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%). RA patients with serum anti-CCP Ab titers above 60 U/mL had a significantly higher frequency of HLA-DRB1*01 (58.3%) and HLA-DRB1*04 alleles (83.3%). 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.

Asymptomatic infection in individuals from the municipality of Barcelos (Brazilian Amazon) is not associated with the anti-Plasmodium falciparum glycosylphosphatidylinositol antibody response

Anti-glycosylphosphatidylinositol (GPI) antibodies (Abs) may reflect and mediate, at least partially, anti-disease immunity in malaria by neutralising the toxic effect of parasitic GPI. Thus, we assessed the anti-GPI Ab response in asymptomatic individuals living in an area of the Brazilian Amazon that has a high level of malaria transmission. For comparative purposes, we also investigated the Ab response to a crude extract prepared from Plasmodium falciparum, the merozoite surface protein (MSP)3 antigen of P. falciparum and the MSP 1 antigen of Plasmodium vivax (PvMSP1-19) in these individuals and in Angolan patients with acute malaria. Our data suggest that the Ab response against P. falciparum GPI is not associated with P. falciparum asymptomatic infection in individuals who have been chronically exposed to malaria in the Brazilian Amazon. However, this Ab response could be related to ongoing parasitaemia (as was previously shown) in the Angolan patients. In addition, our data show that PvMSP1-19may be a good marker antigen to reflect previous exposure to Plasmodium in areas that have a high transmission rate of P. vivax.

A majority of Brazilian patients with rheumatoid arthritis HLA-DRB1 alleles carry both the HLA-DRB1 shared epitope and anti-citrunillated peptide antibodies

The objective of the present study was to evaluate the contribution of the shared epitope (SE), the rheumatoid arthritis (RA) protection model, and the occurrence of anti-cyclic citrullinated peptide (anti-CCP) antibodies in RA patients from a genetically diverse population. One hundred and forty Brazilian RA patients and 161 matched controls were typed for HLA-DRB1 alleles using amplified DNA hybridized with sequence-specific oligonucleotide probes or primers. Patients were stratified according to the presence or absence of SE (DRB1*0401, *0404, *0405, *0101, *1001, and *1402), of the DERAA alleles (DRB1*0103, *0402, *1102, *1103, *1301, *1302, and *1304), and X (all other alleles). Anti-CCP antibodies were measured by ELISA. The combined frequency of SE-positive alleles was significantly greater (76.4 vs 23.6%, P < 0.0001) than the controls. The SE/SE and SE/X genotypes were over-represented (P < 0.0001, OR = 6.02) and DERAA/X was under-represented in RA patients (P < 0.001, OR = 0.49), whereas the frequencies of the SE/DERAA, X/X and X/DERAA genotypes were not significantly different from controls. The frequency of anti-CCP antibodies was higher in SE-positive patients than in SE-negative patients (64.6 vs 44.7%, P = 0.03; OR = 2.25). Although the Brazilian population is highly miscegenated, the results of this study support the findings observed in most genetically homogeneous populations with RA; however, they are not mutually exclusive but rather complementary. The participation of DRB1-DERAA alleles in protection against RA was also observed (OR = 0.4; 95%CI = 0.23-0.68).

Anti-cyclic citrullinated peptide antibodies and rheumatoid factor in leprosy patients with articular involvement

The objective of the present research was to evaluate the usefulness of anti-cyclic citrullinated peptide (anti-CCP) antibodies and the IgM rheumatoid factor (IgM RF) test for the differential diagnosis of leprosy with articular involvement and rheumatoid arthritis (RA). Anti-CCP antibodies and IgM RF were measured in the sera of 158 leprosy patients (76 with and 82 without articular involvement), 69 RA patients and 89 healthy controls. Leprosy diagnosis was performed according to Ridley and Jopling classification criteria and clinical and demographic characteristics of leprosy patients were collected by a standard questionnaire. Leprosy patients with any concomitant rheumatic disease were excluded. Serum samples were obtained from all participants and frozen at _20°C. Measurement of anti-CCP antibodies and IgM RF were performed by ELISA, using a commercial second-generation kit, and the latex agglutination test, respectively. Anti-CCP antibodies and IgM RF were detected in low frequencies (2.6 and 1.3%, respectively) in leprosy patients and were not associated with articular involvement. Among healthy individuals both anti-CCP antibodies and IgM RF were each detected in 3.4% of the subjects. In contrast, in the RA group, anti-CCP antibodies were present in 81.2% and IgM RF in 62.3%. In the present study, both anti-CCP antibodies and IgM RF showed good positive predictive value for RA, helping to discriminate between RA and leprosy patients with articular involvement. However, anti-CCP antibodies were more specific for RA diagnosis in the population under study.

