Immune response of S. Typhi-derived Vi polysaccharide and outer membrane protein a conjugate in mice.

Typhoid fever is a serious concern precisely in developing nations. Still investigators are exploring a better conjugate partner for Vi-polysaccharide to develop a more effective vaccine for typhoid fever. Here, we cloned and expressed S. Typhi outer membrane protein A (OmpA). The conjugation of Vi-polysaccharide with OmpA was carried out by the carbodiimide (EDAC) method employing ADH as a linker. Total Ig and IgG generated against OmpA, and Vi polysaccharide was quantified by ELISA. Vi polysaccharide alone induced very low levels of Vi polysaccharide antibody. Vi-OmpA conjugate (Vi-conjugate) elicited a robust immune response compared to Vi polysaccharide alone and showed booster response. Further, IgG was only evoked by Vi-OmpA conjugate, not with Vi polysaccharide alone. OmpA antibody induction in both the Vi-OmpA conjugate and OmpA were similar level. Taken together, we show that OmpA as a carrier protein conjugated to Vi polysaccharide is immunogenic. We predict OmpA antibodies will contribute protection along with antibodies generated by Vi-polysaccharide. Past and current literature supports that OmpA is highly conserved protein not only among Salmonellae but entire Enterobacteriacea family with 96-100% identity.

Prognostic significance of D-dimer, natural anticoagulants and routine coagulation parameters in acute pancreatitis.

AIM: This study was conducted to assess the predictive value of coagulation abnormalities in determining disease severity and prognosis of acute pancreatitis (AP). METHODS: Patients of AP and 25 healthy volunteers were included in this prospective observational study. The final outcomes were disease severity assessed by Computed Tomography Severity Index, Acute Physiological Assessment and Chronic Health Evaluation--II, presence of organ failure and mortality. Prothrombin time (PT), partial thromboplastin time (PTT), thrombin time (TT), fibrinogen, antithrombin-III (AT-III), protein-C, and protein-S levels were assessed on day 0, 3 and 7 of admission. RESULTS: Of the 38 patients included, 13 died. Mean PT and TT were similar between patients and controls on any given day. PTT showed elevation on day 3 and 7 (p = 0.001) compared to controls, although fibrinogen and D-dimer were significantly higher in patients on all days. Protein C and AT-III were significantly lower in patients and more so in non survivors ( (p = 0.001)) than controls. Multiple logistic regression analysis revealed D-dimer levels > or = 400 - 800 ng/ml and AT- III level of < 71% at admission were associated with high mortality (OR 11.2, AUROC 0.70 and OR 16.6, AUROC 0.82 respectively) as well as predicted organ failure. CONCLUSION: Serum D-dimer and antithrombin-III levels can be used to assess disease severity and predict outcome of patients with acute pancreatitis.

Alteration of Mitochondrial Function in Oxidative Stress in Parkinsonian Neurodegeneration: A Cross-Sectional Study.

CONTEXT: Appropriate mitochondrial function and oxidative balance are critical to neuronal survival. Accumulation of reactive oxygen species leads to oxidative stress that can cause free radical damage to biomolecules of the cell components and the molecules in the cellular milieu that eventually lead to a variety of chronic diseases including neurodegenerative disorders. Mitochondrial dysfunction initiates neuronal apoptosis thereby leading to neurodegenerative diseases including Parkinson's disease (PD). AIM: To evaluate oxidative stress vis-a-vis mitochondrial function (Cytochrome C oxidase activity) in PD patients, Parkinson plus syndrome (PPS) patients in comparison with healthy controls (HCs). SETTINGS AND DESIGN: Cross-sectional Study. METHODS: We assessed oxidative stress by chemiluminescence using luminol, and cytochrome c oxidase activity (CCO) by CCO kit using spectrophotometry in PD patients (n = 80), PPS patients (n = 40), and HCs (n = 40). STATISTICAL ANALYSIS: Data were presented as number (%) or mean ± SD/median as approximate. Quantitative baseline variables were compared among the groups using one-way ANOVA and qualitative variables were compared using Chi-square test. The difference in median was compared using Kruskal-Wallis test followed by Post-hoc Bonferronni correction. RESULTS: Compared to HCs (Median 7.53 ± 15.58 RLU/sec/cell), ROS level in PD (14.13 ± 29.5), and PPS (17.43 ± 15.91) patients was significantly higher (P = 0.0029: HC vs, PD & P = 0.0500: HC vs. PPS). Also, ROS in PD patients (14.13 ± 29.5) was higher that PPS patients (17. 43 ± 15.91) but the difference was not statistically significant (P = 0.84). The CCO activity was found to be diminished in PD (Median: 0.025 ± 0.013 units/ml) and PPS patients (0.027 ± 0.008) in comparison to HCs (0.117 ± 0.049). CONCLUSION: Mitochondrial dysfunction and oxidative stress is associated with PD and PPS and may play an important role in etiopathogenesis. Though the cause-effect conundrum has not been comprehensively probed but addressing oxidative stress and mitochondrial damage may serve as an adjunctive therapy for PD and PPS. Iron metabolism as reflected in the red cell indices may aid in differentiating PD from PPS.

