Influence of polymorphisms in TNF-α and IL1β on susceptibility to alcohol induced liver diseases and therapeutic potential of miR-124-3p impeding TNF-α/IL1β mediated multi-cellular signaling in liver microenvironment

Background and aimsAlcoholic liver disease (ALD) is the leading cause of the liver cirrhosis related death worldwide. Excessive alcohol consumption resulting enhanced gut permeability which trigger sensitization of inflammatory cells to bacterial endotoxins and induces secretion of cytokines, chemokines leading to activation of stellate cells, neutrophil infiltration and hepatocyte injury followed by steatohepatitis, fibrosis and cirrhosis. But all chronic alcoholics are not susceptible to ALD. This study investigated the causes of differential immune responses among ALD patients and alcoholic controls (ALC) to identify genetic risk factors and assessed the therapeutic potential of a microRNA, miR-124-3p.Materials and methodsBio-Plex Pro™ Human Chemokine analysis/qRT-PCR array was used for identification of deregulated immune genes. Sequencing/luciferase assay/ELISA detected and confirmed the polymorphisms. THP1 co-cultured with HepG2/LX2/HUVEC and apoptosis assay/qRT-PCR/neutrophil migration assay were employed as required.ResultsThe combined data analysis of the GSE143318/Bio-Plex Pro™ Human Chemokine array and qRT-PCR array revealed that six genes (TNFα/IL1β/IL8/MCP1/IL6/TGFβ) were commonly overexpressed in both serum/liver tissue of ALD-patients compared to ALC. The promoter sequence analysis of these 6 genes among ALD (n=322)/ALC (n=168) samples revealed that only two SNPs, rs361525(G/A) at -238 in TNF-α/rs1143627(C/T) at -31 in IL1β were independently associated with ALD respectively. To evaluate the functional implication of these SNPs on ALD development, the serum level of TNF-α/IL1β was verified and observed significantly higher in ALD patients with risk genotypes TNF-α-238GA/IL1β-31CT+TT than TNF-α-238GG/IL1β-31CC. The TNF-α/IL1β promoter Luciferase-reporter assays showed significantly elevated level of luciferase activities with risk genotypes -238AA/-31TT than -238GG/-31CC respectively. Furthermore, treatment of conditioned medium of TNF-α/IL1β over-expressed THP1 cells to HepG2/LX2/HUVEC cells independently showed enhanced level of ER stress and apoptosis in HepG2/increased TGFβ and collagen-I production by LX2/huge neutrophil infiltration through endothelial layer. However, restoration of miR-124-3p in THP1 attenuated such inter-cellular communications and hepatocyte damage/collagen production/neutrophil infiltration were prohibited. Target analysis/luciferase-reporter assays revealed that both TNF-α/IL1β were inhibited by miR-124-3p along with multiple genes from TLR4 signaling/apoptosis/fibrogenesis pathways including MYD88, TRAF3/TRADD, Caspase8/PDGFRA, TGFβR2/MCP1, and ICAM1 respectively.ConclusionThus, rs361525(G/A) in TNF-α and rs1143627(C/T) in IL1β gene may be used as early predictors of ALD susceptibility among East Indian population. Impeding overexpressed TNF-α/IL1β and various genes from associated immune response pathways, miR-124-3p exhibits robust therapeutic potential for ALD patients

Combination of critical care pain observation tool and face, legs, activity, cry, consolability scale in assessment of postoperative pain severity in postanesthesia care unit: A prospective cross-sectional analytical study

Context: Effective post-operative pain assessment is mandatory to exclude overdose of analgesics and avoid adverse effects. Patients in PACU have impaired ability to communicate making pain assessment challenging. This study aims to establish agreement between two pain scales, CPOT (Criticalcare Pain Observation Tool) and FLACC (Face, Legs, Activity, Cry, Consolability) and to find out specificity of combination of scales. Methods: Taking sample size of 50 patients of either sex, aged 18-80 years, ASA-PS I-III, undergoing elective surgeries were chosen, study period being June-September 2017. Adequately reversed, extubated patients not receiving sedatives, analgesics and local anaesthetics within 15 minutes before end of operation were included while patients with ASA-PS more than III or on ventilator were excluded. Assessment was done upto 2 hours at 30 minutes interval using CPOT tool and FLACC scale simultaneously by two observers, both being blinded about study. Results: Combination of two scales show high odds ratio (41%) and kappa coefficient (0.78) suggesting excellent agreement. Specificity of combination of scales is very high (95.2%) than individual test. Conclusion: CPOT and FLACC scale together has excellent agreement and their combination are more specific to assess the severity of post-operative pain than when used individually

