nNOS induction and NOSIP interaction impact granulopoiesis and neutrophil differentiation by modulating nitric oxide generation.

Nitric oxide (NO), a versatile free radical and a signalling molecule, plays an important role in the haematopoiesis, inflammation and infection. Impaired proliferation and differentiation of myeloid cells lead to malignancies and Hematopoietic deficiencies. This study was aimed to define the role of nNOS derived NO in neutrophil differentiation (in-vitro) and granulopoiesis (in-vivo) using multipronged approaches. The results obtained from nNOS over-expressing K562 cells revealed induction in C/EBPα derived neutrophil differentiation as evident by an increase in the expression of neutrophil specific cell surface markers, genes, transcription factors and functionality. nNOS mediated response also involved G-CSFR-STAT-3 axis during differentiation. Consistent increase in NO generation was observed during neutrophil differentiation of mice and human CD34+ HSPCs. Furthermore, granulopoiesis was abrogated in the nNOS inhibitor treated mice, depicting a decrease in the numbers of BM mature and progenitor neutrophils. Likewise, in vitro inhibition of nNOS in human CD34+ HSPCs indicated an indispensable role of nNOS in neutrophil differentiation. Expression of nNOS inhibitory protein, NOSIP was significantly and consistently decreased during the final stage of differentiation and was linked with the augmentation in NO release. Moreover, neutrophils from CML patients had more NOSIP and less NO generation as compared to the PMNs from healthy individuals. The present study thus indicates a critical role of nNOS, and its interaction with NOSIP during neutrophil differentiation. The study also highlights the importance of nNOS in the neutrophil progenitor proliferation and differentiation warranting investigations to assess its role in the haematopoiesis-related disorders.

Calcitriol attenuates TLR2/IL-33 signaling pathway to repress Th9 cell differentiation and potentially limits the pathophysiology of rheumatoid arthritis.

There is limited information regarding the TLR2 signaling pathway involved in Th9 cell differentiation. The role of calcitriol in regulating TLR2-mediated Th9 cell development is unknown. Thus, we aimed to unravel the TLR2 signaling pathway in Th9 cells and its regulation by calcitriol. We have used n = 5-6 animals for each murine experiment. Human studies involved five healthy volunteers. Moreover, ten healthy individuals and ten RA patients were included in the study. Murine and human Th9 cells were treated with Calcitriol (100 nM) and Pam3CSK4 (2 µg/mL). The number of IL-9+ve cells was determined by flow cytometry. Real-time PCR was used to assess the gene expression. Serum 25(OH)D3 levels were determined by HPLC. We observed that TLR2 signals via IL-33/ST2 in Th9 cells. Increased TLR2 expression associated with increased IL9 expression and augmented disease severity in RA patients. Calcitriol attenuated TLR2 signaling in murine and human Th9 cells. Low serum vitamin D3 level negatively associated with increased IL-9 and TLR2 expression and disease severity in RA patients. Our data suggest a potential role of calcitriol to ameliorate the disease severity of RA patients.

Identifying the role of ASPN and COMP genes in knee osteoarthritis development.

BACKGROUND: Osteoarthritis (OA) is a common cause of musculoskeletal disability among elders and is characterized by late-onset degeneration of articular cartilage. OA affects various joints, commonly hand, knee, and hip, with clinical features that are unique to each joint. This study was initiated to identify and evaluate the role of the ASPN and COMP genes in the development of knee OA. METHODS: A case-control study was carried out involving 500 cases with knee OA (diagnosed by the American College of Rheumatology) and an equal number of healthy controls. Blood was drawn for genomic DNA isolation. PCR-RFLP and TaqMan assay methods were used to identify the SNPs. mRNA and protein expression of genes were carried out in peripheral blood lymphocytes (PBLs) by RT-PCR and Western immunoblotting. The data obtained were analyzed for the statistical significance between control and case groups. RESULTS: The variant genotype of ASPN and COMP genes was found to be present at a relatively higher frequency in cases than controls. RT-PCR and immunochemical studies revealed increased mRNA and protein expression of such gene in PBLs isolated from cases of knee OA as compared to healthy control. CONCLUSION: The allelic alteration in ASPN and COMP genes in knee OA cases points to the role of these genes in the development of knee OA. Further, increased mRNA and protein expression of ASPN and COMP in peripheral blood samples of patients with the disease suggest that expression profile of candidate gene could be used as a biomarker for predicting the development and progression of knee OA.

