Association of kir2dl2 gene with anti-cyclic citrullinated protein antibodies for serodiagnosis in rheumatoid arthritis

Rheumatoid arthritis is a clinical autoimmune syndrome that causes joint damage. The positive or negative anti-cyclic citrullinated protein (CCP) antibodies serodiagnosis differentiates two subsets of the disease, each with different genetic background. Previous studies have identified associations between KIR genes and rheumatoid arthritis but not with anti-CCP serodiagnosis. Therefore, we investigated the proportion of patients seropositive and seronegative to anti-CCP and its possible association with KIR (killer cell immunoglobulin-like receptor) genes. We included 100 patients with rheumatoid arthritis from western Mexico, who were determined for anti-CCP serodiagnosis by ELISA, and 16 KIR genes were genotyped by PCR-SSP. The proportion of seropositive anti-CCP patients was 83%, and they presented a higher frequency of KIR2DL2 genes than the seronegative group (73.6% vs. 46.2%, p = 0.044) which, in turn, presented a higher KIR2DL2-/ KIR2DL3+ genotype frequency than the first ones (46.2% vs. 17.2%, p = 0.043). These results suggest different KIR genetic backgrounds for each subset of the disease according to anti-CCP serodiagnosis.

La artritis reumatoide es un síndrome clínico autoinmune que causa daño en las articulaciones. El serodiagnóstico positivo o negativo para anticuerpos proteicos anti-cíclicos citrulinados (CCP) diferencia dos subconjuntos de la enfermedad, cada uno con diferente fondo genético. Estudios previos han identificado asociaciones entre los genes killer cell immunoglobulin- like receptor (KIR) y la artritis reumatoide, pero no con el serodiagnóstico de anti-CCP. Por lo tanto, investigamos la proporción de seropositividad y seronegatividad anti-CCP y su posible asociación con genes KIR. Se incluyeron 100 pacientes con artritis reumatoide del occidente de México, a quienes se les determinó su serodiagnóstico anti-CCP por ELISA y también se les realizó genotipificación de 16 genes KIR por PCR-SSP. La proporción de pacientes seropositivos anti-CCP fue del 83% y presentaron una mayor frecuencia génica KIR2DL2 que el grupo seronegativo (73.6% vs. 46.2%, p = 0.044), estos últimos presentaron mayor frecuencia genotípica KIR2DL2-/KIR2DL3+ que los primeros (46.2% vs. 17.2%, p = 0.043). Los resultados sugieren diferente fondo genético KIR para cada subconjunto de la enfermedad, de acuerdo con el serodiagnóstico anti-CCP.

Zinc transporter 8 autoantibodies in patients with type 1 diabetes from a multiethnic population and their first degree relatives / Autoanticorpos transportadores de zinco 8 em pacientes com diabetes tipo 1 de uma população multiétnica e seus parentes de primeiro grau

Objective Zinc transporter 8 autoantibodies (ZnT8A) have been poorly studied in non-Caucasian individuals. We aimed to investigate the prevalence of ZnT8 autoantibodies in patients with T1D and their first degree relatives (FDR) from a multiethnic population, as well as its relation with the insulin (INS) or the protein tyrosine phosphatase non-receptor 22 (PTPN22) gene polymorphisms. Subjects and methods ZnT8A were analyzed in sera from T1D patients (n = 72, mean age of 30.3 ± 11.4 years) of variable duration (15.7 ± 11.8 years) and their FDR (n = 78, mean age of 18.3 ± 9.1 years) by a triple mix Radioligand Binding Assay (RBA) for the ZnT8 autoantibody (ZnT8-RWQ) variants. SNP (single nucleotide polymorphism) for INS and PTPN22 were genotyped. Results The prevalence of ZnT8A was higher in T1D patients than FDR, for ZnT8TripleA (24% vs. 4%,p = 0.001), ZnT8RA (24% vs. 4%, p < 0.001) and ZnT8QA (15% vs. 3%, p = 0.004). All FDR with ZnT8A (n = 3) had at least another positive antibody. Heterozygosis for PTPN22 was associated with a higher frequency of ZnT8TripleA (p = 0.039) and ZnT8RA (p = 0.038). Conclusions ZnT8A is observed in non-Caucasian patients with T1D, even years after the disease onset, as well as in their FDR. In those, there was an overlap between ZnT8A and other T1D antibodies. ZnT8A was associated with PTPN22 polymorphisms. Further longitudinal studies are necessary to elucidate the importance of these findings in the natural history of T1D patients with multiethnic background. .