Intra-Articular hyaluronic acid injection versus RF ablation of genicular nerve for knee osteoarthritis pain: A randomized, open-label, clinical study

Background: Chronic knee osteoarthritis (OA) is one of the most common diseases of advanced age. Available therapies have insufficient evidence and adverse effects. Hyaluronic acid (HA) injection reduces knee pain in certain patients only for short duration. Radiofrequency (RF) neurotomy of genicular nerve branches has been tried recently. Comparison of these two modalities is lacking. The aim of this study was to compare pain relief and daily activities by visual analog scale (VAS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores between intra-articular HA injection and RF neurotomy of genicular nerves. Materials and Methods: Patients were treated with intra-articular HA injection and RF neurotomy of genicular nerves 12 in each group (n = 12). Pain relief and day-to-day activity were compared. Results: There was statistically significant difference and lower VAS and WOMAC scores in the RF group compared to HA group after treatment. Conclusion: As compared to intra-articular HA injection, RF neurotomy of genicular nerves appears to be a promising and more effective therapeutic procedure for patients with chronic knee OA

Oxytocin administration during cesarean delivery: Randomized controlled trial to compare intravenous bolus with intravenous infusion regimen

Background: Oxytocin is routinely administered during cesarean delivery for uterine contraction. Adverse effects are known to occur after intravenous oxytocin administration, notably tachycardia, hypotension, and electrokardiogram (EKG) changes, which can be deleterious in high-risk patients. Aims and Objectives: To compare the hemodynamic changes and uterotonic effect of equivalent dose of oxytocin administered as an intravenous bolus versus intravenous infusion. Study Design: Randomized, double-blind, active controlled trial. Materials and Methods: Eighty parturients undergoing elective cesarean delivery, under spinal anesthesia, were randomly allocated to receive 3 IU of oxytocin either as a bolus intravenous injection over 15 seconds (group B, n = 40) or as an intravenous infusion over 5 minutes (group I, n = 40). Uterine tone was assessed as adequate or inadequate by an obstetrician. Intraoperative heart rate, non-invasive blood pressure, and EKG changes were recorded. These data were compared between the groups. Any other adverse events like chest pain, nausea, vomiting, and flushing were noted. Results: There was significant rise in heart rate and significant decrease in mean arterial pressure in bolus group compared to infusion group. Three patients in bolus group had EKG changes in the form of ST-T depression and 5 patients complained of chest pain. No such complications were found in infusion group. Conclusion: Bolus oxytocin (at a dose of 3 IU over 15 seconds) and infusion of oxytocin (at a dose of 3 IU over 5 minutes) have comparable uterotonic effect. However, the bolus regime shows significantly more adverse cardiovascular events

Combination of self-report method and observational method in assessment of postoperative pain severity in 2 to 7 years of age group: A cross-sectional analytical study

Background: Postoperative pain management is based on assessment of severity of pain. Adult patients can express their pain accurately but difficulty occurs in paediatric population. Children between 2 and 7 years of age may give biased response to any scale of pain assessment as they belong to the preoperational stage of cognitive development. Objectives: To establish the agreement between two pain scale, namely Faces Pain Scale-Revised (FPS-R) and Face, Legs, Activity, Cry, Consolability scale (FLACC) regarding assessment of severity of postoperative pain and to find out true negative in terms of specificity of combination of scale for assessment of postoperative pain. Settings and Design: Postoperative recovery unit, cross-sectional analytical study. Materials and Methods: Four hours after short surgical procedure 95 children were assessed by two pain scale and by two observers simultaneously and data submitted to analyser. Statistical Analysis: IBM SPSS (Version 20.0). P < 0.05 was considered as statistically significant. Results: Combination of these two scales show high odds ratio (39%) and kappa coefficient (0.76) suggesting excellent agreement. Specificity of combination of these scales is very high (95.1%) than individual (FPS-R-17.85%, FLACC-2.2%). Spearman′s correlation coefficient (ρ) was computed to ascertain the correlation between two scales and a significant positive correlation was found (ρ = 0.727, P = 0.00). Conclusion: FPS-R and FLACC scale has excellent agreement to diagnose the severity of postoperative pain in 2-7 years of age group and combination of these two scales has high specificity to assess the severity of postoperative pain than individual

Distinct distribution pattern of hepatitis B virus genotype C and D in liver tissue and serum of dual genotype infected liver cirrhosis and hepatocellular carcinoma patients.

