Replication of PTPRC as genetic biomarker of response to TNF inhibitors in patients with rheumatoid arthritis.

Genetic biomarkers could be useful for orienting treatment of patients with rheumatoid arthritis (RA), but none has been convincingly validated yet. Putative biomarkers include 14 single nucleotide polymorphisms that have shown association with response to TNF inhibitors (TNFi) in candidate gene studies and that we assayed here in 755 RA patients. Three of them, in the PTPRC, IL10 and CHUK genes, were significantly associated with response to TNFi. The most significant result was obtained with rs10919563 in PTPRC, which is a confirmed RA susceptibility locus. Its RA risk allele was associated with improved response (B=0.33, P=0.006). This is the second independent replication of this biomarker (P=9.08 × 10(-8) in the combined 3003 RA patients). In this way, PTPRC has become the most replicated genetic biomarker of response to TNFi. In addition, the positive but weaker replication of IL10 and CHUK should stimulate further validation studies.

Juvenile, adult and late-onset systemic lupus erythematosus: a long term follow-up study from a geographic and ethnically homogeneous population.

OBJECTIVES: This paper aims to identify clinical and serological differences, damage accrual and mortality, in juvenile, adult and late-onset SLE. METHODS: We conducted our study with patients fulfilling SLE classification criteria taken from the Hospital Gregorio Marañon Autoimmune Systemic Rheumatic Diseases' Registry (1986 to 2012). Clinical characteristics, laboratory data and therapies used during the course of the disease were analysed with patients divided into 3 groups: juvenile-onset (≤ 18 years), adult-onset (19-50) and late onset (>50 years). RESULTS: Four hundred and forty-five patients were included. Renal disease and cutaneous manifestations were more frequent in the juvenile-onset group at disease onset. During follow-up, juvenile-onset group presented a higher incidence of renal disease, malar rash, Raynaud's phenomenon, cutaneous vasculitis, and neuropsychiatric manifestations than the other two groups. Arthritis and lymphopoenia were more frequent in the adult-onset group. Arterial hypertension and neoplasm were more frequent in the late-onset group. Low serum complement, anti-dsDNA, anti-U1RNP and anti-Sm antibodies were more common in the juvenile-onset group. Patients with late-onset SLE had more damage accrual. Thirty-seven patients (8.3%) died during the study. All-cause mortality was significantly higher in the late-onset group. Age at disease onset >50 years was an independent risk factor for damage accrual (OR, 2.2; 95%CI, 1.1-4.6; p=0.029) and mortality (OR, 2.6; 95%CI, 1.1-6.3; p=0.03). CONCLUSIONS: We found significant differences in clinical and serological profiles between juvenile, adult and late-onset SLE. The most significant of which was a higher prevalence of neuropsychiatric and renal complications as well as different autoantibody signatures for the juvenile-onset group.

Investigating the link between disease activity and infliximab serum levels in rheumatoid arthritis patients.

OBJECTIVES: The aim of this study was to examine the extent to which infliximab (IFX) serum levels impact disease activity in rheumatoid arthritis (RA) patients. METHODS: In this cross sectional study, serum samples were taken prior to drug infusion from 60 RA patients who had been undergoing IFX therapy > 12 months as a first line of biological treatment. Patient IFX levels were tested and then associated with clinical disease activity. Three DAS28 cut-off points, <2.6, <3.2 and <5.1 were used to determine whether detectable IFX levels were any predictor of clinical disease activity. Logistic regression analysis was run to check other possible factors associated with RA clinical outcomes such as MTX concomitant use, CRP and ESR. RESULTS: Sixteen (27%) out of the 60 patients tested negative; 28 (46%) presented subtherapeutic and 16 (27%) therapeutic IFX levels. Median IFX levels were higher in patients either in remission or showing low disease activity than in those with moderate and high disease activity (p=0.014). Significant association was found between IFX levels and clinical disease activity (p=0.001). Detectable levels of IFX shows better sensitivity and specificity to identify patients with DAS28<3.2 than to identify patients with DAS28<2.6 or DAS28<5.1. Conversely, the best DAS28 cut-off to identify detectable/undetectable IFX was 3.19, with AUC under ROC curve 0.804 (Sd.E 0.070), 76% specificity and 83% sensitivity (p<0.001). MTX use, CRP and ESR did not interfere with this association. Seven out of the 8 patients with anti-IFX antibodies presented DAS28>3.2 (p=0.005). CONCLUSIONS: DAS28 and IFX serum levels were shown to have an inverse correlation. Undetectable IFX serum levels were associated to RA patients presenting DAS28>3.2 meaning that DAS28 <3.2 may be useful to clinicians to evaluate patient response to drug therapy.