Objetivo Os autoanticorpos transportadores de zinco 8 (ZnT8A) foram pouco estudados em indivíduos não caucasianos. Nosso objetivo foi investigar a prevalência de autoanticorpos ZnT8 em pacientes com T1D e seus parentes de primeiro grau (PPG) em uma população multiétnica, assim como a sua relação com os polimorfismos genéticos da insulina (INS) ou proteína tirosina fosfatase não receptora tipo 22 (PTPN22). Sujeitos e métodos ZnT8A foram analisados no soro de pacientes com T1D (n = 72, idade média de 30,3 ± 11,4 anos) de duração variável (15,7 ± 11,8 anos) e seus PPG (n = 72, idade média de 30,3 ± 11,4 anos) usando-se um ensaio de competição com radioligantes (RBA) para variantes dos autoanticorpos ZnT8 (ZnT8-RWQ). Os polimorfismos de nucleotídeo único para a INS e PTPN22 foram genotipados. Resultados A prevalência de ZnT8A foi mais alta em pacientes T1D do que nos PPG, para ZnT8TriploA (24% contra 4%, p = 0,001), ZnT8RA (24% contra 4%, p < 0,001) e ZnT8QA (15% contra 3%, p = 0,004). Todos os PPG com ZnT8A (n = 3) apresentaram positividade para pelo menos outro anticorpo. A heterozigose para PTPN22 foi associada a uma frequência mais alta de ZnT8TriploA (p = 0,039) e de ZnT8RA (p = 0,038). Conclusões Os ZnT8A foram observados em pacientes não caucasianos com T1D, mesmo depois de anos do início da doença, assim como em seus PPG. Nos parentes, houve uma sobreposição entre os ZnT8A e outros anticorpos para T1D. Os ZnT8A mostraram-se associados aos polimorfismos PTPN22. São necessários outros estudos longitudinais para se elucidar a importância desses achados na história natural de pacientes com T1D com antecedentes étnicos variados. .

Proteomic data show an increase in autoantibodies and alpha-fetoprotein and a decrease in apolipoprotein A-II with time in sera from senescence-accelerated mice

We evaluated changes in levels by comparing serum proteins in senescence-accelerated mouse-prone 8 (SAMP8) mice at 2, 6, 12, and 15 months of age (SAMP8-2 m, -6 m, -12 m, -15 m) to age-matched SAM-resistant 1 (SAMR1) mice. Mice were sacrificed, and blood was analyzed by 2-dimensional electrophoresis combined with mass spectrometry. Five protein spots were present in all SAMP8 serum samples, but only appeared in SAMR1 samples at 15 months of age except for spot 3, which also showed a slight expression in SAMR1-12 m sera. Two proteins decreased in the sera from SAMP8-2 m, -6 m, and -12 m mice, and divided into 2 spots each in SAMP8-15 m sera. Thus, the total number of altered spots in SAMP8 sera was 7; of these, 4 were identified as Ig kappa chain V region (M-T413), chain A of an activity suppressing Fab fragment to cytochrome P450 aromatase (32C2_A), alpha-fetoprotein, and apolipoprotein A-II. M-T413 is a monoclonal CD4 antibody, which inhibits T cell proliferation. We found that M-T413 RNA level was significantly enhanced in splenocytes from SAMP8-2 m mice. This agreed with serum M-T413 protein alterations and a strikingly lower blood CD4+ T cell count in SAMP8 mice when compared to the age-matched SAMR1 mice, with the latter negatively correlating with serum M-T413 protein volume. Age-related changes in serum proteins favored an increase in autoantibodies and alpha-fetoprotein and a decrease of apolipoprotein A-II, which occurred in SAMP8 mice at 2 months of age and onwards. These proteins may serve as candidate biomarkers for early aging.