The impact of co-infection of several hepatitis B virus (HBV) genotypes on the clinical outcome remains controversial. This study has for the first time investigated the distribution of HBV genotypes in the serum and in the intrahepatic tissue of liver cirrhotic (LC) and hepatocellular carcinoma (HCC) patients from India. In addition, the genotype-genotype interplay and plausible mechanism of development of HCC has also been explored.The assessment of HBV genotypes was performed by nested PCR using either surface or HBx specific primers from both the circulating virus in the serum and replicative virus that includes covalently closed circular DNA (cccDNA) and relaxed circular DNA (rcDNA) of HBV from the intrahepatic tissue. The integrated virus within the host chromosome was genotyped by Alu-PCR method. Each PCR products were cloned and sequences of five randomly selected clones were subsequently analysed.HBV/genotype D was detected in the serum of all LC and HCC patients whereas the sequences of the replicative HBV DNA (cccDNA and rcDNA) from the intrahepatic tissue of the same patients revealed the presence of both HBV/genotype C and D. The sequences of the integrated viruses exhibited the solo presence of HBV/genotype C in the majority of LC and HCC tissues while both HBV/genotype C and D clones were found in few patients in which HBV/genotype C was predominated. Moreover, compared to HBV/genotype D, genotype C had higher propensity to generate double strand breaks, ER stress and reactive oxygen species and it had also showed higher cellular homologous-recombination efficiency that engendered more chromosomal rearrangements, which ultimately led to development of HCC.Our study highlights the necessity of routine analysis of HBV genotype from the liver tissue of each chronic HBV infected patient in clinical practice to understand the disease prognosis and also to select therapeutic strategy

Table_1_Influence of polymorphisms in TNF-α and IL1β on susceptibility to alcohol induced liver diseases and therapeutic potential of miR-124-3p impeding TNF-α/IL1β mediated multi-cellular signaling in liver microenvironment.docx

Background and aimsAlcoholic liver disease (ALD) is the leading cause of the liver cirrhosis related death worldwide. Excessive alcohol consumption resulting enhanced gut permeability which trigger sensitization of inflammatory cells to bacterial endotoxins and induces secretion of cytokines, chemokines leading to activation of stellate cells, neutrophil infiltration and hepatocyte injury followed by steatohepatitis, fibrosis and cirrhosis. But all chronic alcoholics are not susceptible to ALD. This study investigated the causes of differential immune responses among ALD patients and alcoholic controls (ALC) to identify genetic risk factors and assessed the therapeutic potential of a microRNA, miR-124-3p.Materials and methodsBio-Plex Pro™ Human Chemokine analysis/qRT-PCR array was used for identification of deregulated immune genes. Sequencing/luciferase assay/ELISA detected and confirmed the polymorphisms. THP1 co-cultured with HepG2/LX2/HUVEC and apoptosis assay/qRT-PCR/neutrophil migration assay were employed as required.ResultsThe combined data analysis of the GSE143318/Bio-Plex Pro™ Human Chemokine array and qRT-PCR array revealed that six genes (TNFα/IL1β/IL8/MCP1/IL6/TGFβ) were commonly overexpressed in both serum/liver tissue of ALD-patients compared to ALC. The promoter sequence analysis of these 6 genes among ALD (n=322)/ALC (n=168) samples revealed that only two SNPs, rs361525(G/A) at -238 in TNF-α/rs1143627(C/T) at -31 in IL1β were independently associated with ALD respectively. To evaluate the functional implication of these SNPs on ALD development, the serum level of TNF-α/IL1β was verified and observed significantly higher in ALD patients with risk genotypes TNF-α-238GA/IL1β-31CT+TT than TNF-α-238GG/IL1β-31CC. The TNF-α/IL1β promoter Luciferase-reporter assays showed significantly elevated level of luciferase activities with risk genotypes -238AA/-31TT than -238GG/-31CC respectively. Furthermore, treatment of conditioned medium of TNF-α/IL1β over-expressed THP1 cells to HepG2/LX2/HUVEC cells independently showed enhanced level of ER stress and apoptosis in HepG2/increased TGFβ and collagen-I production by LX2/huge neutrophil infiltration through endothelial layer. However, restoration of miR-124-3p in THP1 attenuated such inter-cellular communications and hepatocyte damage/collagen production/neutrophil infiltration were prohibited. Target analysis/luciferase-reporter assays revealed that both TNF-α/IL1β were inhibited by miR-124-3p along with multiple genes from TLR4 signaling/apoptosis/fibrogenesis pathways including MYD88, TRAF3/TRADD, Caspase8/PDGFRA, TGFβR2/MCP1, and ICAM1 respectively.ConclusionThus, rs361525(G/A) in TNF-α and rs1143627(C/T) in IL1β gene may be used as early predictors of ALD susceptibility among East Indian population. Impeding overexpressed TNF-α/IL1β and various genes from associated immune response pathways, miR-124-3p exhibits robust therapeutic potential for ALD patients.</p