Augmentation of iNOS expression in myeloid progenitor cells expedites neutrophil differentiation.

Neutrophils play important role in immunity and inflammation through diverse mechanisms. Reports from this lab and others have demonstrated involvement of NO in neutrophil adhesion, chemotaxis, bacterial killing, reactive oxygen species generation, neutrophil extracellular traps' formation, and apoptosis. Constitutive expression of iNOS in human neutrophils has also been documented. The role of NO-iNOS in neutrophil differentiation however remains ill-defined. The present study was undertaken to understand the role of NO generated from iNOS in the neutrophil differentiation by using iNOS-overexpressing K562 cells (K562iNOS ) and iNOS-deficient murine progenitor cells (lineage negative cells; lin-ve ). We observed that iNOS overexpression led to increased neutrophilic differentiation in K562 cells; more specifically an early and accelerated neutrophilic differentiation was spotted in K562iNOS . These observations were further validated using iNOS knockout lin-ve cells or hematopoietic progenitor cells that exhibited delayed neutrophil differentiation in comparison to its wild-type counterpart. In addition, a significant increase in the gene expression of iNOS during neutrophilic differentiation of CD34+ hematopoietic stem and progenitor cells derived from human bone marrow further substantiates importance of iNOS in neutrophil differentiation. Moreover, a significant increase in NO generation during neutrophil differentiation was observed and enhanced neutrophil differentiation with NO donor was also observed, implying the importance of NO in neutrophil differentiation. Collectively, using alternative approaches, we demonstrated that neutrophil differentiation is significantly influenced by iNOS or NO, suggesting the possibility of exploiting this novel link for therapeutic aspects of NO generated from iNOS and neutrophil differentiation in hematopoiesis-related disorders.

Significance of methylation status of MASPIN gene and its protein expression in prognosis of gallbladder cancer.

AIM: We investigated prognostic significance of methylation status of MASPIN gene and its protein expression in normal subjects, cholelithiasis and gallbladder cancer (GBC) patients. METHODS: MASPIN protein expression and its promoter methylation in gallbladder tissue of cholelithiasis (n = 36), GBC (n = 46) and controls (n = 25) were investigated. Clinicopathological parameters and prognosis of patients with GBC were correlated with protein expression of MASPIN. Survival analysis of GBC patients was performed using Kaplan-Meier method. RESULTS: Significant increased (P < 0.0001) protein expression of MASPIN was observed in GBC as compared to cholelithiasis, whereas negligible expression was found in normal tissues. Methylation-specific PCR revealed statistical significant (P = 0.005) difference in methylation status of MASPIN promoter between gallstone and GBC patients. Significant association was observed between methylation profile with protein expression of MASPIN (P = 0.004), stage (P = 0.011) and cellular differentiation (P = 0.012) for GBC. Also, significant (P = 0.004) difference in survival was observed for malignant patients having demethylated MASPIN promoter. Further significant negative correlation (Pearson's coefficient [r] = -0.617; P < 0.0001) was observed between MASPIN expression and survival of cancer patients after surgery. Overall survival was significantly shorter (P ≤ 0.0001; hazard ratio [HR] for death = 2.84; 95% confidence interval [CI], 0.09865-0.3683) in patients of GBC with MASPIN expression ≥169.56 pg/mg (median survival; 10 months) with compared to those with expression <169.56 pg/mg (median survival; 16 months). CONCLUSION: Overexpression of MASPIN protein may play an important role in malignant progression and is correlated with a poor prognosis. Also MASPIN DNA methylation can be used as a novel therapeutic target for treating GBC.

Effectiveness of bone marrow-derived mononuclear stem cells for neurological recovery in participants with spinal cord injury: A randomized controlled trial.