Volume, conductivity, and scatter parameters of leukocytes as early markers of sepsis and treatment response.

INTRODUCTION: Morphologic changes in the size and granularity of leukocytes seen in sepsis could be measured using the volume, conductivity, and scatter (VCS parameters) from the automated hematology analyzers. The objective of this study is to find the clinical usefulness of VCS parameters as possible indicators of sepsis and to determine the effect of treatment on these parameters. METHODS: This observational study was conducted in a tertiary level hospital in India. Hemogram and VCS parameters obtained from LH 750 (Beckman coulter, Fullerton, CA) from 134 proven blood culture-positive cases of sepsis were reviewed on the day of culture positivity (day 0), day 3, and day 7 were analyzed and compared with those of samples from otherwise healthy 100 participants. Statistical analysis of data was done, and cutoff value was established using receiver-operator characteristic curve. RESULTS: Out of 134 culture-positive cases, 55.2% (n = 74) Gram-negative and 44.8% (n = 60) Gram-positive bacteria were isolated. The mean neutrophil volume (MNV) and mean monocyte volume (MMV) were higher in the sepsis group compared to that of the control group (165.43 ± 18.21 vs. 140.59 ± 7.6, P = 0.001 for MNV and 179.8 ± 14.16 vs. 164.54 ± 9.6, P = 0.001 for MMV). A significant decrease in MNV and MMV was observed with the initiation of the treatment. Significant changes in scatter and conductivity parameters were also noticed. A cutoff value of 150.2 for MNV gave a sensitivity and specificity of 79.1% and 95%, respectively, with an area under the curve (AUC) of 92.3%. With a cutoff of 168.3, MMV had a sensitivity of 80.6% and specificity of 77.5%, AUC of 83%. CONCLUSION: VCS parameters such as MNV and MMV can be easily obtained by an automated hematology analyzer and could be used for early detection and therapeutic response in sepsis.

Serum S100B levels in patients with depression.

BACKGROUND: The biochemical basis of depression has been related to blood-brain barrier (BBB) allowing/restricting a number of components to enter the brain milieu from the peripheral plasma milieu. S100B has been associated with BBB damage and is used as a marker of its integrity. Several studies have reported that depressive patients have increased levels of S100B in serum and cerebrospinal fluid. MATERIALS AND METHODS: Forty-two confirmed cases of depression, 13-25 years of ages were recruited from the Department of Psychiatry, All India Institute of Medical Sciences during the period from January 2013 to June 2014 along with 42 healthy controls of comparable age and sex. Psychometric evaluation of the patients and controls was done to assess the severity of depression using Beck's Depression Inventory-II and Hamilton Depression Rating Scale. Medical assessment and laboratory investigations were done. Serum S100B levels were measured using Sandwich ELISA. The results obtained were statistically analyzed. RESULTS: Levels of serum S100B were significantly elevated in patients with major depression as compared to controls. Significantly higher levels of S100B were seen only in females as compared to their healthy counterparts. Serum S100B was higher in depressed participants with the recurrent disorder than those with single episode. No correlation of levels of this marker was seen with clinical severity of the patients. It was found that with increased duration of illness for which the patient was being treated with antidepressants, the patients had higher levels of S100B. CONCLUSIONS: Serum S100B can be used as a biomarker of depression.

Alterations in mitochondrial membrane potential in peripheral blood mononuclear cells in Parkinson's Disease: Potential for a novel biomarker.

BACKGROUND: Progressive apoptosis in the dopaminergic neurons of substantia nigra lead to Parkinson's disease. Since neurons require substantially higher supply of energy, their mitochondria have a pivotal status in neuronal survival. These organelles have a key role to play in apoptosis and any impairment thereof may lead to apoptosis mediated cell death. OBJECTIVES: To evaluate and compare the mitochondrial membrane potential (Δψ) in Parkinson's disease patients and healthy controls. METHODS: We evaluated the mitochondrial membrane potential (Δψ) in the peripheral blood mononuclear cells by Flow cytometry using a lipophillic cationic dye JC-1 in Parkinson's disease patients (N = 61) and healthy controls (N = 37). RESULTS: JC-1 fluorescence was measured and represented as percentage positivity i.e., Mean±SEM in FL-2 (representing non-apoptotic aggregates) and FL-1 (indicating apoptotic cell population having depolarized or damaged mitochondria) channels. The ratio of % FL-2 and % FL-1, which is an indicator of cellular mitochondrial membrane potential, was found to be significantly higher in healthy controls (Mean±SEM = 60.48±18.42) as compared to patients (Mean±SEM = 24.30±4.671) in both stimulated and unstimulated conditions. CONCLUSIONS: Mitochondrial membrane potential is altered and hence its evaluation in peripheral blood mononuclear cells may serve as an early marker of apoptosis in PD and, therefore, may pave way for early interventions. Since Δψ has a role in the maintenance of electrochemical gradient, the disruption of which may lead to neuronal apoptosis, Δψ is intricately nested within etiopathogenesis of PD and may prove to be useful in design of diagnostics, prognostics and therapeutics for PD.

