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

Analysis of different deleted regions in chromosome 11 and their interrelations in early and late onset breast tumors: association with cyclin D1 amplification and survival

Previous studies have shown that younger women exhibit more aggressive pathologic features of breast cancer (BC) in comparison to older women; young age could be an independent predictor of adverse prognosis. In order to find any existing differences in the molecular progression of BC in both younger and older women, chromosome 11 (chr.11) was taken as a tool, due to its frequent deletion and amplification, particularly of CyclinD1 (CCND1) locus in BC. In the present work, the comparative analysis in the frequency of deletion in different regions in chr.11 and CCND1 amplification in BC in the two age groups was studied, as well as the interrelation and prognostic significance of these chromosomal alterations. The chr. 11 alterations were also studied in types of breast lesions other than carcinoma to see the prevalence of the alterations in these diseases. For this purpose, comparative deletion mapping of chr.11 using 17 microsatellite markers and CCND1 amplification was examined in 30 early-onset (≤40 years) and 33 late-onset (>40 years) breast carcinomas, as well as 11 other types of breast lesions. The frequency of deletion and CCND1 amplification was much higher in carcinomas than with other types of breast lesions. A total of six highly deleted regions, namely, 11p15.5, 11p11.2, 11q13.2, 11q22.3-23.1, 11q23.3-24.1, and 11q25, were identified in carcinomas of the two age groups. The 11q13.2 deletion and CCND1 amplification was comparatively higher in the carcinoma of younger women. The following significant associations were observed for (a) LOH at 11q25 with LOH at 11q13.2, 11q22.3-23.1, 11q23.3-24.1 and CCND1 amplification, respectively, and (b) LOH at 11p15.5 with LOH at 11q22.3-23.1 in carcinoma of younger women. On the other hand, the significant associations in older women were (a) LOH at 11q25 with LOH at 11q22.3-23.1, 11q23.3-24.1, respectively, and (b) LOH at 11q22.3-23.1 with LOH at 11q23.3-24.1. Deletion at 11q13.2 was also associated with reduced overall survival in the younger group, indicating its prognostic significance. It is evident from our data that the pattern of chromosomal alterations are not exactly same in the carcinomas in the two age groups. Differential interrelationship of the chromosomal alterations and prognosis in these two age groups indicate that the molecular pathogenesis of the carcinomas is not similar

Molecular study of clonality in multifocal and bilateral breast tumors

The clonal origin of multiple tumors in the same individual has long been debated. The main aim of this study is to find out whether multiple tumors in same individuals originated from a single clone. In our previous work (Pathol. Res. Pract. 199 (2003) 313-321), the deletion at chromosome1p36 was found to occur early because of common allelic loss in the bilateral tumors. In order to further investigate the findings about the clonality of tumors, eight tumors from four patients (two synchronous bilateral breast carcinoma [biBC], one case with breast carcinoma in one breast and multiple calcified fibroadenoma nodules in another breast, and one case with multifocal fibroadenosis in one breast) were subjected to polymerase chain reaction (PCR) to detect (a) loss of heterozygosity (LOH) and microsatellite size alterations (MA) using microsatellite markers distributed over five chromosomal arms 11p/q, 13q and 17p/q, and (b) Cyclin D1 amplification. Some markers were intragenic for BRCA1, BRCA2, BRCAX, ATM, TP53, and RB1. Although a few cases were studied, our findings suggest that in at least a proportion of patients multiple tumors may arise from a single clone

Differential Association of BRCA1 and BRCA2 Genes with Some Breast Cancer–Associated Genes in Early and Late Onset Breast Tumors