The presence of anti-citrullinated protein antibodies (ACPA) and rheumatoid factor on patients with rheumatoid arthritis (RA) does not interfere with the chance of clinical remission in a follow-up of 3 years

Autoantibodies in early rheumatoid arthritis (RA) have important diagnostic value. The association between the presence of autoantibodies against cyclic citrullinated peptide and the response to treatment is controversial. To prospectively evaluate a cohort of patients with early rheumatoid arthritis (< 12 months of symptoms) in order to determine the association between serological markers (rheumatoid factor (RF), anti-citrullinated protein antibodies) such as anti-cyclic citrullinated peptide antibodies (anti-CCP) and citrullinated anti-vimentin (anti-Sa) with the occurrence of clinical remission, forty patients diagnosed with early RA at the time of diagnosis were evaluated and followed for 3 years, in use of standardized therapeutic treatment. Demographic and clinical data were recorded, disease activity score 28 (DAS 28), as well as serology tests (ELISA) for RF (IgM, IgG, and IgA), anti-CCP (CCP2, CCP3, and CCP3.1) and anti-Sa in the initial evaluation and at 3, 6, 12, 18, 24, and 36 months of follow-up. The outcome evaluated was the percentage of patients with clinical remission, which was defined by DAS 28 lower than 2.6. Comparisons were made through the Student t test, mixed-effects regression analysis, and analysis of variance (significance level of 5%). The mean age was 45 years, and a female predominance was observed (90%). At the time of diagnosis, RF was observed in 50% of cases (RF IgA-42%, RF IgG-30%, and RF IgM-50%), anti-CCP in 50% (no difference between CCP2, CCP3, and CCP3.1) and anti-Sa in 10%. After 3 years, no change in the RF prevalence and anti-CCP was observed, but the anti-Sa increased to 17.5% (P = 0.001). The percentage of patients in remission, low, moderate, and intense disease activity, according to the DAS 28, was of 0, 0, 7.5, and 92.5% (initial evaluation) and 22.5, 7.5, 32.5, and 37.5% (after 3 years). There were no associations of the presence of autoantibodies in baseline evaluation and in serial analysis with the percentage of clinical remission during follow-up of 3 years The presence of autoantibodies in early RA has no predictive value for clinical remission in early RA.

Influence of anti-infliximab antibodies and residual infliximab concentrations on the occurrence of acquired drug resistance to infliximab in rheumatoid arthritis patients

Infliximab (IFX) can be immunogenic for humans and lead to the formation of antibodies against IFX (anti-IFX Ab), which could induce acquired IFX resistance.

Anti-HIV state but not apoptosis depends on IFN signature in CD4+ T cells

To gain insights into the molecular mechanisms underlying early host responses to HIV in the CD4(+) T cell target population, we examined gene expression in CD4(+) T cells isolated 24 h after ex vivo HIV infection of lymphocyte aggregate cultures derived from human tonsils. Gene profiling showed a distinct up-regulation of genes related to immune response and response to virus, notably of IFN-stimulated genes (ISGs), irrespective of the coreceptor tropism of the virus. This mostly IFN-alpha-dependent gene signature suggested the involvement of plasmacytoid dendritic cells, a principal component of the antiviral immune response. Indeed, depletion of plasmacytoid dendritic cells before HIV inoculation abrogated transcriptional up-regulation of several ISGs and resulted in increased levels of HIV replication. Treatment with a blocking anti-IFN-alphaR Ab yielded increased HIV replication; conversely, HIV replication was decreased in pDC-depleted cultures treated with IFN-alpha. Among up-regulated ISGs was also TRAIL, indicating a potential role of the IFN signature in apoptosis. However, a blocking anti-TRAIL Ab did not abrogate apoptosis of CD4(+) T cells in CXCR4-tropic HIV-infected cultures, suggesting the involvement of pathways other than TRAIL mediated. We conclude that acute HIV infection of lymphoid tissue results in up-regulation of ISGs in CD4(+) T cells, which induces an anti-HIV state but not apoptosis.

