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Molecular And Cellular Medicine

Molecular and Cellular Medicine

The scientific research in the department is focused towards understanding the molecular basis of liver disease and translating the findings into clinical applications for better diagnosis as well as treatment strategies. The research studies are carefully designed to meet the challenging biological problems by fostering a strong collaboration between laboratory scientists and clinicians. The scientific proposals are hypothesis driven, designed around “key-questions” and use a multiprong approach of animal models, in vitro systems, together with clinical specimens. The research areas are centred around the following major themes:

  • Mechanisms of immune paresis and sepsis in liver failure
  • Regenerative Medicine and Genome Editing
  • Hepatomics : Microbiome, Metagenome, Metabolome and Epigenome
  • Biology of HBV and HEV
  • Extracellular vesicles in liver disease
  • Animal physiology
  • Vascular biology and portal hypertension
  • Hepatocellular Carcinoma
  • Developing animal models of liver disease
 

National Recognitions

  • DST funded core Laboratory for Non-Transplant Therapeutic Strategies for Advanced Liver Disease.
  • ILBS-Ayush Project on Pre-clinical Trials of Ayur Formulations for Fatty Liver Disorder
Molecular and Cellular Medicine Photo
 

Highlights of the Research Activities

  • Development of thiourea drugs as novel antivirals (Dr. Vijay Kumar)

    There is active research in developing small molecules targeting HBV replication. The work has shown that a candidate molecule 1-(3-(1H-imidazol-1-yl) propyl)-3-(2,4-difluorophenyl) thiourea (coded as 'IR-415') exhibited excellent antiviral efficacy in cell culture model. Twenty derivatives of IR-415 referred to as DSA-00 were synthesized and evaluated for their antiviral activity against hepatitis B virus (HBV) in cell culture. The thiourea derivative caused significant inhibition in viral DNA replication and secretion of hepatitis B surface antigen were observed. Consistent with these antiviral properties, our molecular docking studies predicted a high affinity interaction of these derivatives with HBx protein. Importantly, DSA-00 and its derivatives exhibited minimal toxicity at higher concentrations. Thus, these derivatives have the potential to be developed as new therapeutics for mono or combination therapy for the management of HBV infection (Singh et al., Chemistry Select, 2023). We have now filed a patent on the same.

  • Immune predictors of hepatitis B surface antigen seroconversion in patients with hepatitis B reactivation (Dr. Nirupma Trehanpati)

    In Hepatitis B, our group has observed that after HBV reactivation chances of seroclearance are more with appearance of anti-HBS titers more than 10IU/ml. However, after HBV reactivation non seroconverters had increased inhibitory markers on HBV specific CD4 and CD8 T cells. Leptin and CX3CL1 caused exhaustion of CD8, Tfh and B cells. In non seroconverters, there was more of atypical memory B cells (ATM B) and short lived plasma B cells, which may hampered seroclearence in them. Use of checkpoint inhibitors molecules could serve as potential therapy for non- seroconverters in HBV reactivation

    Molecular and Cellular Medicine Photo

    Further we have shown that in chronic HBV patients, stopping nucleoside analogue (NA) therapy induces immune activation and develop flare in about 40% of HBeAgnegative patients. After relapse, introducing Peg- IFN therapy to such patients causes immune restoration by increasing CD8 effector memory cells and CD19 B cells with HBsAg loss in one fourth of them (Islam et al. Heptology Communications, 2023).

    Molecular and Cellular Medicine Photo
  • Dysregulation in amino acid and NAD+ metabolism in palmitate treated hepatocytes and plasma of non-alcoholic fatty liver disease spectrum (Dr. Gayatri Ramakrishna)

    Altered metabolism is a hallmark feature associated with fatty liver disease and palmitic acid is the most abundant saturated fatty acid, therefore, we studied the metabolic in hepatocytes treated with palmitic acid and also the differentially expressed plasma metabolites in spectrum of nonalcoholic fatty liver. The metabolites were analyzed by liquid chromatography-mass spectrometry (LC-MS) platform. Metabolomic analysis of lipid treated hepatocyte cell lines showed differential changes in phenylalanine and tyrosine pathways, fatty acid metabolism and bile acids. The key metabolites tryptophan, kynurenine and carnitine differed significantly between subjects with NAFL, NASH and those with cirrhosis. As the tryptophan-kynurenine axis is also involved in denovo synthesis of NAD+, we found significant alterations in the NAD+ related metabolites in both palmitic acid treated and also fatty liver disease with cirrhosis. The study underscores the importance of amino acid and NAD+ supplementation as promising strategies in fatty liver disorder. (Aggarwal et al, BBRC, 2023)