Caracterización de los autoanticuerpos en la anemia hemolítica autoinmune secundaria al tratamiento con interferón alfa / Characterization of autoantibodies in autoimmune hemolytic anemia following treatment with interferon alfa

Se estudiaron 13 pacientes con leucemia mieloide crónica y anemia hemolítica autoinmune inducida por el interferón alfa, a quienes se les realizó la detección de inmunoproteínas y la caracterización de las subclases de IgG en los hematíes mediante la prueba de antiglobulina directa (PAD) y la técnica de microplacas. Se aplicó además un ELISA para la cuantificación de inmunoglobulinas en los hematíes. Se detectó la presencia de IgG y C3 en el 53,84 por ciento de los casos, IgG sola en el 23,07 por ciento y en el 15,38 por ciento se identificaron autoanticuerpos IgG e IgA. En 11 pacientes se demostró la presencia de IgG1 y en un caso se identificaron además autoanticuerpos de la subclase IgG3. El ELISA detectó autoanticuerpos en concentraciones de 183 moléculas de IgG por hematíe en un paciente con PAD negativa. En los pacientes con hemólisis de alto grado se encontró una concentración de autoanticuerpos entre 1 500 y 3 180 moléculas de IgG por hematíe, mientras que en los casos con hemólisis de bajo grado se comportó entre 183 y 1 000 moléculas. Se observó una correlación negativa entre las cifras de Hb y los valores de haptoglobina plasmática con el número de moléculas de IgG por hematíe y una correlación positiva entre este último con el conteo de reticulocitos

We studied 13 patients with chronic myeloid leukemia and autoimmune hemolytic anemia induced by interferon alfa. They underwent tests for immune protein detection and characterization of IgG subclasses in RBCs by direct antiglobulin test (PAD) and the microplate technique. Also they were applied ELISA test for quantifying immunoglobulins in the red blood cells. It was detected the presence of IgG and C3 in 53.84 percent of cases, IgG alone in 23.07 percent and in 15.38 percent were identified IgG and IgA autoantibodies. In 11 patients the presence of IgG1 was showed and also in one case the subclass IgG3 autoantibodies was identified. The ELISA detected antibodies at concentrations of 183 IgG molecules per erythrocyte in a patient with negative PAD. In high-grade hemolysis patients, it was found a concentration of autoantibodies between 1 500 and 3 180 molecules of IgG per erythrocyte, while in low-grade hemolysis patients it behaved between 183 and 1 000 molecules. There was a negative correlation between Hb and plasma haptoglobin values with the number of IgG molecules per erythrocyte and a positive correlation between the latter with the reticulocyte count

Fc γ R IIB gene polymorphisms in Indian systemic lupus erythematosus (SLE) patients.

Background & objectives: Receptors for the Fc fragment of immunoglobulin G (Fc γ Rs) represent the link between humoral and cellular immune responses. Polymorphisms in Fc γ Rs have been identified as genetic factors influencing susceptibility to various autoimmune diseases. This study was aimed to identify Fc γ R IIB genotypes in Indian systemic lupus erythematosus (SLE) patients and to correlate these with clinical presentation and autoantibody profile. Methods: Eighty consecutive clinically diagnosed SLE patients were included. SLE patients were classified according to the American College of Rheumatology (ACR) criteria. Disease activity was assessed by Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). PCR-RFLP method was used to detect Fc γ R IIB polymorphism. Results: Of the 80 SLE patients, 53 were LN and 27 were SLE without nephritis. The mean SLEDAI score at evaluation was 6.5 ± 5.8. Among SLE patients Fc γ R IIB genotype frequency was 61.2 per cent for Ile/Thr, 20.0 per cent for Thr/Thr and 18.8 per cent for Ile/Ile as compared to 65, 12.5 and 22.5 per cent respectively among normal population. There was no significant difference for Fc γ R IIB genotypes between SLE and normals. The allele frequency for Thr allele in SLE patients was slightly higher (0.51) than in normals (0.45). Thr allele frequency in LN patients was slightly higher (0.53) than in SLE patients without nephritis (0.49). Though a higher percentages of symptoms like renal manifestations (81.3%), arthritis (62.5%) and oral ulcer (56.3%) were noted in patients with Thr/Thr genotypes, no significant difference was noted when these patients were compared with Ile/Ile and Ile/Thr genotypes. Interpretation & conclusions: The findings of this study indicate towards an involvement of Thr allele with SLE disease severity and clinical presentation in Indian SLE patients. Future study on a large sample is needed to support this finding to understand the association of Fc γ R IIB 232Thr/Thr genotype as a susceptibility factor in SLE.