BACKGROUND: Complete lesion after spinal cord injury (SCI) remains irreversible with little hope of neurological recovery. Newer interventions such as re-stimulation of damaged neurons using artificial agents and the use of stem cells for neuronal regeneration have shown promising results. AIM: This study was undertaken for evaluating the neurological status of acute SCI participants after stem cell augmentation and comparing them with other treatment methods. SETTING AND DESIGN: Randomized controlled trial in the northern Indian population. MATERIALS AND METHODS: A total 193 SCI participants of complete paraplegia with unstable T4-L2 injury having thoracolumbar injury severity score ≥4 were enrolled in this study. Participants were randomly allocated for three different treatment modalities, namely, conventional with stem cell augmentation (Group-1), conventional (Group-2), and conservative (Group-3). Neurological recovery after 1 year was evaluated through the ASIA Impairment Scale (AIS)-grading, sensory, and motor scores. STATISTICAL ANALYSIS: T-test for sensory-motor score analysis of each group and analysis of variance for comparison of same variables between the groups. RESULTS: After 1-year significant difference was observed in the AIS-grade, sensory and motor scores in-Group 1 (P < 0.001). In Group-1 versus 2, the mean difference at 1 year for AIS grade, sensory and motor scores were 0.40 (P = 0.010, 95% confidence interval [CI] 0.075-0.727), 8.52 (P = 0.030, 95% CI 0.619-16.419), and 4.55(P = 0.003, 95% CI 1.282-7.815), respectively. In Group-1 versus 3, 1.03, 19.02 and 7.22 (P < 0.001 for each of the parameters) and in Group-2 versus 3, 0.63 (P < 0.001), 10.49 (P = 0.009), and 2.68 (P = 0.019), respectively. CONCLUSIONS: Significant motor neurological recovery and AIS-grade promotion was observed in Group-1 as compared to Group-2 and 3.

Proton NMR based serum metabolic profile correlates with the neurological recovery in treated acute spinal cord injury (ASCI) subjects: A pilot study.

BACKGROUND: Acute Spinal Cord Injury (ASCI) is still having substantial morbidity and mortality despite of advanced therapeutics. Major obstacles are paucity of monitoring tools or biomarkers for severity determination, recovery and prognostication. A prospective case control pilot study with serum 1H NMR spectroscopic metabolic profiling was carried out to evaluate metabolites perturbations and its relationship with recovery and to see role of stem cells in facilitating neurological recovery. METHODOLOGY: Twenty subjects with ASCI were classified on the basis of therapeutic modality into surgical fixation alone (Group-1, n = 10), stem cell adjuvant (Group-2, n = 10) and healthy controls (Group-0, n = 10). Serum samples were collected at admission (baseline) and after six months (follow-up). NMR data of serum sample were quantified and subjected to Wilcoxon and ANOVA tests. Further validation was performed using supervised OSC-PCA and OPLS-DA by incorporating substantial control samples. RESULT: Twenty-eight metabolites were identified; well resolved resonances of fifteen metabolites were quantified wherein seven were statistically significant. Predominantly amino acids and ketone bodies played vital role in the differentiation of groups. CONCLUSIONS: Serum NMR spectroscopy reveals certain metabolites perturbations having clear correlation with pattern of recovery in treated ASCI subject. Stem cells treatment group had comparatively effective recovery.

Expression of Genes and Their Polymorphism Influences the Risk of Knee Osteoarthritis.

INTRODUCTION: Genetic factors including the level of expression of the fingerprint of genes involved in the development of bones and cartilage such as GDF-5 or ESR-α or CALM-1 are known to be strong determinants of the osteoarthritis (OA) in Caucasian and Oriental populations. Because of high prevalence of OA in Indian population and availability of limited genetic data, we determined whether similar genetic factors are involved in Indians as well. METHODS: A case control study was carried out involving 500 patients of knee OA and equal number of healthy controls. Genotyping analyses in whole blood, mRNA, and protein expressions in peripheral blood lymphocytes (PBLs) were performed using established protocols. RESULTS: Our results showed a significantly decreased level of mRNA and protein expressions for GDF-5, ESR-α, and CALM-1 genes in PBLs of OA cases when compared to healthy controls. The frequency of variant genotypes of these genes was also increased significantly in cases of OA compared to controls. CONCLUSION: Our results demonstrated that the decrease in expression of GDF-5, ESR-α, and CALM-1 in PBLs and association of polymorphism in these genes may be important in predicting the severity and thereby the progression of OA in Indian population.

Association between Single Nucleotide Polymorphisms of SMAD3 and BMP5 with the Risk of Knee Osteoarthritis.