  • Bone Marrow Rejuvnation: A Novel Approch to Prevent Liver Failure and Onset of Decompensatation in Cirrhosis (Dr Anupam Kumar)

    Poor liver clearance of injury and infection is associated with an increased hepatic burden of intestinal bacteria and uncontrolled liver injury in cirrhosis, particularly in the transition to decompensated cirrhosis. Demand-adapted hematopoiesis plays an integral role in the resolution of injury and infection. How this process changes during chronic liver injury is not known. Using progressive model of chronic liver injury the group has shown that Increased OXPHOS with loss of glycolysis disrupts the balance between HSC self-renewal and differentiation, leading to increased myelopoiesis and loss of Long-term HSC reserve in chronic liver injury. This loss of hematopitic stem cell reserve accounts for regeneration failure and transition to decompensate cirrhosis. As proof- of-principal the group has also showed that intra BM infusion of hBM induces the repopulation of native LT-HSC, accelerates fibrosis resolution, potentiates hepatocyte regeneration and prevent onset of hepatic decompensatation in cirrhosis (Nautiyal et al, Scientific Reports (under revision).

  • Fecal microbiota transplantation compared with prednisolone in severe alcoholic hepatitis patients: a randomized trial (Dr Shevtank)

    Fecal microbiota transplant (FMT) has evolved as a key therapy for severe alcoholic hepatitis patients. Yet its efficacy is dependent on dietary and healthy donor profiles. In a recent study, the team investigated the effect of dietary modifications in healthy individuals under going Panchkarma ayurvedic therapy. It was identified that whereas the use of ghee modified the microbiota to some extent, it stabilized it overall without inducing variations in the plasma metabolic profile (Fig). The microbiota profile was further stabilized with increase of protein intake of more than 32gms. This provides a key lead in establishing the healthy gut microbiota profile, which can be stored for long use or used a fresh from the general 'healthy' individuals. An animal model of alcoholic hepatitis was tested for use of dietary interventions in donors for an improved FMT. The results clearly suggest that abstinence of alcohol shifts the bacteria away from the healthy type, whereas egg or vegetarian protein based FMT shifted the composition towards the healthy type. Additionally, a Center of Excellence grant, from Ministry of AYUSH, Govt. of India, was awarded to ILBS (PI:Dr SK Sarin), wherein dietary composition for establishing a 'healthy Indian diet' is being worked on. Initial findings of the study have shown promising results in the use of locally grown foods in India, which are likely to provide to the world a sustainable alternative to the current Mediterranean diet. The lab today is the center for microbiota nd metagenomic investigations in multiple liver diseases.

    Molecular and Cellular Medicine Photo
  • Hepatitis B Virus x Protein Increases Cellular OCT3/4 and MYC and Facilitates Cellular Reprogramming (Dr. Sanal MG)

    Hepatitis B virus x (HBx) is a multifunctional protein coded by the Hepatitis B virus that is involved in various cellular processes such as proliferation, cell survival/apoptosis, and histone methylation. HBx was reported to be associated with liver "cancer stem cells." The stemness inducing properties of HBx could also facilitate the generation of pluripotent stem cells from somatic cells. It is well established that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) using a cocktail of transcription factors called Yamanaka's factors (YFs) (OCT4, SOX2, KLF4, and MYC). The reprogramming process proceeds step-by-step with reprogramming factor chromatin interactions, transcription, and chromatin states changing during transitions. HBx is a "broad spectrum trans-activator" and therefore could facilitate these transitions. Our work showed that We found that the addition of HBx to YF improves iPSC derivation, and it increases the efficiency of iPSC generation from "difficult or hard-to-reprogram samples" such as high passage/senescent fibroblasts. Further, we show that HBx can substitute the key transcription factor MYC in the YF cocktail to generate iPSC. The cellular levels of OCT3/4 and MYC were increased in HBx expressing cells. Our results have practical value in improving the efficiency of pluripotent stem cell derivation from "difficult to reprogram" somatic cells, in addition to providing some insights into the mechanisms of liver carcinogenesis in chronic hepatitis B. To conclude, HBx improves the reprogramming efficiency of YFs. HBx increases the cellular levels of OCT3/4 and MYC (Sanal et al, Cell Reprogram, 2023).