Acute Traumatic Endotheliopathy in Isolated Severe Brain Injury and Its Impact on Clinical Outcome.

STUDY DESIGN: Prospective observational cohort. OBJECTIVE: To investigate the difference in plasma levels of syndecan-1 (due to glycocalyx degradation) and soluble thrombomodulin (due to endothelial damage) in isolated severe traumatic brain injury (TBI) patients with/without early coagulopathy. A secondary objective was to compare the effects of the degree of TBI endotheliopathy on hospital mortality among patients with TBI-associated coagulopathy (TBI-AC). METHODS: Data was prospectively collected on isolated severe TBI (sTBI) patients with Glasgow Coma Scale (GCS) ≤8 less than 12 h after injury admitted to a level I trauma centre. Isolated sTBI patients with samples withdrawn prior to blood transfusion were stratified by conventional coagulation tests as coagulopathic (prothrombin time (PT) ≥ 16.7 s, international normalized ratio (INR) ≥ 1.27, and activated partial thromboplastin time (aPTT) ≥ 28.8 s) and non-coagulopathic. Twenty healthy controls were also included. Plasma levels of thrombomodulin and syndecan-1 were estimated by ELISA. With receiver operating characteristic curve (ROC) analysis, we defined endotheliopathy as a syndecan-1 cut-off level that maximized the sum of sensitivity and specificity for predicting TBI-AC. RESULTS: Inclusion criteria were met in 120 cases, with subjects aged 35.5 ± 12.6 years (88.3% males). TBI-AC was identified in 50 (41.6%) patients, independent of age, gender, and GCS, but there was an association with acidosis (60%; p = 0.01). Following isolated sTBI, we found insignificant changes in soluble thrombomodulin (sTM) levels between patients with isolated TBI and controls, and sTM levels were lower in coagulopathic compared to non-coagulopathic patients. Elevations in plasma syndecan-1 (ng/mL) levels were seen compared to control (31.1(21.5-30.6) vs. 24.8(18.5-30.6); p = 0.08). Syndecan-1(ng/mL) levels were significantly elevated in coagulopathic compared to non-coagulopathic patients (33.7(21.6-109.5) vs. 29.9(19.239.5); p = 0.03). Using ROC analysis (area under the curve = 0.61; 95% Confidence Interval (CI) 0.50 to 0.72), we established a plasma syndecan-1 level cutoff of ≥30.5 ng/mL (sensitivity % = 55.3, specificity % = 52.3), with a significant association with TBI-associated coagulopathy. CONCLUSION: Subsequent to brain injury, elevated syndecan-1 shedding and endotheliopathy may be associated with early coagulation abnormalities. A syndecan-1 level ≥30.5 ng/mL identified patients with TBI-AC, and may be of importance in guiding management and clinical decision-making.

Do gene polymorphism in IL-1ß, TNF-α and IL-6 influence therapeutic response in patients with drug refractory epilepsy?

PURPOSE: Pro-inflammatory cytokines may play an important pathophysiological role in patients with epilepsy. To understand the role of genes encoding pro-inflammatory cytokines in epilepsy, this study aimed to evaluate the polymorphisms of the promoter regions of IL-1ß-511C>T (rs16944), TNF-α-308G>A (rs1800629) and IL-6-174G>C (rs1800795) genes and to look into the interaction between these genes in influencing seizure susceptibility, seizure frequency and response to therapy. METHODS: The comparative frequency of polymorphism was determined in rs16944, rs1800629 and rs1800795 using PCR-RFLP in a group of 120 persons with epilepsy (PWE) and 110 ethnically matched healthy subjects of comparable age and sex in the North Indian population. RESULTS: Alleles and genotypes of rs16944, rs1800629 and rs1800795 were not found to influence the odds ratio of having susceptibility to epilepsy. Also gene-gene interaction of possible nine combinations of these genes did not show any positive association with epilepsy. The genotype and allelic frequency of rs1800795 showed a significant association (p<0.05) in seizure frequency (number of seizures/6-months) and drug refractory epilepsy. However, the genotype and allelic frequency of rs16944 and rs1800629 were not found to have such effect. CONCLUSION: This study demonstrates that the rs16944, rs1800629 and rs1800795 polymorphism does not act as a strong susceptibility factor for epilepsy in North Indian population. The genotypic association of rs1800795 with seizure frequency and drug-refractory epilepsy raises the issue that a specific set of polymorphic genes can influence seizures and therapeutic response in epilepsy.