INTRODUCTION: The role of genetic factors influencing osteoarthritis (OA) susceptibility is well documented and several candidate genes have been identified to be associated with it. Among these genes are Bone Morphogenetic Protein 5 (BMP5) and Smad family member 3 (SMAD3), all involved in Transforming Growth Factor (TGF) signaling pathway. The knee is the commonly affected joint, and knee OA has an especially high prevalence in Asian population. AIM: To investigate associations between Single Nucleotide Polymorphisms (SNPs) rs12901499 in SMAD3 and rs921126 in the BMP5 gene with knee OA susceptibility in and around Lucknow, Uttar Pradesh, India. MATERIALS AND METHODS: SNPs rs12901499 in SMAD3 and rs921126 in BMP5 were genotyped in patients with knee OA and age- sex matched OA-free controls from our population. A total of 450 patients with knee OA and 458 controls were enrolled in the study. Venous blood samples were obtained from all cases as well as controls for PCR-RFLP (Polymerase Chain Reaction- Restriction Fragment Length Polymorphism). Data was collected and entered in excel sheets. Statistical analyses of the data were performed using statistical software package SPSS version 16.0. Chi-square, Student's t-test and logistic regression tests were used to analyse the data. RESULTS: GA and GG genotypes of both SNPs (rs12901499 and rs921126), and variant G, were associated with a significantly increased risk of knee OA. A significantly increased risk of knee OA was associated with the genotype GG and GA of rs12901499 (p < 0.03 and p <0.004 respectively) and rs921126 (p< 0.0001 and p<0.001 respectively) compared with the AA genotype. In addition, those bearing at least one G allele (GG + GA) had a significantly increased risk of knee OA compared with those without the G allele (AA) in rs921126 (p< 0.0001). However, in rs12901499, significant association with the risk of knee OA was not found (p<0.4). On age and gender based stratification, the association between the risk of OA and rs921126 GG mutant compared with AA homozygotes was strong in both gender (adjusted OR= 2.93 for male and 2.25 for female) and in those aged >55 years (adjusted OR= 3.4), similarly in rs12901499, GG mutant compared with AA homozygote was strong in female (adjusted OR= 1.5) and in those aged >55 years (adjusted OR= 1.5). CONCLUSION: The results showed that both in SMAD3 rs12901499 and BMP5 921126, G allele is significantly associated with knee OA. A to G change and variant G genotype may contribute to knee OA risk in our study population of Lucknow.

Expression of MMP-8 in Pressure Injuries in Spinal Cord Injury Patients Managed by Negative Pressure Wound Therapy or Conventional Wound Care: A Randomized Controlled Trial.

PURPOSE: The purpose of this study was to assess the level of matrix metalloproteinase-8 (MMP-8) and wound-healing outcome measures (length, width, and depth, exudate amount, and tissue type) in pressure injuries (PIs) of spinal cord-injured patients treated with negative pressure wound therapy (NPWT) using a novel negative pressure device versus PI treated with wet to moist gauze (conventional wound care). DESIGN: Randomized controlled trial. SUBJECTS AND SETTING: Forty-four spinal cord-injured patients with stage 3 and 4 sacral PI participated in the study. The study setting was the Department of Orthopedic Surgery at King George's Medical University, in Lucknow, India. METHODS: Twenty two subjects were randomly allocated to undergo NPWT via a novel negative pressure device, and 22 participants received conventional wound dressing (wet to moist gauze dressings). Pressure injuries were treated for 9 weeks or until wound closure. Levels of MMP-8 were analyzed in the tissues of PIs at weeks 0, 3, 6, and 9 by enzyme-linked immunosorbent assay. RESULTS: Significantly lower levels of MMP-8 were observed in the NPWT group at week 6 and week 9. There were no significant changes in the length and width of PIs between the groups till week 3. Significant reduced length and width were observed in PIs of patients in the NPWT group at week 6 (P = .04) and week 9 (P = .001). Similarly, significant reduction in the depth of PIs was observed in the NPWT group at week 9 (P < .05). At the end of 9 week, levels of MMP-8 showed a positive correlation with reduction in the length, width, and depth of PIs in the NPWT group while in the conventional dressing group, negative correlation was observed in association with MMP-8 and the length, width, and depth of PIs. Exudate levels were significantly lower in the NPWT group compared with the conventional dressing group from week 3 (2.96 ± 0.21 vs 2.62 ± 0.49); this difference persisted through week 9 (1.35 ± 0.75 vs 0.14 ± 0.35). Conversion of slough into red granulation tissue was significantly higher in the NPWT group after week 6 (P = .001). CONCLUSION: Reduced levels of MMP-8 and an increased rate of healing were found in patients allocated to treatment with a novel negative pressure device as compared to wet to moist gauze conventional dressing. The novel NPWT device used in this study reduced exudate production and enhanced the rate of formation of red granulation tissue.