Accumulating evidence indicating more aggressive features of breast carcinoma (BC) in young women than their older counterparts have raised the question of whether these differences are present at the genetic level. Methods: For this purpose, we performed a comparative analysis of the frequency of deletions of BRCA1, BRCA2, BRCAX, TP53, ATM, and RB1 and amplification of Cyclin D1 and also studied the interrelation and prognostic significance of these genetic alterations in 30 early onset (�40 years) and 33 late onset (�40 years) cases of BC. These gene alterations were also studied in 11 other types of breast lesions. Results: A differential pattern of alterations (deletion/amplification) was observed in the two age groups, with the sequence in younger women being BRCA1 (72%), TP53 (71%), ATM (64%), BRCA2 (62%), RB1 (60%), Cyclin D1 (43%), and BRCAX (24%) and that in the older group being TP53 (66%), RB1 (63%), BRCA1 (56%), ATM (53%), BRCA2 (45%), Cyclin D1 (24%), and BRCAX (23%). Similar, differential correlations were also seen with several clinicopathological parameters, prognosis, and combinations of alterations among these genes in the two age groups. Conclusions: Differential frequencies and interrelationships of genetic alterations and prognoses in these two age groups indicate that the molecular pathways for the development of tumors in both age groups may not be similar, though the ultimate effect is deregulation of cell cycle checkpoints and defects in the DNA repair pathway

Differential alterations of the genes in the CDKN2A-CCND1-CDK4-RB1 pathway are associated with the development of head and neck squamous cell carcinoma in Indian patients

Purpose: The aim of this study was to analyse the alterations of the genes in the CDKN2A/CCND1/CDK4/RB1 pathway in the G1-S phase of the cell cycle during development of head and neck squamous cell carcinoma (HNSCC). Methods: The alterations of these genes were analysed in 22 dysplastic lesions, 26 stage-I/II and 33 stage-III/IV HNSCC tumours of Indian patients. Results: The alterations [mutation, hypermethylation, homozygous deletion and loss of heterozygosity/microsatellite size alteration (LOH/MA)] in the CDKN2A were found to be highest in 57% of the samples, followed by CCND1 amplification and LOH/MA at the RB1 locus in 14% and 8.5% of the samples, respectively. No dominant CDK4 Arg24Cys mutation was seen in our samples. Comparatively high frequency of CDKN2A alterations (except homozygous deletion) was found in dysplastic head and neck lesions and remained almost constant or increased during progression of the tumour, whereas the homozygous deletion of CDKN2A and the alterations in CCND1 and RB1 genes were seen mainly in the later stages of the tumour. Conclusions: Our study suggested that mutation/hypermethylation/allelic alterations (LOH/MA) of CDKN2A were associated with the development of dysplastic head and neck lesions. All the other alterations might provide some cumulative effect during progression of later stages of the tumour to have selective growth advantages

Deletion mapping of chromosome 1 in early onset and late onset breast tumors - a comparative study in Eastern India

Younger women exhibit more aggressive pathologic features of breast cancer (BC) compared to their older counterparts. Young age has been shown to be an independent predictor of adverse prognosis. These findings have raised the question of whether these differences are also present at the genetic level. Twenty-five early onset (age ≤ 40 years) tumors including 4 bilateral tumors, and 26 late onset(> 40 years) breast tumors, including 2 bilateral tumors, were xamined for loss of heterozygosity (LOH) at chromosome 1 using 11 polymorphic microsatellite markers. A comparative study revealed high frequencies of LOH in chr.1p36 (61%), 1p31.3 (40%), 1p21.3 (50%) and 1q22–23.2 (56%) in a younger group, and chr. 1p36 (46%), 1p34.2 (48%), and 1q22–23.2 (52%) in an older group. These differences in LOH frequency in these two age groups were significant for chr. 1p21.3(p = 0.025) only. These data suggest that the deletion pattern in early onset breast tumors is not fully identical to late onsetbreast tumors. Similar differential deletion patterns of LOH in the 5 highly deleted regions were seen in premenopausal and postmenopausal groups. An association was seen between LOH at chr.1p34.2 and chr. 23.2 and higher grade of the tumors in older women. Among the highly deleted regions, the deletion at chr.1p36 was found to occur early in both groups because of common allelic loss in the bilateral tumors

