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

BACKGROUND: Paediatric 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). METHODS: 2D-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. RESULTS: Out 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. CONCLUSIONS: We 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

Immune responses in patients with HIV infection after vaccination with recombinant Hepatitis B virus vaccine

BACKGROUND: Patients with HIV infection are at risk of co-infection with HBV, as the routes of transmission are shared and thus immunization with HBV vaccine could be protective in them. The aim of the present study was to assess the efficacy of recombinant vaccine in treatment-naive HIV positive patients and healthy controls, and to dissect out differences if any, in different limbs of immune response. METHODS: Forty HIV positive patients and 20 HIV negative controls, negative for HBsAg, HBsAbs and HBcAbs were vaccinated with three doses of 40μg and 20μg of vaccine respectively. Patients were divided into high CD4 and low CD4 group based on CD4+ lymphocytes of 200 and < 200/mm3 respectively. Group II consisted of healthy controls. Detection of phenotypic markers was done by flowcytometry. Cytokine estimation was done by sandwich ELISA. HBsAbs were estimated in serum by ELISA. RESULTS: After vaccination, CD(4)+, CD(8)+ and CD(3)+ cells increased significantly in all the groups. There was no increase in NK cell activity in patients with high CD(4)+ lymphocytes and only a marginal increase in patients with low CD(4)+ lymphocytes (170 to 293/mm3) whereas a marked increase was observed in controls (252 to 490/mm3). After vaccination, although an increase in memory cells was observed in HIV positive patients, yet HBsAb levels were significantly lower than controls (P < 0.05) indicating a functional defect of memory cells in HIV/AIDS patients. Basal IFN-γ levels were also significantly lower in HIV/AIDS patients (P < 0.01). Although the levels increased after vaccination, the peak level remained lower than in controls. HBsAb titers were much lower in HIV positive patients compared to controls. (High CD(4)+ group: 8834 mIU/ml, low CD(4)+ group: 462 mIU/ml Vs. Controls: 16,906 mIU/ml). IL-4 and IL-10 were low in patients. CONCLUSION: Despite a double dose in patients, IL-4 and IL-10, which regulate antibody response, were also lower in patients, and this together with low CD(4)+ counts and lack of T help, accounted for low HBsAb levels. Vaccination in patients with CD(4)+ lymphocytes < 50/mm(3) was ineffective. Thus early immunization is advocated in all HIV positive patients at a stage when they are still capable of mounting an adequate immune respons

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

Abstract Background Paediatric 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). Methods 2D-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. Results Out 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. Conclusions We 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.</p

Renal histology in pauci-immune rapidly progressive glomerulonephritis: 8-year retrospective study

Context: The need to perform reporting of renal biopsies of antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitides in a more uniform manner required relook at our eight-year data. Aims: To document detailed renal histopathology of pauci-immune rapidly progressive glomerulonephritis (RPGN) and also to seek any significant differences in renal histology of C-ANCA-positive, P-ANCA-positive, and ANCA-negative patients. Materials and Methods: A detailed analysis of the histopathologic features of renal biopsies of 48 patients in whom a diagnosis of pauci-immune glomerulonephritis was concluded on renal biopsy and who presented clinically as rapidly progressive renal failure was done. Statistical Analysis Used: One-way ANOVA and Pearson Chi square tests. Results: Compared with ANCA +ve patients, the ANCA -ve patients were much younger (46.85 ± 16.12 years vs 34.28±15.94 years). No significant differences were found between renal lesions of C-ANCA, P-ANCA, and ANCA-negative patients, except for diffuse tubular atrophy which was more severe and more frequently present with P-ANCA positivity (P value=0.013). Conclusions: Pauci-immune RPGN (irrespective of ANCA status) is a relatively rare disorder in patients who are undergoing the renal biopsy at our institute, constituting 2% of all renal biopsies submitted. It is mandatory to have ANCA serology status during reporting of a kidney biopsy showing pauci-immune crescentic or necrotizing glomerulonephritis. Also, if a uniform reporting strategy is followed throughout the country, the studies from this vast country will be comparable