Proton NMR metabolic profiling of CSF reveals distinct differentiation of meningitis from negative controls.

BACKGROUND: Cerebrospinal fluid (CSF) is an essential bio-fluid of the central nervous system (CNS), playing a vital role in the protection of CNS and performing neuronal function regulation. The chemical composition of CSF varies during onset of meningitis, neurodegenerative disorders (positive controls) and in traumatic cases (negative controls). METHODS: The study design was broadly categorized into meningitis cases, negative controls and positive controls. Further differentiation among the three groups was carried out using Principal Component Analysis (PCA) followed by supervised Partial Least Square Discriminant Analysis (PLS-DA). RESULTS: The statistical analysis of meningitis vs. negative controls using PLS-DA model resulted in R2 of 0.97 and Q2 of 0.85. There was elevation in the levels of ketone bodies, total free amino acids, glutamine, creatine, citrate and choline containing compounds (choline and GPC) in meningitis cases. Similarly, meningitis vs. positive controls resulted in R2 of 0.80 and Q2 of 0.60 and showed elevation in the levels of total free amino acids, glutamine, creatine/creatinine and citrate in the meningitis group. Four cases of HIV were identified by PLS-DA model as well as by clinical investigations. CONCLUSION: On the basis of metabolic profile it was found that negative control CSF samples are more appropriate for differentiation of meningitis than positive control CSF samples.

CYR61-An angiogenic biomarker to early predict the impaired healing in diaphyseal tibial fractures.

BACKGROUND: Angiogenesis is a prerequisite for fracture repair, whereas insufficient blood supply is likely to result in impaired healing. In the present study, we aimed to determine the correlation of simple tibial fracture healing outcome with serial estimation of CYR61 expressions in the early phase of healing. METHODS: In total, 107 adult fractured patients and 97 healthy controls were analysed. Peripheral blood samples were taken from controls (at once) and fractured patients at 4th, 7th, 10th, 15th, 20th and 28th days of post-fracture follow-ups to quantify the CYR61 mRNA and protein expression by qRT-PCR and Western blotting assay, respectively. Clinic-radiological follow-up was done at 6th, 10th, 16th, 20th, and 24th weeks of post-fracture follow-ups using RUST scores to analyse the fracture healing progression and their final outcomes. RESULTS: By considering controls as Group I (n = 97), as per the clinico-radiological status at 24th week, fracture patients were divided into two groups: Group II (normal healing, n = 91) and Group III (impaired healing, n = 16). Both CYR61 mRNA and protein expressions were lower (baseline) in Group I than in Groups II and III; however, a significant difference was observed only with the Group II. In both groups, expressions of CYR61 mRNA as well as protein gradually upregulated from the baseline to a peak and then declined. Both, the CYR61 mRNA as well as protein expressions were significantly higher at all follow-ups in Group II than in Group III. Mean RUST scores between Group II and Group III showed a significant statistical difference at each follow-up. Significant correlation was found between the CYR61 expressions and the RUST score (fracture healing progression). CONCLUSION: We conclude that CYR61 expression provides an early prediction of the healing outcomes of simple diaphyseal tibial fractures. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Such an approach would benefit not only the patients' wellbeing but also the entire health care system in terms of the cost implications associated with long lasting treatment interventions and hospitalisation. However, the authors recommend further multicentric study with a large sample size to increase the validity, reliability, and generalisability of our observation and inferences.

Elucidation of dietary risk factors in osteoarthritis knee­a case-control study.

