analyzed: (1) cyclobutane dimers including cis-syn isomers and two diastereomers of the trans-syn isomers; (2) 6-4 photoadducts and the corresponding Dewar valence isomers; (3) photohydrates comprising two diastereomers and (4) a new photoproduct resembling nucleobase amine adducts, which occurs only for dTpdC. The quantum yields of formation of these photoproducts and for some secondary photoreactions were measured by kinetic analysis of the photoproduct yield as a function of photon fluence. These results indicate that cis-syn cyclobutane dimers are the photoproducts formed with highest efficiency with dT[p]dC dimers being formed with 50-75% the efficiency of dT[p]dT dimers. The 6-4 photoadducts are formed with 5-10% the efficiency of cis-syn cyclobutane dimers and the 6-4 photoadduct of dTpdC is formed two to three times more efficiently than that of dTpdT. Photohydrates are also formed efficiently due to an equilibrium between stacked and unstacked complexes of the dinucleoside monophosphates. It is shown that three of these photoproducts, namely the cyclobutane dimers of dTpdC, the 6-4 photoadducts and the possible nucleobase amine adduct, undergo photolysis in the UV-B region resulting in either photoreversion or secondary photoreaction.

Kinetic analysis of the deamination reactions of cyclobutane dimers of thymidylyl-3',5'-2'-deoxycytidine and 2'-deoxycytidylyl-3',5'-thymidine.

The cyclobutane dimer photoproducts of dTpdC and dCpdT have been produced by acetophenone photosensitization and separated by reverse-phase HPLC. Each dinucleoside monophosphate was shown to produce one cis,syn isomer and two trans,syn isomers. Three of these photoproducts, namely, the cis,syn isomers of dTpdC and dCpdT and one trans,syn isomer (the syn-anti glycosidic isomer) of dTpdC were selected to study the deamination kinetics. Analysis of the pH dependence indicates that the deamination proceeds by the hydrolysis of the imido amide group of the 5,6-saturated cytosine base with the formation of a carbinolamine intermediate. Determination of the kinetic parameters showed that, for these three cyclobutane dimers, the rate-determining step at physiological pH is a cyclobutane dimers, the rate-determining step at physiological pH is a nucleophilic attack of hydroxide ion on the protonated 5,6-saturated cytosine base. The kinetic analysis showed that the cis,syn isomers deaminate approximately 3 times faster than the trans,syn isomer, which is due to a large difference in pKa of the 5,6-saturated cytosine moiety. An electrostatic interaction between the iminium group of cytosine and the carbonyl group of thymine is proposed to account for the increase in pKa for the cis,syn isomers relative to the trans,syn isomer. A similar interaction is proposed to explain the relative difference in reactivity between the cis,syn isomers and the trans,syn isomer with regard to the breakdown of the carbinolamine intermediate.

Phosphoramidon-sensitive effects of big endothelins in the perfused rabbit kidney.

The enzymatic conversion of human big endothelins (1, 2, and 3) to their respective active metabolites (endothelin-1, -2, and -3) was investigated in the perfused rabbit kidney through the pressor- and eicosanoid-releasing properties of these peptides. Intra-arterial bolus injections of endothelin-1 and -2 (5-50 pmol), endothelin-3 (100-250 pmol), and big endothelin-1 and -2 (100-250 pmol) into the kidney produced dose-dependent increases of perfusion pressure, whereas big endothelin-3 was inactive at doses up to 1,000 pmol. Endothelin-1 and -2 (10 nM), endothelin-3 (100 nM), and big endothelin-1 and -2 (100 nM) are potent enhancers of prostacyclin release without inducing any release of thromboxane B2 in the perfused kidney. In contrast, big endothelin-3 did not trigger the release of eicosanoids. A metalloprotease inhibitor, phosphoramidon (100 microM, 60 minutes), reduced the prostanoid release and pressor responses induced by big endothelin-1 and -2 without affecting the response induced by endothelin-1, -2, and -3. These results suggest the presence of a phosphoramidon-sensitive endothelin converting enzyme that converts the precursors of endothelin-1 and -2, but not of endothelin-3, in the renal vasculature of the rabbit.

Yields of radiation-induced main chain scission of poly U in aqueous solution: strand break formation via base radicals.

The G values for single-strand breaks G(ssb) in polyuridylic acid (poly U) have been measured by low-angle laser light scattering in aqueous solutions under various conditions (e.g. in the presence of N2O, Ar and t-butanol). In N2O-saturated solutions at room temperature and pH 5.6, the G(ssb) is 2.3. The efficiency of ssb formation was found to be 41 per cent for OH radicals, 19 per cent for H atoms and congruent to zero for e-aq. On the basis of 20 per cent and less than 5 per cent attack on the sugar moiety by OH radicals and H atoms, respectively, the large G(ssb) values obtained cannot be explained solely as resulting from radicals produced by reaction of OH radicals and H atoms on the sugar moiety. It is therefore proposed that base radicals produced by the reaction of OH radicals or H atoms with the uracil moiety can also lead to chain break formation in poly U via radical transfer to the sugar moiety.