Shashi Anand

Gene expression of cytokines (TNF-α, IFN-γ), serum profiles of IL-17 and IL-23 in paediatric systemic lupus erythematosus:

Objective: Paediatric systemic lupus erythematosus (pSLE) exhibits an aggressive clinical phenotype and severe complications commonly renal involvement. This could be reflective of the ongoing chronic pro-inflammatory cytokine milieu. We examined relative gene expression of tumour necrosis factor-alpha (TNF-α), interferon-γ (IFN-γ) and serum levels of interleukin-17 (IL-17) and IL-23 and their association with SLEDAI (SLE disease activity index) score and organ manifestations in pSLE. Methods: We enrolled 40 pSLE patients (age 5–16 years, on treatment) and 20 age-matched healthy controls. Relative gene expression levels of IFN-γ and TNF-α in the peripheral blood were determined by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). β actin gene was used for normalization of gene expression. Serum levels of IL-17 and IL-23 were determined by solid phase sandwich ELISA. Statistical analysis were carried out for comparing (Mann-Whitney U test) and correlating data (Univariate, multivariate analysis and Pearson correlation test) with SLEDAI scores and clinical manifestations. Results: Over-expression of TNF-α and IFN-γ was found in 90% (36/40) and 80% (32/40) of pSLE patients, respectively. The relative gene expression of TNF-α and IFN-γ were significantly correlated with renal manifestations (p < 0.05). Further, relative expression of IFN-γ gene correlated significantly with skin manifestations and SLEDAI (p < 0.05). Serum levels of IL-17 (766.95 ± 357.83 pg/ml) and IL-23 (135.4 ± 54.23 pg/ml) in pSLE were significantly higher than in controls (IL-17, 172.7 ± 39.19 pg/ml and IL-23, 21.15 ± 10.99 pg/ml) (p < 0.05). Patients with cutaneous (p = 0.002) and haematological involvement (p = 0.003) had high serum IL-17 levels. Serum IL-17 levels correlated with SLEDAI (r = 0.447; p < 0.05). Conclusions: In this preliminary study, we observed a persistent, strong pro-inflammatory cytokine milieu in pSLE patients which reflects ongoing inflammatory damage in different organs. The gene expression profile of these cytokines may be used for assessing organ involvement in pSLE. IL-17 may also serve as a prognostic marker in pSLE. However, longitudinal studies on treatment of naïve patients are required to corroborate these findings.

Cardiac abnormalities in acute organophosphate poisoning

Background. Potentially lethal cardiac complications can occur in patients with acute organophosphate poisoning (OPP) and may be overlooked. Patients and Methods. Thirty-six patients with acute OPP were studied. Clinical features and the nature of compound involved were recorded. The QT interval was plotted against heart rate to determine the risk for Torsades de Pointes using the Fossa nomogram. Echocardiography was undertaken in 29 patients. Twenty-four-hour Holter monitoring was performed on day 1 in five patients. Thirteen died. Necropsy was performed and hearts were studied both grossly and microscopically. Results. Gross examination of the heart in 13 cases revealed cardiac discoloration or blotchiness in 12, patchy pericarditis in six, auricular thrombus in six, right ventricular hypertrophy in four, and dilatation in three. On histopathology, all 13 cases had myocardial interstitial edema and vascular congestion, eight had patchy interstitial inflammation, two had patchy myocarditis, and six had a mural thrombus. Sinus tachycardia was the most common electrocardiographic abnormality. The others were corrected QT interval prolongation, ST–T changes, U waves, and ventricular premature contractions. Echocardiography in 29 patients showed minor abnormalities in 10. On Holter monitoring, episodic tachycardia and ST–T changes were observed in four, QT prolongation in three, and episodic bradycardia in two. Conclusions. Patchy myocardial involvement as a result of direct cardiac toxicity could be one of the factors responsible for serious cardiac complications. As myocardial involvement is patchy, it may not be manifest clinically or on echocardiography. Continuous cardiac monitoring should be undertaken to detect dynamic cardiac changes.

Serum proteomic profiling in granumomatosis with polyangiitis using two-dimensional gel electrophoresis along with matrix assisted laser desorption ionization time of flight mass spectrometry.

The present study is a proteomic approach to find differentially expressed proteins in sera of limited and systemic subsets of active disease versus their remitting state in patients with granulomatosis with polyangiitis (GPA) and their correlation with disease activity.