  • Gut Lymphangiogenesis in Liver Cirrhosis (Dr. Savneet Kaur)

    Dr Savneet's lab focuses on studying the mechanisms of portal hypertension in liver cirrhosis and developing therapies that ameliorate detrimental consequences, such as ascites and infections, associated with portal hypertension. Given the established role of vascular endothelial growth factor-C (VEGF-C) in improving growth and functions of lymphatic vessel (LVs), in a recent study we demonstrated that VEGF-C treatment could ameliorate the functions of LVs and also complications of cirrhosis and portal hypertension. The study involved developing a nano formulation comprising LV-specific growth factor, recombinant human VEGF-C (Cys156Ser) protein (E-VEGF-C) and delivering it orally in different models of rat cirrhosis to target mLVs. The work showed that in cirrhotic rats, mLVs were dilated and leaky with impaired drainage. Treatment with E-VEGF-C induced proliferation of mLVs, reduced their diameter, and improved functional drainage. Ascites and portal pressures were significantly reduced in E-VEGF-C rats compared with vehicle rats. Both endogenous and exogenous bacterial translocation were limited to MLNs in E- VEGF-C rats with reduced levels of endotoxins in ascites and blood in comparison with those in vehicle rats. The work concluded that E-VEGF-C treatment ameliorates mesenteric lymph drainage and portal pressure and strengthens cytotoxic T-cell immunity in MLNs in experimental cirrhosis. It may thus serve as a promising therapy to manage ascites and reduce pathogenic gut bacterial translocation in cirrhosis (Juneja et al, J Hep Reports, 2023). A patent has also been filed for the new nanoformulation of VEGF-C developed.

    Molecular and Cellular Medicine Photo
  • Fingolimod: A Novel Therapy for Hepatopulmonary Syndrome (Dr. Sukriti)

    Dr Sukriti's lab specializes in the study of tiny membrane-bound structures released by cells, known as extracellular vesicles. Her team employs advanced techniques to decipher their role in liver disease. A recent publication from her team in Journal of Hepatology showed hepatopulmonary syndrome (HPS) which is characterized by a defect in arterial oxygenation induced by pulmonary vascular dilatation in liver disease patients. Fingolimod, a sphingosine-1-phosphate (S1P) receptor modulator, suppresses vasodilation by reducing nitric oxide (NO) production. Low level of plasma sphingosine-1- phosphate (S1P) is associated with severe pulmonary vascular shunting and hence, it can serve as a marker of disease severity in patients with hepatopulmonary syndrome (HPS). Fingolimod, a functional agonist of S1P, reduces hepatic inflammation, improves vascular tone thus retards the progression of fibrosis in preclinical animal model of HPS. Fingolimod is being proposed as a potential novel therapy for management of patients with HPS (Sukriti et al, J Hep, 2023).

    Molecular and Cellular Medicine Photo
  • Biomolecular map of albumin identifies signatures of severity and early mortality in acute liver failure (Dr Jaswinder Maras)

    Alterations in albumin structure and function have been shown to correlate with outcomes in cirrhosis. Dr Jaswinder's team undertook a biomolecular analysis of albumin to determine its correlation with hepatocellular injury and early mortality in ALF. In patients with ALF, albumin is more oxidised and glycosylated especially in non-survivors (p <0.05). In non-survivors, albumin was more often bound (p <0.05, false discovery rate <0.01) to proteins associated with inflammation, advanced glycation end product, metabolites linked to arginine, proline metabolism, bile acid, and mitochondrial breakdown products. Increased bacterial taxa (Listeria, Clostridium, etc.) correlated with lipids (triglycerides [4:0/12:0/12:0] and phosphatidylserine [39:0]) and metabolites (porphobilinogen and nicotinic acid) in non-survivors (r2 >0.7). Multi-omics signature-based probability of detection for non-survival was >90% and showed direct correlation with albumin functionality and clinical parameters (r2 >0.85). Probability-of-detection metabolites built on the top five metabolites, namely, nicotinic acid, l-acetyl carnitine, l-carnitine, pregnenolone sulfate, and N-(3-hydroxybutanoyl)-l-homoserine lactone, showed diagnostic accuracy of 98% (AUC 0.98, 95% CI 0.95-1.0) and distinguish patients with ALF predisposed to early mortality (log-rank <0.05). On validation using high-resolution mass spectrometry and five machine learning algorithms in test cohort 1 (plasma and paired one-drop blood), the metabolome panel showed >92% accuracy/sensitivity and specificity for prediction of mortality. In conclusion In ALF, albumin is hyperoxidised and substantially dysfunctional. Our study outlines distinct 'albuminome' signatures capable of distinguishing patients with ALF predisposed to early mortality or requiring emergency liver transplantation (Sharma et al, J Hep, 2023).