BACKGROUND AND OBJECTIVE: Nutritional imbalance, combined with endocrine abnormalities, may be involved in the pathogenesis of osteoarthritis (OA). This study was conducted to determine the association of OA with dietary factors, such as quantity and quality of nutrient intake. METHODS: This case-control study enrolled 180 knee osteoarthritis (KOA) subjects who met the American College of Rheumatology definition of KOA, with an equal number of matched controls. Outcome measures, such as dietary nutrient intake and its frequency, were recorded using a food frequency questionnaire. RESULTS: Compared to controls, cases were older individuals with a higher body mass index (BMI). Physical activity scores were lower in female cases compared to male cases and controls. A significantly higher intake of phosphorus and fat was observed in overall cases (fat in females only). A significantly lower intake of vitamin C and vitamin D was observed in overall cases and the significance of vitamin D persisted on gender-wise bifurcation. On multiple logistic regression analysis, the intake of vitamin D (odds ratio [OR] = 0.79) and vitamin C (OR = 0.97) was inversely associated with the presence of KOA in the observation group, especially in females. Generally, the intake of food servings/day, green leafy vegetables (GLVs), and fats/oils was higher, whereas the intake of fruits, milk/milk products, and meat/poultry was lower in cases compared to controls. CONCLUSION: Low intake of vitamin D and vitamin C is a possible risk factor for KOA. Certain food groups, such as fruits, milk/milk products, and meat/poultry are beneficial for KOA. Further studies are needed to elucidate the associations between diet and KOA.

Correlation of shock index and modified shock index with the outcome of adult trauma patients: a prospective study of 9860 patients.

BACKGROUND: Triage at emergency department is performed to identify those patients who are relatively more serious and require immediate attention and treatment. Despite current methods of triage, trauma continues to be a leading cause of morbidity and mortality. AIMS: This study was to evaluate the predictive value of shock index (SI) and modified shock index (MSI) for hospital mortality among adult trauma patients. MATERIALS AND METHODS: In this prospective longitudinal study, all adult patients who sustained trauma enrolled as per as inclusion/exclusion criteria. After the collection of data, SI and MSI were calculated accordingly. All parameters were again recorded hourly and calculations were done at six-hour intervals. Further, to achieve a value that can be analyzed, we determined threshold value for vital signs, which set the threshold values as heart rate at 120 beats per minute, systolic blood pressure at less than 90, and SI at cut-off 0.5-0.9 and MSI at less than 0.7 to more than 1.3. RESULTS: We analyzed 9860 adult trauma patients. Multivariate regression analysis demonstrated that heart rate more than 120 beats per minute, systolic blood pressure less than 90 mmHg, and diastolic blood pressure (DBP) less than 60 mmHg correlate with hospital stay and mortality rate. MSI <0.7 and >1.3 had higher odds of mortality as compared to other predictors. CONCLUSIONS: MSI is an important marker for predicting the mortality rate and is significantly better than heart rate, systolic blood pressure, DBP and SI alone. Therefore, modified SI should be used in the triage of serious patients, including trauma patients in the emergency room.

TNF-α, IL-6, and IL-8 cytokines and their association with TNF-α-308 G/A polymorphism and postoperative sepsis.

INTRODUCTION: Early prediction of postoperative sepsis remains an enormous clinical challenge. Association of TNF-α-308 G/A polymorphism with sepsis remains controversial. We, therefore, investigated this polymorphism with serum levels of cytokines TNF-α, IL-6, and IL-8 in relation to development of sepsis following major gastrointestinal surgery. METHODS: Two hundred and thirty-nine patients undergoing major gastrointestinal surgery were enrolled. Polymorphism was studied through the analysis of restriction fragments of Nco1-digested DNA with the polymerase chain reaction. All patients were followed for 1 month following surgery for evidence of sepsis. Levels of serum cytokines TNF-α, IL-6, and IL-8 were measured preoperatively and postoperatively by enzyme-linked immunosorbent assay (ELISA). RESULTS: Forty-seven (19.66 %) patients developed postoperative sepsis. Patients with postoperative sepsis were significantly (p = 0.002) more likely to possess AA homozygous genotype with higher capacity to produce cytokines TNF-α (p < 0.0001), IL-6 (p < 0.0001), and IL-8 (p < 0.0001) as compared to other genotypes. When compared with patients carrying at least one G allele, the AA genotype was associated with a significantly higher probability (odds ratio (OR) = 4.17; p = 0.003; 95 % confidence interval (CI) = 1.5-11.48) of developing sepsis. Compared with the GG genotype, AA was associated with a significantly higher probability (OR = 5.18; p = 0.0008; 95 % CI = 1.82-14.76) of sepsis development. CONCLUSION: TNF-α-308 G/A polymorphism is significantly associated with the development of postoperative sepsis and with increased expression of cytokines TNF-α, IL-6, and IL-8.