A comparative proteomic study of sera in paediatric systemic lupus erythematosus patients and in healthy controls using MALDI-TOF-TOF and LC MS–A pilot study

BackgroundPaediatric systemic lupus erythematosus (pSLE) exhibits an aggressive clinical phenotype with severe complications and overall poor prognosis. The aim of this study was to analyse differential expression of low molecular weight (LMW) serum protein molecules of pSLE patients with active disease in comparison to sera of healthy age matched controls. Further, some of the differential expressed spots were characterised and identified by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and liquid chromatography (LC-MS).Methods2D-PAGE was performed using pooled sera of active pSLE and age matched healthy controls. Gels were silver-stained and differentially expressed protein spots were detected by automated image master platinum 2D software. 79 ± 17 protein spots were detected for control gels and 78 ± 17 protein spots for patient gels. Of these eleven protein spots were selected randomly and characterized by MALDI-TOF MS (five protein spots) and LC MS (six protein spots) techniques.ResultsOut of the 11 protein spots, 5 protein spots were significantly upregulated viz., leiomodin 2 (LMOD2); epidermal cytokeratin 2; immunoglobulin kappa light chain variable region; keratin 1 and transthyretin (TTR). Three protein spots were significantly down regulated e.g., apolipoprotein A1 (APOA1); chain B human complement component C3c; campath antibody antigen complex. Two protein spots (complement component C3; retinol binding protein (RBP) were found to be expressed only in disease and one protein spot cyclohydrolase 2 was only expressed in controls.ConclusionsWe conclude that 2-D maps of patients with active pSLE and controls differ significantly. In this pilot study, using proteomic approach we have identified differential expressed proteins (of LMW) e.g., RBP, LMOD 2, TTR, Component C3c Chain B and APO A1. However, in future, further studies need to confirm the physiological and pathological role of these proteins in similar cohorts of pSLE.

HLA DRB1 Alleles Discriminate the Manifestation of Autoimmune Hepatitis as Type 1 or Type 2 in North Indian Population

BACKGROUND Autoimmune hepatitis is a polygenic disorder of unknown etiology, where genetic factors affect the occurrence and clinical phenotype of the disease. It has been reported as a rare disease entity in the Indian subcontinent. This study was undertaken to investigate the association of HLA alleles with autoimmune hepatitis type 1 and type 2 in north Indian population and to analyze if distinct human leukocyte antigen (HLA) alleles help in characterization of the subtypes of autoimmune hepatitis. METHODS Sixty-eight patients with autoimmune hepatitis and 128 healthy controls were recruited in the study. Out of 68 patients, 55 were diagnosed with autoimmune hepatitis type 1 and 13 with autoimmune hepatitis type 2. The patients and the controls were typed for HLA class II alleles by PCR-SSP method. RESULTS HLA DRB1*04 and DRB1*08 were found to be significantly associated with autoimmune hepatitis type 1 in north Indian population. It was also observed that DRB1*04, DRB1*13 were significantly associated with pediatric autoimmune hepatitis type 1 and DRB1*08 was significantly associated with adult autoimmune hepatitis type 1. DRB1*14 was significantly associated with autoimmune hepatitis type 2. CONCLUSION The study indicates that autoimmune hepatitis in north Indian population is associated with HLA alleles that may help to discriminate the subtypes as autoimmune hepatitis type 1 and type 2. The study also highlights the ethnic variations in the Indian subcontinent in context to the genetic association of HLA with autoimmune diseases.

Cellular immune response and cytokine profile among hepatitis C positive living donor renal transplant recipients.

Background. Hepatitis C virus (HCV) infection is prevalent among renal transplant recipients. Methods. Twenty-five recipients of living donor renal transplantation with HCV (group I) and without HCV (group II) were serially monitored at three time points, that is, pretransplant, day 10, and 6 months posttransplant. Phenotypic characterization of T-cell subsets and natural killer cells was performed by flow cytometric immunophenotyping. Cytometric bead array immunoassay was used to simultaneously measure six cytokines (interleukin [IL]-2, IL-4, IL-5, IL-10, tumor necrosis factor-&agr;, and interferon-&ggr;) from phytohemagglutin-stimulated culture supernatants, and transforming growth factor (TGF)-&bgr;1 levels were determined by ELISA. Real-time polymerase chain reaction method was used to determine the serum viral load among group I patients at three time points. Results. Group I patients on day 10 posttransplant showed a significant increase in T cells subsets with reduced interferon-&ggr; and increased TGF-&bgr;1 levels. A significantly increased CD8+ T cells and TGF-&bgr; levels were seen at 6-month posttransplant among group I patients. Multivariate linear regression analysis showed TGF-&bgr; and tumor necrosis factor-&agr; as the most significant predictors affecting early (day 10) and late (6 months) posttransplant viral load, respectively. Conclusion. After an initial increase in the viral load immediately posttransplantation, there is a reduction in viral load. A concomitant timed dissection of the immune response shows a complex interactive environment in which, despite immunosuppression, not only the antiviral immune response persists but the virus is also able to modulate the host immune response for its survival. Per se, HCV does not adversely affect the allograft or patient outcome in this case–control study.