Crystal structure of an anti-carbohydrate antibody directed against Vibrio cholerae O1 in complex with antigen: Molecular basis for serotype specificity

The crystal structure of the murine Fab S-20-4 from a protective anti-cholera Ab specific for the lipopolysaccharide Ag of the Ogawa serotype has been determined in its unliganded form and in complex with synthetic fragments of the Ogawa O-specific polysaccharide (O-SP). The upstream terminal O-SP monosaccharide is shown to be the primary antigenic determinant. Additional perosamine residues protrude outwards from the Ab surface and contribute only marginally to the binding affinity and specificity. A complementary water-excluding hydrophobic interface and five Ab–Ag hydrogen bonds are crucial for carbohydrate recognition. The structure reported here explains the serotype specificity of anti-Ogawa Abs and provides a rational basis toward the development of a synthetic carbohydrate-based anti-cholera vaccine.

Auto-antibodies do not influence development of atherosclerotic plaques in rheumatoid arthritis

Background: Many questions remain unanswered about premature atherosclerosis in rheumatoid arthritis (RA). Besides inflammation, some studies have suggested the role of autoantibodies on its pathogenesis. Objective: The aim of this study was to investigate the presence of antibodies against phospholipids, beta2-glycoproteinl (beta2-gpl), lipoprotein lipase, and heat shock proteins (Hsp) in RA patients and to evaluate their possible association with subclinical carotid atherosclerosis. Methods: Seventy-one RA patients and 53 age- and sex-matched controls were selected to perform anticardiolipin antibodies (aCL) (IgG and IgM), anti-beta2-gpl (IgG, IgM, and IgA), anti-lipoprotein lipase (anti-LPL), anti-Hsp 60, and anti-Hsp 65 by ELISA tests. Intima-medial thickness (IMT) of common carotid and presence of plaques were assessed by high-resolution B-mode ultrasonography. Exclusion criteria were smoking, diabetes, and arterial hypertension. Lipoproteins, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and fibrinogen levels, as well as health assessment questionnaire (HAQ) and disease activity score (DAS) 28 were also evaluated. Results: Age (48.93 +/- 12.31 vs. 45.37 +/- 9.37 years; p = 0.20) and body mass index (BMI) (p = 0.69) were similar in RA and controls, as well as female gender (p = 0.56). The mean IMT was similar between RA and controls (0. 721 +/- 0.16 vs. 0.667 +/- 0.14 turn, p = 0.07) but the frequency of plaques was higher in RA (14.1% vs. 1.9%; p = 0.02). In RA patients, IMT measurements did not differ according to the presence or absence of these antibodies: IgG aCL (0.62 +/- 0.64 vs. 0.72 +/- 0.17 mm, p = 0.24), IgM aCL (0.65 +/- 0.79 vs. 0.73 +/- 0.17 mm, p = 0.33), anti-Hsp 60 (0.78 +/- 0.20 vs. 0.71 +/- 0.16 mm, p = 0.27), anti-Hsp 65 (0.73 +/- 0.16 vs. 0.72 +/- 0.17 mm, p = 0.77), IgG anti-beta2-gpl (0.73 +/- 0.16 vs. 0.71 +/- 0.17 mm, p = 0.72), and anti-CCP (0.71 +/- 0.16 vs. 0.76 +/- 0.20 mm, p = 0.36). In addition, IMT did not correlate with antibodies titers: IgG aCL (r = -0.09, p = 0.47), IgM aCL (r = - 0.15, p = 0.21), anti-Hsp 60 (r = 0.10, p = 0.42), anti-Hsp 65 (r = 0.05, p = 0.69), IgG anti-beta2-gpl (r = - 0.07, p = 0.57), IgM anti-beta2-gpl (r = - 0.05, p = 0.69), IgA anti-beta2-gpl (r = 0.03, p = 0.79), and anti-CCP (r = - 0.07, p = 0.57). RA patients with plaques had a significantly higher age compared to those without plaques (p = 0.001), as well as higher mean IMT (p < 0.001), total cholesterol (p = 0.001), and LDL (p = 0.003). Conclusions: In RA a clear association between all autoantibodies studied herein and increased IMT or presence of plaques was not observed. The great prevalence of carotid atherosclerosis in RA was related to age, total and LDL cholesterol, as identified in normal population. (c) 2008 Elsevier Masson SAS. All rights reserved.