Image_1_Influence of polymorphisms in TNF-α and IL1β on susceptibility to alcohol induced liver diseases and therapeutic potential of miR-124-3p impeding TNF-α/IL1β mediated multi-cellular signaling in liver microenvironment.tif

Background and aimsAlcoholic liver disease (ALD) is the leading cause of the liver cirrhosis related death worldwide. Excessive alcohol consumption resulting enhanced gut permeability which trigger sensitization of inflammatory cells to bacterial endotoxins and induces secretion of cytokines, chemokines leading to activation of stellate cells, neutrophil infiltration and hepatocyte injury followed by steatohepatitis, fibrosis and cirrhosis. But all chronic alcoholics are not susceptible to ALD. This study investigated the causes of differential immune responses among ALD patients and alcoholic controls (ALC) to identify genetic risk factors and assessed the therapeutic potential of a microRNA, miR-124-3p.Materials and methodsBio-Plex Pro™ Human Chemokine analysis/qRT-PCR array was used for identification of deregulated immune genes. Sequencing/luciferase assay/ELISA detected and confirmed the polymorphisms. THP1 co-cultured with HepG2/LX2/HUVEC and apoptosis assay/qRT-PCR/neutrophil migration assay were employed as required.ResultsThe combined data analysis of the GSE143318/Bio-Plex Pro™ Human Chemokine array and qRT-PCR array revealed that six genes (TNFα/IL1β/IL8/MCP1/IL6/TGFβ) were commonly overexpressed in both serum/liver tissue of ALD-patients compared to ALC. The promoter sequence analysis of these 6 genes among ALD (n=322)/ALC (n=168) samples revealed that only two SNPs, rs361525(G/A) at -238 in TNF-α/rs1143627(C/T) at -31 in IL1β were independently associated with ALD respectively. To evaluate the functional implication of these SNPs on ALD development, the serum level of TNF-α/IL1β was verified and observed significantly higher in ALD patients with risk genotypes TNF-α-238GA/IL1β-31CT+TT than TNF-α-238GG/IL1β-31CC. The TNF-α/IL1β promoter Luciferase-reporter assays showed significantly elevated level of luciferase activities with risk genotypes -238AA/-31TT than -238GG/-31CC respectively. Furthermore, treatment of conditioned medium of TNF-α/IL1β over-expressed THP1 cells to HepG2/LX2/HUVEC cells independently showed enhanced level of ER stress and apoptosis in HepG2/increased TGFβ and collagen-I production by LX2/huge neutrophil infiltration through endothelial layer. However, restoration of miR-124-3p in THP1 attenuated such inter-cellular communications and hepatocyte damage/collagen production/neutrophil infiltration were prohibited. Target analysis/luciferase-reporter assays revealed that both TNF-α/IL1β were inhibited by miR-124-3p along with multiple genes from TLR4 signaling/apoptosis/fibrogenesis pathways including MYD88, TRAF3/TRADD, Caspase8/PDGFRA, TGFβR2/MCP1, and ICAM1 respectively.ConclusionThus, rs361525(G/A) in TNF-α and rs1143627(C/T) in IL1β gene may be used as early predictors of ALD susceptibility among East Indian population. Impeding overexpressed TNF-α/IL1β and various genes from associated immune response pathways, miR-124-3p exhibits robust therapeutic potential for ALD patients.</p