Effect of the human leukocyte antigen HLA-DRB1 and anti-cyclic citrullinated peptide on the outcome of rheumatoid arthritis patients

Our objective was to determine whether the presence of the human leukocyte antigen HLA-DRB1 locus is associated with production of anti-cyclic citrullinated peptide antibodies (anti-CCP Abs) and to what extent they are associated with increased susceptibility to and severity of rheumatoid arthritis (RA) in Egyptian patients. Twenty-nine RA patients gave informed consent to participate in a case-control study that was approved by the Ain Shams University Medical Ethics Committee. RA disease activity and severity were determined using the simplified disease activity index and Larsen scores, respectively. We used a wide scale national study on the pattern of HLA typing in normal Egyptians as a control study. Anti-CCP Abs and HLA-DRB1 typing were determined for all subjects. The alleles most strongly associated with RA were HLA-DRB1 [*01 , *04 and *06] (41.4 percent). RA patients with serum anti-CCP Ab titers above 60 U/mL had a significantly higher frequency of HLA-DRB1*01 (58.3 percent) and HLA-DRB1*04 alleles (83.3 percent). Significant positive correlations were found between serum and synovial anti-CCP Ab titer, RA disease activity, and severity (r = 0.87, 0.66 and 0.63, respectively; P < 0.05). HLA-DRB1 SE+ alleles [*01 and *04] were highly expressed among Egyptian RA patients. The presence of these alleles was associated with higher anti-CCP Ab titer, active and severe RA disease. Early determination of HLA-DRB1 SE+ alleles and serum anti-CCP Ab could facilitate the prediction of the clinical course and prognosis of RA when first evaluated leading to better disease control.

Asociación de la variante GHRd3 del receptor de la hormona de crecimiento con autoinmunidad en la diabetes tipo 1 / Association of GHRd3 variant of growth hormone receptor gene with autoimmunity in type 1 diabetes

Background: Growth Hormone Receptor (GRH) is expressed in the liver, pancreas, stomach and small intestine. A high expression of GHR mRNA in the mucosal gut suggests a possible role of this receptor on digestive and immune functions. Aim: To investigate the putative effects of the GHRd3 variants on the cytokine profile and distribution of auto-antibodies in children with type 1 diabetes (T1D). Material and Methods: Unrelated unaffected controls (n =192) and incident cases of children with T1D (n =127) were analyzed for GHRd3 polymorphism, cytokine profile and a panel of auto-antibodies. Results: The allele frequency for d3 was 24.8 percent in type 1 diabetics and 34.1 percent in controls (p =NS). Among type 1 diabetic children, the carriers of the GHRd3 polymorphism had significantly higher levels of interleukin-lB than homozygous for the wild type genotype (5.7 and 17.7, pg/ml respectively p <0.015). Carriers of d3 variant had a higher frequency of positive anti-insulin antibodies (anti-IAA) than children without this variant (39.6 and 17.7 percent respectively, p <0.01). Conclusions: The observed frequency of the GHR d3/d3 genotype was comparable to other reports. A relationship between d3 variant and anti-IAA antibodies and interleukin-1ß was observed.

Recurrence of papillary thyroid cancer suspected by high anti-thyroglobulin antibody levels and detection of peripheral blood thyroglobulin mRNA / Recorrência de carcinoma papilífero de tireóide suspeitada devido a presença de títulos elevados de anticorpo antitireoglobulina e detecção de mRNA de tireoglobulina em sangue periférico

The amplification of thyroglobulin (TG) mRNA in peripheral blood of patients with thyroid cancer has been studied for almost one decade, but its real contribution for diagnosis of cancer relapse has not yet been established. In the present paper we report the case of a patient with papillary thyroid cancer with undetectable stimulated serum thyrogobulin levels after thyroid ablation. Follow-up showed the presence of high titers of anti-thyroglobulin antibodies and the presence of TG mRNA in a peripheral blood sample, while cervical ultrasound and thorax and cervical computerized tomography were negative. Reinvestigation confirmed lymph node metastases. Anti-TG antibodies progressively decreased after surgery for metastatic lymph nodes resection followed by radioiodine therapy. Although our recent findings show that patients with positive TG mRNA do not have increased risk of cancer recurrence after 24 months of follow-up, the presence of TG mRNA along with high anti-TG antibodies were important indicators that determined further extensive investigation of tumour relapse in this patient, since positron emission tomography scan was not available at our Institution. A methodological standardization that can distinguish specific from non-specific TG mRNA amplification might be of great interest for the follow-up of differentiated thyroid cancer, especially in patients with high levels of anti-TG antibodies.