Autoantibodies as predictors of biological therapy for early rheumatoid arthritis

Introduction: The association between serological markers with the need of biological therapy for early rheumatoid arthritis (ERA) is not known, with few available data addressing this question. Objectives: To prospectively evaluate a cohort of patients with ERA (less than 12 months of symptoms) in order to determine the possible association between serological markers (rheumatoid factor (RF), anti-cyclic citrullinated peptide antibodies (anti-CCP), and citrullinated anti-vimentin (anti-Sa) with parameters of therapeutic outcome (this later defined by the need of introducing biological therapy). Patients and methods: Forty patients with early RA were evaluated at the time of diagnosis and have been followed for 3 years, in use of standardized therapeutic treatment. Demographic and clinical data were recorded, as well as serology tests (ELISA) for RF (IgM, IgG and IgA), anti-CCP (CCP2, CCP3 and CCP3.1) and anti-Sa in the initial evaluation and at 3, 6, 12, 18, 24 and 36 months of follow-up. As outcomes of the RA development, the need or not for biological therapy during the follow-up period were considered. Comparisons were made through the Student t test, mixed-effects regression analysis and analysis of variance (significance level of 5%). Results: The mean age was 45 (+/- 12) years; a female predominance was observed (90%). At the time of diagnosis, RF was observed in 50% of cases (RF IgA - 42%, RF IgG - 30% and RF IgM - 50%), anti-CCP in 50% (no difference between CCP2, CCP3 and CCP3. 1) and anti-Sa in 10%. After 3 years, no change in the RF prevalence neither in the anti-CCP was observed, but the anti-Sa increased to 17.5% (p = 0.001). Biological therapy was necessary in 22.5% of patients. The mean RF IgA and anti-CCP 2 levels during the 3 years were higher among patients who needed biological therapy (p <0.05 for both). Conclusion: Higher titles of RF and anti-CCP over time were associated with the need for biological therapy.

Differential gene expression of peripheral blood mononuclear cells from rheumatoid arthritis patients may discriminate immunogenetic, pathogenic and treatment features

This study aimed to evaluate the association between the differential gene expression profiling of peripheral blood mononuclear cells of rheumatoid arthritis patients with their immunogenetic (human leucocyte antigen shared-epitope, HLA-SE), autoimmune response [anti-cyclic citrullinated peptide (CCP) antibodies], disease activity score (DAS-28) and treatment (disease-modifying antirheumatic drugs and tumour necrosis factor blocker) features. Total RNA samples were copied into Cy3-labelled complementary DNA probes, hybridized onto a glass slide microarray containing 4500 human IMAGE complementary DNA target sequences. The Cy3-monocolour microarray images from patients were quantified and normalized. Analysis of the data using the significance analysis of microarrays algorithm together with a Venn diagram allowed the identification of shared and of exclusively modulated genes, according to patient features. Thirteen genes were exclusively associated with the presence of HLA-SE alleles, whose major biological function was related to signal transduction, phosphorylation and apoptosis. Ninety-one genes were associated with disease activity, being involved in signal transduction, apoptosis, response to stress and DNA damage. One hundred and one genes were associated with the presence of anti-CCP antibodies, being involved in signal transduction, cell proliferation and apoptosis. Twenty-eight genes were associated with tumour necrosis factor blocker treatment, being involved in intracellular signalling cascade, phosphorylation and protein transport. Some of these genes had been previously associated with rheumatoid arthritis pathogenesis, whereas others were unveiled for future research.