Tumor necrosis factor-ß Nco1 polymorphism and susceptibility to sepsis following major elective surgery.

BACKGROUND: Post-operative sepsis remains a substantial cause of morbidity and mortality. In injured patients, that a polymorphism of the gene for tumor necrosis factor-ß (TNF-ß) has been related to the development of sepsis. Genetic factors may also have a role in etio-pathogenesis of sepsis following surgery. We investigated the relationship of the polymorphism of the gene for TNF-ß and the serum concentration of TNF-α to the development of sepsis after elective major surgery. METHODS: The study population consisted of 211 patients undergoing major elective surgery. The NcoI polymorphism of TNF-ß was studied in genomic DNA through the analysis of restriction fragments of Nco1-digested DNA with the polymerase chain reaction (PCR). All patients were followed for 1 mo after surgery for any evidence of sepsis. Serum concentrations of TNF-α were measured pre- and post-operatively by enzyme linked immunosorbent assay (ELISA). Genotypes of TNF-ß and the production of TNF-α were related to the occurrence of sepsis. RESULTS: Post-operative sepsis developed in 21.8% (n=46) of the patients. The overall mortality was 4.2% (n=9). The overall allele frequency of the TNF-ß genotype was 0.32 for TNFB1 and 0.68 for TNFB2. Within the TNF-ß genotype, 11.84% (n=25) of the patients were homozygous recessive for TNFB1, 41.23% (n=87) were heterozygous, with TNFB1/TNFB2, and 46.91% (n=99) were homozygous dominant for TNFB2. The incidence of post-operative sepsis was significantly (p=0.01) higher in patients homozygous for the TNFB2 allele. When compared with patients carrying at least one TNFB1 allele (TNFB1 homozygous and heterozygous genotype), the TNFB2 homozygous genotype was associated with an odds ratio (OR) of 2.60 (p=0.005; 95% CI 1.32-5.15) for the development of sepsis. As compared with that for the heterozygous genotype, the OR for the homozygous TNFB2 genotype was 3.00 (p=0.003; 95% CI 1.39-6.44). In patients with post-operative sepsis, serum concentrations of TNF-α were significantly higher (p=0.02) in TNFB2 homozygous individuals than in those of individuals of the other TNF-ß genotypes. CONCLUSION: The development of sepsis was associated with a greater capacity to produce TNF-α after surgery. The Nco1 polymorphism of the TNF-ß gene was associated with the development of post-operative sepsis with an increased serum concentration of TNF-α. In patients without post-operative sepsis, polymorphism of the TNF-ß gene was not related to different levels of TNF-α production. This indicates an association between polymorphism of the TNF-ß gene and post-operative sepsis, suggesting the TNFB2/B2 genotype as a high-risk factor for the development of sepsis after elective surgery.

Elevated expression of maspin mRNA as a predictor of survival in stage II and III gallbladder cancer cases.

BACKGROUND: Maspin expression is a potential prognostic factor for various malignancies but its relation with gallbladder cancer is unknown and needs to be investigated needs to be investigated. We therefore here focused on maspin mRNA expression in normal, gall stone disease and gallbladder cancer subjects, with particular attention to prognostic importance in individuals with malignancies. MATERIALS AND METHODS: This study was carried out at the Department of Surgical Gastroenterology, King George's Medical University, Lucknow, India. Gallbladder samples from normal (n=25), gall stone disease (n=25) and cancer patients (n=38) were analysed for maspin mRNA expression by semi-quantitative reverse transcriptase PCR and quantitative real time PCR. Statistical analysis was carried out using the Students t test or ANOVA. Survival analysis was conducted according to the Kaplan-Meier method and correlations were assessed using the Pearson correlation method. p<0.05 was considered statistically significant. RESULTS: Significant increase (p=0.028) in expression of maspin mRNA was observed in gallbladder cancer as compared to gall stone disease, whereas no expression was found in normal tissues. Significant correlation (Pearson's coefficient(r)=-0.798, p<0.0001) was observed between relative quantification of maspin mRNA and survival of cancer patients after surgery, with significantly shorter (p=0.002) survival in patients having relative quantification >1.5 as compared to those having relative quantification <1.5. Similarly, significant differences in patient survival for maspin mRNA expression was observed for stage II (p=0.025) and III (p=0.011) cancer. CONCLUSIONS: Higher expression of maspin mRNA in gallbladder cancer has prognostic significance for stage II and III cancer, which needs to be investigated further.