Is Psoriasis an autoimmune disease: interpretations from an immunofluorescence based study

Psoriasis is a multisystem, immune‐mediated inflammatory disease. Some authors have proposed an autoimmune basis for psoriasis; however, till date, it has not been definitely established. This study was conducted to explore the autoimmune nature of psoriasis.

A molecular marker of disease activity in autoimmune liver diseases with histopathological correlation; FoXp3/RORγt ratio

Autoimmune liver diseases (AILDs) encompass a group of diseases with variable clinicopathological manifestations. Th17 and Treg cells have roles in the pathogenesis of AILDs with a balance shifted towards a relative increase in activity of the Th17 cells. In this study, the balance between the transcription factors of Treg and Th17 cells (FoXp3 and RORγt) was sought as a molecular marker of disease activity and to highlight the pathogenesis. The peripheral blood samples of 46 treatment‐naive patients were collected and RNA was extracted. Real time PCR was performed and the ratio of gene expression was calculated. Histopathology of 18 patients was obtained and the activity score of these biopsies were also corroborated with their respective molecular (FoXp3/RORγt) (FRGT=FoXp3‐ROR Gamma T) ratio. The FRGT ratio in healthy individuals was close to 1 and in disease the ratio changed significantly. This ratio (FRGT) was not significantly different in different varieties of AILD or in adult or paediatric form of the disease. However, the ratio remained consistently below 1 (mean 0.3) in acute disease and high (mean 224.7) in chronic or asymptomatic form of the disease (p < 0.001). The histopathological activity score also significantly correlated with the ratio. This signified the relative excess of Th17 (RORγt) in active disease as compared to Treg (FoXp3) and the reverse in chronic form. This ratio can be an important peripheral molecular marker to assess the disease activity without the necessity of performing a liver biopsy.

Serial measurements of hepatitis C viral load by real-time polymerase chain reaction among recipients of living-donor renal transplants: a short-term follow-up study from a single center.

There is a high prevalence of hepatitis C virus (HCV) infection among immunosuppressed patients including renal transplant recipients. The study investigated serum viral loads for up to 6 months posttransplantation among these patients. Serum viral load was serially monitored using real-time polymerase chain reaction (PCR) in 25 HCV-positive renal transplant recipients pretransplantation as well as day 10 and 6 months posttransplantation. A liver biopsy specimen obtained under vision at the time of transplantation was analyzed for viral load as well as for histological changes. There was increased viremia at day 10 followed by a significant (2 log) reduction at 6 months posttransplantation. Pretransplantation serum and intrahepatic viral load showed significant positive correlations (r = 0.727; P = .001), the latter also reflecting liver fibrosis score (r = 0.423; P = .05). The findings suggested that serum viral load reflects intrahepatic viral load, which in turn correlates with liver fibrosis. At 6 months posttransplantation, the modulatory effects of immunosuppressive drugs and of the host immune response may lead to a reduced viral load.

Allo-specific immune response profiles indicative of acute rejection in kidney allografts using an in vitro lymphocyte culture-based model.

BackgroundAbility to predict the manner in which a recipient’s immune system would respond to a transplanted graft by analyzing cytokine profiles of the “allograft antigen sensitized” recipient lymphocytes in vitro might provide a means to identify patients at risk to adverse clinical endpoints.MethodsCytokine/chemokine gene expression profiles of peripheral blood mononuclear cells co-cultured with allograft antigen-pulsed macrophages were studied in 49 renal transplant recipients—12 with acute cellular rejection (ACR) with or without antibody-mediated rejection (AMR), 7 with AMR (without ACR), and 30 with stable allografts (SA). An 86-gene inflammatory cytokines and receptors PCR array was used to measure fold changes in gene expression between pulsed and un-pulsed cultures.ResultsOn linear discriminant analysis and multivariate analysis of variance, a gene set comprising C3, CCL3, IL1B, TOLLIP, IL10, CXCL5, ABCF1, CCR3, IL10RB, CXCL1, and IL1R1 differentiated the ACR–AMR from the SA group. Similarly, a gene set comprising IL10, C3, IL37, IL1B, CCL3, CARD18, and TOLLIP differentiated the AMR from the SA group. No significant difference was found between the ACR–AMR vs AMR groups.ConclusionDistinct post in vitro stimulation cytokine profiles at the time of transplantation thus correlated with the occurrence of post-transplantation rejection episodes which indicated feasibility of this in vitro model to assess the recipient’s anti-graft response at an early stage.