A amplificação de mRNA de tireoglobulina (TG) no sangue periférico de pacientes com câncer de tireóide tem sido estudada por quase uma década, mas a sua real contribuição para o diagnóstico do câncer ainda não foi estabelecida. No presente trabalho, relatamos o caso de uma paciente com carcinoma papilífero de tireóide com níveis séricos de TG indetectáveis após ablação com radioiodo. No seguimento apresentou títulos elevados de anticorpos anti-TG e a presença de mRNA TG em uma amostra de sangue periférico, enquanto a ultra-sonografia e as tomografias computorizadas de tórax e cervical foram negativas. Os níveis de anticorpos anti-TG diminuíram progressivamente após ressecção cirúrgica dos linfonodos seguida de terapia com radioiodo. Embora nossos achados recentes mostrem que pacientes com mRNA TG não apresentam risco aumentado de recorrência do câncer após 24 meses de seguimento, a presença de mRNA TG associada a altos títulos de anticorpos anti-TG foram importantes indicadores que determinaram o prosseguimento da investigação para recorrência tumoral nesta paciente, uma vez que não dispunhamos, na ocasião, e PET-Scan em nossa instituição. Uma padronização metodológica que permita distinguir entre amplificação de mRNA TG específica e inespecífica poderá ser de grande interesse no seguimento do carcinoma diferenciado da tireóide, especialmente naqueles com títulos elevados de anticorpos anti-TG.

Biomarkers of susceptibility to type 1 diabetes with special reference to the Indian population.

Type 1 diabetes (T1D) is a polygenic autoimmune disease. Susceptibility to T1D is strongly linked to a major genetic locus that is the major histocompatibility complex (MHC) and several other minor loci including insulin, CTLA4 that contribute to diabetes risk in an epistatic way. MHC harbours genes whose primary function is to govern immune responsiveness. Being the most polymorphic genomic region known in humans, MHC serves as a very exciting minigenome model for studying susceptibility to T1D. We have observed enormous diversity in HLA class I and class II genes in the north Indian population and identified several 'novel alleles' and 'unique haplotypes'. For example, multiple DR3+ve autoimmunity favouring haplotypes have been identified, some of which are unique to the Asian north Indian T1D patients. Our molecular studies have revealed that (i) the classical Caucasian autoimmunity favouring AH8.1 (HLA-A1 B8 DR3) is rare in the Indian population and has been replaced by a variant AH8.1v that differs from the Caucasian AH8.1 at several gene loci, (ii) AH8.2 (HLA-A26 B8 DR3) is the most common DR3 positive haplotype in this population and resembles the Indian AH8.1v rather than Caucasian AH8.1, and (iii) there are additional HLA-DR3 haplotypes HLA-A24 B8 DR3 (AH8.3), A3 B8 DR3 (AH8.4) and A31 B8 DR 3 (AH 8.5) that occur in the Indian population. The studies have led to a hypothesis that AH8.1 and AH8.1v might have co-evolved from a common ancestor but preferential divergence of AH8.2 over AH8.1 leading to survival advantage might have been driven by vigorous pathogenic challenges encountered by the Indian population. These studies have important implications in our understanding of disease pathogenesis, identification of high risk individuals, disease diagnosis, disease management and immunological therapeutic approaches.

The association of the HLA class II antigens with clinical and autoantibody expression in Malaysian Chinese patients with systemic lupus erythematosus.

The frequency of the HLA class II antigens/alleles (HLA-DR, DQ and DP) were studied in 70 Malaysian Chinese patients with systemic lupus erythematosus (SLE) to examine the contribution of these genes to disease susceptibility, their clinical expression and Immunological responses. This was done using modified PCR-RFLP technique. These samples were then compared with 66 ethnically matched controls. We found a strong association of the DQA1*0102 (p corr = 0.032, rr = 3.39), DQB1*0501 (p corr = 0.003, rr = 4.55), *0601 (p corr = 0.006, rr = 4.22) and DPB1* 0901(p corr = 0.02, rr = 4.58) with SLE. Clinically, we found a strong association of DR2 and DQA1*0301 with renal involvement and DQA1*0102 with alopecia. Immunologically, statistical analysis (Chi-square test ) showed a strong association of DQA1*0102 with anti-Ro/La antibodies while DQA1*0301 was observed to be strongly associated with antibodies to ds DNA. DQA1*0102 was found more frequently in those with a later disease onset (30 years of age or above). From these data we suggest that the HLA class II genes play a role in conferring disease susceptibility and clinical and immunological expression.