  • Interspecies transcriptomic comparison identifies a potential porto-sinusoidal vascular disorder rat model suitable for in vivo drug testing (Dr Dinesh M Tripathi)

    Porto-sinusoidal vascular disorder (PSVD) involves a group of rare vascular liver diseases of unknown aetiology that affect intrahepatic vessels at the level of portal venules and/or sinusoids in the absence of cirrhosis. PSVD typically affects young individuals, and the progression of the disease leads to the development of portal hypertension (PH) and its associated life-threatening complications, which are associated with high morbidity and mortality worldwide.Information on the pathophysiology of PSVD is scarce, and although several pro-thrombotic, immunologic, toxic, infectious and/or hereditary alterations have been suggested to play a role in the pathogenesis of PSVD,1 none of them have been clearly confirmed. This lack of knowledge is directly responsible for the inexistence of pathophysiological-oriented treatments for the disease, which, clinically, implies that PSVD patients' medical treatment is limited to the management of PH-related complications when they appear.7 Therefore, there is an urgent need to develop new therapeutic strategies able to stop or halt the progression of the disease. We have presented the first transcriptional study comparing PSVD animal models to human disease and showed that the easy-to-implement Selfox rat model fulfils the diagnostic criteria for early- to mid-stage PSVD while also displaying key histological and transcriptomic features shared among advanced PSVD patients. While we acknowledge the limitations of this model, we think that it might provide a good basis from which to build upon. Considering there is no treatment for the disorder and scarce information on its pathophysiology, we believe that this model can be a good PSVD preclinical model for drug testing in progressing PSVD while the field deepens in the unravelling of PSVD pathophysiology (Campreciós et al, Liver Int. 2024).

CENTRE FOR COMPARATIVE MEDICINE, ANIMAL HOUSE FACILITY

The animal house experimental facility has now been renamed centre for comparative medicine to facilitate hepatobiliary research. The facility is registered with 'The Committee for Control and Supervision of Experiments on Animals (CCSEA)', for research for education purpose, research for commercial purpose, breeding for in-house use and trade of small animals. The registration number is 1562/GO/ReRcBiBt/S/11/CPCSEA valid till 27/09/2023. Spread over an area of 15,000 square feet area the facility is equipped with state of the art facilities for animal housing and experimentation. A qualified and trained team of veterinarians and staff personnel oversee all the animal health concerns, and provide all necessary veterinary and husbandry care to ensure that healthy animals are available for research. All research proposals are carried out only after obtaining the approval of Institutional Animal Ethics Committee (IAEC). The Centre is actively involved in various animal models of human liver disease.

Salient Highlights, CCM ILBS, 2023

  • CCSEA registration of CCM, ILBS has been renewed along with re-constitution of the IAEC in October 2023 for five years.
  • Three(03) meetings of Institutional Animal Ethics Committee (IAEC) held in 2023.
  • Research Projects approved in IAEC in 2023 are fourteen(14).
  • Laboratory Animal Bio methodology, Welfare and Ethics Course offered twice a year and trained 05 Ph.D. students.
  • Successfully conducted two workshops and a seminar:
    • Rodent Surgery Workshop (RS101), 1-3 June 2023 attended by 20 participants.
    • Hands-on Workshop on Basic Training in Animal Handling & Experimentation from 1-4 November, 2023 attended by 18 participants.
    • Seminar on World Animal Day was conducted of 4th October 2023
  • Existing animal Models of Liver Diseases at ILBS: Jet-Lag induced NAFLD, Gallstone mice model, Vagotomy rat model, CBDL induced model of Hepatopulmonary syndrome (HPS), 2/3rd Partial Hepatectomy mice & rat model, PPVL (Partial Portal vein ligation) of non-cirrhotic portal hypertension, Cirrhotic models of portal hypertension, HCC mouse models : xenograft, orthotropic,syngenic, DEN induced HCC etc.
  • Contribution of CCM team veterinarians in Publications/Abstract/Oral/Poster in 2023:
  • Oral Turbo Talk on “Triple Humanized Mouse Model: Next Generation Tool For Malaria Research” in the International Conference on Vector Borne Diseases (NAVBD) at GOA University,15th-17th Feb,2023.
  • Oral Presentation by Dr. Anupma Kumari in LASACON 2023 held in Bangalore 7-8 November, 2023.