A comparative analysis of nasogastric and intravenous fluid resuscitation in patients with malignant obstructive jaundice prior to endoscopic biliary drainage.

BACKGROUND: An alternative to intravenous is nasogastric fluid administration through normal functioning gut. Though not common, this practice has significance in mass causalities and elective situations. AIM: The study was designed to compare nasogastric and intravenous fluid resuscitation in malignant obstructive jaundice (OJ) and their effect on endotoxemia. MATERIALS AND METHODS: Sixty patients with malignant OJ undergoing endoscopic biliary drainage were randomized into two groups. A total of 4 l of fluid (Ringer's lactate) was administered to Group A through nasogastric tube and to Group B through intravenous route for 48 h. Vital parameters, serum bilirubin, serum creatinine, creatinine clearance rate, electrolytes, and endotoxemia were monitored. RESULTS: Significant improvement in blood pressure (Group A, P = 0.014; Group B, P = 0.020) and significant decrease in serum bilirubin level (Group A, P = 0.001; Group B, P > 0.0001) was observed in both groups after resuscitation. Significantly decreased (P = 0.036) post hydration endotoxin level was observed in Group A as compared to Group B. Febrile events were significantly higher (P = 0.023) in Group B as compared to Group A (6 vs 0). Electrolyte abnormalities were found more in Group B, however statistically insignificant. CONCLUSION: In OJ patient undergoing biliary drainage, preoperative fluid resuscitation through nasogastric tube may be helpful in reducing postoperative septic complications and endotoxemia.

Does vitamin D improve osteoarthritis of the knee: a randomized controlled pilot trial.

BACKGROUND: Animal, epidemiologic, and human clinical studies suggest a putative role for vitamin D in osteoarthritis (OA). Inadequate sunlight exposure and lower serum levels of 25(OH)D appear in some reports to be associated with an increased risk for progression of knee OA. QUESTIONS/PURPOSES: We asked whether treatment with vitamin D would (1) reduce knee pain (WOMAC and VAS), (2) improve function (WOMAC), and (3) change levels of relevant biochemical markers in patients with knee OA with vitamin D insufficiency. METHODS: This randomized controlled pilot trial prospectively enrolled 107 patients with knee OA with vitamin D insufficiency (25(OH)D ≤ 50 nmol/L) to receive oral vitamin D or placebo. The primary outcome measures were pain and function, and the secondary were biochemical markers. At baseline, the two groups were comparable. The patients were followed for 1 year. RESULTS: At 12 months, knee pain had decreased in the vitamin D group by mean -0.26 (95% CI, -2.82 to -1.43) on VAS and -0.55 (95% CI, -0.07 to 1.02) on the WOMAC, whereas in the placebo group, it increased by mean 0.13 (95% CI, -0.03 to 0.29) on the VAS and 1.16 (95% CI, 0.82 to 1.49) on the WOMAC (effect size = 0.37 and 0.78). Likewise knee function improved in the vitamin D group by mean -1.36 (95% CI, -1.87 to -0.85) over the placebo group which had a mean 0.69 (95% CI, -0.03 to 1.41; effect size = 0.06). There were significant biochemical changes in serum total calcium, 25(OH)D and alkaline phosphatase. CONCLUSIONS: The results above suggest there is a small but statistically significant clinical benefit to vitamin D treatment in patients with knee OA, although we recommend a long-term study to determine whether these changes are clinically important and whether they will be sustained with time. Further studies with long-term radiologic evaluations are needed.