The fluorolysis assay, a highly sensitive method for measuring the cytolytic activity of T cells at very low numbers

We have developed a highly sensitive cytolysis test, the fluorolysis assay, as a simple nonradioactive and inexpensive alternative to the standard Cr-51-release assay. P815 cells were stably transfected with a plasmid expressing the enhanced green fluorescent protein (EGFP) gene. These target cells were coated with or without cognate peptide or anti-CD3 Ab and then incubated with CD8(+) T cells to allow antigen-specific or nonspecific lysis. The degree of target cell lysis was measured using flow cytometry to count the percentage of viable propidium iodide(-) EGFP(+) cells, whose numbers were standardized to a reference number of fluorochrome-linked beads. By using small numbers of target cells (200-800 per reaction) and extended incubation times (up to 2 days), the antigen-specific cytolytic activity of one to two activated CD8(+) T cells of a CTL line could be detected. The redirected fluorolysis assay also measured the activity of very few ( greater than or equal to6) primary CD8(+) T cells following polyclonal activation. Importantly, antigen-specific lysis by small numbers ( greater than or equal to 25) of primary CD8(+) T cells could be directly measured ex vivo. This exquisite sensitivity of the fluorolysis assay, which was at least 8-33-folds higher than an optimized 51 Cr-release assay, allows in vitro and ex vivo studies of immune responses that would otherwise not be possible due to low CTL numbers or frequencies. (C) 2002 Elsevier Science B.V. All rights reserved.

Essential role of CD8 palmitoylation in CD8 coreceptor function.

To investigate the molecular basis that makes heterodimeric CD8alphabeta a more efficient coreceptor than homodimeric CD8alphaalpha, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8beta and that this allows partitioning of CD8alphabeta, but not of CD8alphaalpha, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8beta abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3zeta in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8beta is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.

Additives and Protein-DNA Combinations Modulate the Humoral Immune Response Elicited by a Hepatitis C Virus Core-encoding Plasmid in Mice

Humoral and cellular immune responses are currently induced against hepatitis C virus (HCV) core following vaccination with core-encoding plasmids. However, the anti-core antibody response is frequently weak or transient. In this paper, we evaluated the effect of different additives and DNA-protein combinations on the anti-core antibody response. BALB/c mice were intramuscularly injected with an expression plasmid (pIDKCo), encoding a C-terminal truncated variant of the HCV core protein, alone or combined with CaCl2, PEG 6000, Freund's adjuvant, sonicated calf thymus DNA and a recombinant core protein (Co.120). Mixture of pIDKCo with PEG 6000 and Freund's adjuvant accelerated the development of the anti-core Ab response. Combination with PEG 6000 also induced a bias to IgG2a subclass predominance among anti-core antibodies. The kinetics, IgG2a/IgG1 ratio and epitope specificity of the anti-core antibody response elicited by Co.120 alone or combined with pIDKCo was different regarding that induced by the pIDKCo alone. Our data indicate that the antibody response induced following DNA immunization can be modified by formulation strategies.

Extracellular cyclophilins contribute to the regulation of inflammatory responses.

The main regulators of leukocyte trafficking during inflammatory responses are chemokines. However, another class of recently identified chemotactic agents is extracellular cyclophilins, the proteins mostly known as receptors for the immunosuppressive drug, cyclosporine A. Cyclophilins can induce leukocyte chemotaxis in vitro and have been detected at elevated levels in inflamed tissues, suggesting that they might contribute to inflammatory responses. We recently identified CD147 as the main signaling receptor for cyclophilin A. In the current study we examined the contribution of cyclophilin-CD147 interactions to inflammatory responses in vivo using a mouse model of acute lung injury. Blocking cyclophilin-CD147 interactions by targeting CD147 (using anti-CD147 Ab) or cyclophilin (using nonimmunosuppressive cyclosporine A analog) reduced tissue neutrophilia by up to 50%, with a concurrent decrease in tissue pathology. These findings are the first to demonstrate the significant contribution of cyclophilins to inflammatory responses and provide a potentially novel approach for reducing inflammation-mediated diseases.