Natural Clay-Modified Piezocatalytic Membrane for Efficient Removal of Coliform Bacteria from Wastewater

In the modern era, water pollution, especially from industries, agricultural farms, and residential areas, is caused by the release of a large scale of heavy metals, organic pollutants, chemicals, etc., into the environment, posing a serious threat to aquatic ecosystems and nature. Moreover, untreated sewage waste discharged directly into nearby water bodies can cause various diseases to mankind due to the high load of fecal coliform bacteria. This work demonstrates the development of a biocompatible, cost-effective, highly robust, efficient, flexible, freestanding, and reusable membrane using naturally formed biocompatible kaolinite clay-doped poly(vinylidene fluoride-hexafluoropropylene) (PVDF-HFP) for effective piezodynamic destruction of coliform bacteria. In this study, Escherichia coli (E. coli) and Enterococcus faecalis (E. faecalis) have been used to evaluate the mechanical stimulus-responsive antibacterial efficacy of the nanocomposite membrane. The membrane can effectively eradicate nearly 99% viable E. coli and 97% E. faecalis within a span of 40 min under mechanical stimulation (soft ultrasound ∼15 kHz). To further understand the mechanism, an evaluation of reactive oxygen species and bacterial FESEM was performed. These studies revealed that bacterial cells suffered severe visible cell damage after 40 min of piezocatalysis, elucidating the fact that the synthesized membrane is capable of producing a superior piezodynamic antibacterial effect

Genetic Association and Gene-Gene Interaction Reveal Genetic Variations in ADH1B, GSTM1 and MnSOD Independently Confer Risk to Alcoholic Liver Diseases in India

<div><p>Genetic susceptibility is an important modifier of clinical outcome and natural history of progression in Alcoholic liver disease (ALD). While the significance of ethnicity in this evolution is very clear, subtle inter-individual genetic variant(s) might be important and thus we investigated those in an Indian population. Fourteen markers were genotyped within two alcohol metabolism genes [Alcohol dehydrogenase (<i>ADH</i>) gene clusters (<i>ADH1B</i> and <i>ADH1C</i>) and Aldehyde dehydrogenase (<i>ALDH2</i>)], one microsomal ethanol oxidizing enzyme cytochrome p450 (<i>CYP2E1</i>) and three oxidative stress response (OSR) genes (<i>MnSOD</i>, <i>GSTT1</i> and <i>GSTM1</i>) among 490 Bengali individuals (322 ALD and 168 control) from Eastern and North-Eastern India and validation was performed in a new cohort of 150 Bengali patients including 100 ALD and 50 advanced non-alcoholic steatohepatitis (NASH). Out of 14 genetic variants, carriage of 5 genotypes (rs2066701CC in <i>ADH1B</i>, rs1693425TT in <i>ADH1C</i>, rs4880TT in <i>MnSOD</i> and <i>GSTT1</i>/<i>GSTM1</i> null, p-value <0.05) were noted significantly higher among ALD patients while inter or intra group gene-gene interaction analysis revealed that addition of risk genotype of any OSR gene enhanced the possibility of ALD synergistically. Multiple logistic regression analysis showed independent association of rs2066701CC, rs4880TT and <i>GSTM1</i> null genotype with ALD while lower frequencies of those genotypes in advanced NASH patients further confirmed their causal relation to ALD. Thus these findings suggest that the three variants of <i>ADH1C</i>, <i>MnSOD</i> and <i>GSTM1</i> can be used to identify individuals who are at high risk to develop ALD and may be helpful in proper management of Indian alcoholics.</p></div

Determination of risk factors associated with disease stages.

<p>Risk genotypes were validated in steatohepatitis (with fibrosis/ compensated cirrhosis) and decompensated chronic liver disease (DCLD) stages.</p