Ritu Aggarwal

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.

HLA-B ⁄ 1502 is associated with carbamazepine induced Stevens-Johnson syndrome in North Indian population

The evidence of association between HLA-B(∗)1502 and anticonvulsant induced Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) from the Indian population is scant. Patients with a history of SJS/TEN secondary to carbamazepine or phenytoin were enrolled. The control group comprised of patients who had received carbamazepine/phenytoin for ⩾ 6 months without any adverse cutaneous event. Low-resolution DNA typing for HLA-B and high resolution HLA-B(∗)15 typing was performed. Seventeen patients with history of SJS/TEN secondary to carbamazepine (9) or phenytoin (8) and 50 tolerant controls (carbamazepine-37; phenytoin-13) were enrolled. The mean age of patients and controls was 33.9 ± 11.6 and 28.1 ± 9.9 years, respectively. HLA-B(∗)1502 was observed in 2/9 (22.2%) carbamazepine-SJS/TEN patients and none of the 37 carbamazepine tolerant controls (p = 0.035). HLA-B(∗)1502 was not observed in any of the 8 phenytoin-SJS/TEN patients or the 13 phenytoin tolerant controls. Our data suggests that HLA-B(∗)1502 is a risk factor for carbamazepine induced SJS/TEN. Therefore, HLA-B(∗)1502 testing should be performed prior to initiating carbamazepine in North Indian population.

Mechanism of action of celecoxib on normal and acid-challenged gastric mucosa

Selective COX-2 inhibitor, celecoxib, delays the healing of gastric ulcers by inhibiting prostaglandins synthesis. Therefore, the effect of celecoxib on normal and acid-challenged gastric mucosa was studied. Wistar rats were distributed into four groups: group-1 (vehicle treated), group-2 (celecoxib treated), group-3 (given 0.6N HCl) and group-4 (HCl+celecoxib treated). The gastric mucosa was assessed histopathologically and by evaluating gastric adherent mucus. To assess the role of oxidative stress, the levels of free radicals and antioxidants were measured. The histopathological examination showed mild inflammation in group-2, moderate inflammation in group-3 and severe inflammation in group-4. The results showed an increase in malondialdehyde and a decrease in gastric adherent mucus, nitrite, reactive thiols and glutathione in groups-2-4 as compared to control group. Activity of superoxide dismutase, catalase and glutathione-s-transferase was increased in all the groups except the group-1. The present study suggested that celecoxib aggravated the gastric damage caused by acid which may be mediated by altering the balance between free radicals and antioxidants.

Role of Oxidative Stress in Celecoxib-Induced Renal Damage in Wistar Rats

Celecoxib, a selective cyclo-oxygenase-2 (Cox-2) inhibitor, prevents the formation of prostaglandins, responsible for maintenance of renal function. Celecoxib administration may lead to renal damage. Since free radicals and antioxidant mechanisms play a significant role in renal injury; this study was designed to evaluate the role of oxidative stress in celecoxib-induced renal damage. The administration of celecoxib resulted in moderate and mild tubulointerstitial nephritis in chronic and acute group. The renal function tests were significantly altered only in the chronic group. The results in both the acute and the chronic group showed (1) a significant increase in the lipid peroxidation and in the activities of superoxide dismutase, catalase and glutathione-S-transferase and (2) a decrease in nitrite, reactive thiols and glutathione. In conclusion, our study suggests that chronic administration of celecoxib may have a damaging effect on kidney, as evident through altered histopathology and renal functions. This damage may be mediated by oxidative stress.

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.

Prevalence of human papillomavirus, Epstein-Barr virus, and cytomegalovirus in fine needle aspirates from lung carcinoma: A case-control study with review of literature.

Oncogenic viruses have recently been allied with lung carcinoma, however, the causal association has not been established till date. The study was conducted to determine the prevalence of high‐risk Human papillomavirus (HPV; subtypes 16, 18, 31, 33 and 45), Epstein‐Barr virus (EBV) and cytomegalovirus (CMV) in lung carcinoma using polymerase chain reaction (PCR) on fine needle aspirates.