Evaluation of lymphoproliferative responses by carboxy fluorescein succinimidyl ester assay in heart recipients with infections.

The analysis of proliferative responses using 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) in flow cytometry is widely used to assess lymphocyte function. The aim of this study was to evaluate nonspecific and specific lymphoproliferative responses using CFSE in heart recipients before and after transplantation and their association with the development of infection. We used four-color flow cytometry to measure the response of peripheral CD3+, CD4+, and CD8+ T cells to phytohemagglutinin mitogen (PHA), tetanus toxoid, hepatitis B, and influenza vaccines using a CFSE proliferation assay in 12 heart recipients and 8 healthy control subjects. Recipients were prospectively evaluated. Immunological studies were performed before and at 3 months after transplantation. A 12-month clinical follow-up examination sought to detect the prevalence of severe infectious complications. Heart recipients (infected [n = 7] and uninfected [n = 5]) disclosed significantly lower percentages of proliferative responses than healthy controls against PHA at both study points. Baseline CD3+, CD4+, and CD8+, antitetanus proliferative responses were significantly lower in infected heart recipients than controls. Patients who developed infections displayed significantly lower percentages of CD3+CFSE and CD8+CFSE cells to PHA mitogen at 3 months after transplantation versus those without infections. In conclusion, nonspecific T-cell reactivity to PHA was lower in heart recipients with posttransplantation infections.

Immunization with an HIV-1 immunogen induces CD4+ and CD8+ HIV-1-specific polyfunctional responses in patients with chronic HIV-1 infection receiving antiretroviral therapy.

Development of polyfunctional T lymphocyte responses is critical in the immunological response against HIV-1. Fifty-four HIV-1 infected patients receiving antiretroviral treatment (ART) and immunization with an HIV-1 immunogen or placebo, periodically every 3 months throughout a period of 36 months, were evaluated for the purposes of analysing the development of HIV-1-specific CD4+ and CD8+ responses. A significant increase of proliferating and IFN-gamma producing CD8+ HIV-1-specific T cells, of HIV-1-specific precursor frequencies for CD8+ and for CD4+ T cells and of Gag/pol-specific memory CTL precursors (CTLp) was observed in the immunogen group in comparison to placebo. IL-2 intracellular expression and IFN-gamma and TNF-alpha co-expression in HIV-1-specific CD8+ T cells were also substantially increased in the immunized group. A negative correlation between viral load and CD3+CD4+CFSElow HIV-1-specific lymphoproliferative response and frequency of Gag/pol-specific CTLp was solely observed in the HIV-1 immunogen group. Long-term immunization in patients receiving ART helps to develop HIV-1-specific polyfunctional T cell responses.

Multiple functional Sp1 domains in the minimal promoter region of the neuronal nicotinic receptor alpha5 subunit gene.

The alpha5 subunit is a component of the neuronal nicotinic acetylcholine receptors, which are probably involved in the activation step of the catecholamine secretion process in bovine adrenomedullary chromaffin cells. The promoter of the gene coding for this subunit was isolated, and its proximal region was characterized, revealing several GC boxes located close to the site of transcription initiation (from -111 to -40). Deletion analysis and transient transfections showed that a 266-base pair region (-111 to +155) gave rise to approximately 77 and 100% of the maximal transcriptional activity observed in chromaffin and SHSY-5Y neuroblastoma cells, respectively. Site-directed mutagenesis of five different GC motifs indicated that all of them contribute to the activity of the alpha5 gene, but in a different way, depending on the type of transfected cell. Thus, in SHSY-5Y cells, alteration of the most promoter-proximal of the GC boxes decreased alpha5 promoter activity by approximately 50%, whereas single mutations of the other GC boxes had no effect. In chromaffin cells, by contrast, modification of any of the GC boxes produced a similar decrease in promoter activity (50-69%). In both cell types, however, activity was almost abolished when four GC boxes were suppressed simultaneously. Electrophoretic mobility shift assays using nuclear extracts from either chromaffin or SHSY-5Y cells showed the specific binding of Sp1 protein to fragment -111 to -27. Binding of Sp1 to the GC boxes was also demonstrated by DNase I footprint analysis. This study suggests that the general transcription factor Sp1 plays a dominant role in alpha5 subunit expression, as has also been demonstrated previously for alpha3 and beta4 subunits. Since these three subunits have their genes tightly clustered and are expressed in chromaffin cells, probably as components of the same receptor subtype, we propose that Sp1 constitutes the key factor of a regulatory mechanism common to the three subunits.