Characterization of Toll-like receptor transcriptome in squamous cell carcinoma of cervix: A case–control study

OBJECTIVE Human papillomavirus (HPV) is a proven etiological agent for cervical cancer However, not all HPV infections result in cervical cancer. The mechanisms of host immune system to prevent/control HPV infection remain poorly understood. Toll-like receptors (TLRs) are a system of innate immune defense. HPV has been demonstrated to modulate TLR expression and interfere in TLR signaling pathways, leading to persistent viral infection and carcinogenesis. The aim was to study the relative gene expression of TLRs in cervical squamous cell carcinoma (SCC). METHODS Gene expression profile of TLRs 1 to 9 was examined in 30 cervical SCCs and an equal number of normal cervical tissue samples using a PCR array platform. Gene expression studies for TLRs 3 and 7 were validated by western blotting. RESULTS HPV was detected in all cases and in none of the controls (p<0.0001). HPV16 was the preponderant (83.3%) subtype. A significant downregulation in the relative gene expression of TLR3 (p<0.0001), TLR4 (p<0.0005) and TLR5 (p<0.0001) was observed in cases. A significant upregulation for TLR1 was observed (p=0.006). Although TLRs 2, 7, 8 and 9 were upregulated and TLR6 was downregulated, it was not significant. The western blot performed with antibodies against TLRs 3 and 7 confirmed the findings of the gene expression studies. CONCLUSIONS A significant downregulation in the gene expression of TLRs 3, 4 and 5 and upregulation of TLR1 was observed in cervical SCC as compared to controls. Study results evoke the proposition for investigating TLRs 3, 4 and 5 agonists for therapeutic exploration.

Incipient primary biliary cirrhosis/autoimmune hepatitis overlap or hepatitic form of primary biliary cirrhosis: a case report

A 42 year old asymptomatic female detected as incipient Primary Biliary Cirrhosis/Autoimmune Hepatitis overlap during routine checkup. The biochemical profile showed evolution from a mildly deranged liver function test in 2004 along with increased erythrocyte sedimentation rate to a 4 times elevation of alkaline phosphatase in 2006 with mildly deranged alanine transaminase. Autoimmune markers demonstrable were Anti mitochondrial antibody M2 and sp100. Histopathology showed dual features, dominant findings were of autoimmune heptatitis. Features consistent with Primary Biliary Cirrhosis were minimal with an occasional portal tract showing paucity of bile ducts and occasional bile duct proliferation. Human leucocyte antigen DR/DQ genotype was as follows: DRB1*03, DRB1*07, DQB1*02, DQB1*04.

Interleukin-5, interleukin-6, interleukin-8 and tumour necrosis factor-alpha levels obtained within 24-h of admission do not predict high-risk infection in children with febrile neutropenia

PURPOSE Biomarkers that can predict the severity of febrile neutropenia (FN) are potential tools for clinical practice. OBJECTIVE The objective of this study is to evaluate the reliability of plasma interleukin (IL) levels as indicators of high-risk FN. MATERIALS AND METHODS Children with haematological malignancies and FN were enrolled prospectively. A blood sample was obtained within 24-h of admission for estimation of IL-5, IL-6, IL-8 and tumour necrosis factor-alpha (TNF-α) level by the enzyme-linked immunosorbent assay. Patients were stratified into three groups. Group I (low-risk): No focus of infection; Group II: Clinical/radiological focus of infection; Group III: Microbiologically proven infection or FN related mortality. Groups II and III were analysed as high-risk. The cytokines were assessed at three different cut-off levels. RESULTS A total of 52 episodes of FN in 48 patients were evaluated. The mean age was 6 years (range: 2-13). Primary diagnosis included acute lymphoblastic leukaemia (82%), non-Hodgkins lymphoma (13%) and acute myeloid leukaemia (5%). Absolute neutrophil count was < 200 cells/μl in half and 200-500 in 23%. Majority were categorised as Group I (69%), followed by Group II (16%) and III (15%). The range of IL-5 was too narrow and similar in the two risk-groups to be of any relevance. The best sensitivity of TNF-α and IL-6 for high-risk group was 78% and 70%, respectively. The highest specificity observed was 35%. The negative predictive value of IL-6, IL-8 and TNF-α exceeded 80%. CONCLUSION IL-5, IL-6, IL-8 and TNF-α failed as predictors of clinically localised or microbiologically documented infection in children with chemotherapy induced FN. However, IL-6, IL-8 and TNF-α could be useful in excluding the possibility of high-risk infection.