Team :

Molecular and Cellular Medicine Photo
 

Confocal Microscopy Facility

The Confocal Microscope Facility at ILBS is a state of the art cellular imaging facility generously supported under the FIST programme of Dept of Science and Technology (DST-FIST). The Confocal microscope is a versatile imaging platform for biological research specimens such as 2D cell culture, 3D spheroids and paraffin embedded tissue specimens.

Molecular and Cellular Medicine Photo

Key Features of Instrumentation:

  • Model: Leica SP8
  • Point Scanning
  • Excitation Lasers :
  • Soild State Laser : Diode 405,
  • Argon Line:458, 488, 514
  • Solid State:DPSS 561,
  • Helium Line:HeNe 633nm
  • Spectral Detection:3PMTs and 2 HyDs ( ultrasensitive detectors)
  • Objectives:Dry objectives:10X, 20X, 40X
  • Oil Immersion:63X and 100X
  • Multi-dimensional acquisition for time series, z stack and lambda scan
Molecular and Cellular Medicine Photo
 

Biosafety Level-3 Containment Facility

Biosafety Level-3 (BSL-3) facility of ILBS is a state of the art facility of the Department of Molecular and Cellular Medicine, whose construction started in 2018 and was finally commissioned in May 2019. BSL-3 containment facility operations and experimentations are monitored by dedicated Operation and Experimentations (comprising of external experts) committees, respectively. In February 2020 the BSL3 facility was solely dedicated for SARS-CoV2 testing by the Department of Virology.

The BSL-3 facility is governed under the statutory regulatory guidelines Review Committee on Genetic Manipulation (RCGM), Department of Biotechnology, Ministry of Science and Technology. Institutional Biosafety Committee (IBSC) provides on-site regulatory oversight and compliance apart from BSL-3 Containment Facility Operation Committee's periodic internal inspection and audit. The BSL-3 Containment Facility of ILBS is certified by RCGM BT/IBKP/070/2020-BSL3 valid up to 29/11/2024. BSL-3 operations and experimentations are managed as per the Standard Operating Procedure for working in BSL-3 facility and The Manual: Practices and Procedures for the Biosafety Level 3 Containment Facility. Facility has restricted entry for only authorized person through access card/biometry along with manual entry-exit logs. Personnel are granted access upon successfully completion of BSL-3 training. Containment of BSL- 3 is achieved and maintained with negative pressure gradient round the clock by Building Management System. Facility is on uninterrupted power supply (UPS), equipped with emergency shutdown mechanism and provision of complete fumigation. In-vitro as well as mouse experiment can be performed in BSL-3 facility. Currently the BSL3 facility is being used by department of clinical virology for SARS-CoV2 RT-PCR testing and other investigators for their research projects involving corona virus work. Training program was organized in August 2023 and total 10 participants were trained apart from regular induction training to the newly enrolled PhD scholar's, twice a year.

Equipment - Biosafety Cabinets, Deep Freezer, CO2 Incubators, Centrifuge, Inverted microscope, Individually ventilated cages, Isoflurane anaesthesia machine, double door autoclave and other emergency equipment i.e. emergency shower, eye wash and fire extinguishers

Biosafety Level-3 Containment Facility Training

BSL3 Training Aug, 2023

cGMP Facility

Emerging science of cell and gene therapy like stem cell therapy, immune cell therapy and hepatocyte transplant holds great promise in the management of liver patients. However their usages in India are limited due to required infrastructure and expertise. To promote the use of these therapies for the benefit of patients with liver failure and HCC, ILBS had developed an state- of-the-art Therapeutic Cells Production (cGMP) Facility. The facility has approximately 200 Sqr. meter ISO Class-6 clean room with 2 production suits, one media preparation suit, one pre- processing, one analysis suit and one Cryostorage suit fully equipped with GMP compliance Cell therapy system (CTS). At present the facility is actively involved in production of clinical grade umbilical cord mesenchymal stem cells for the treatment of patients with liver failure. On compassionate ground these cells have been used in some steroid non-responsive/ineligible alcoholic hepatitis patients having no transplant option and shown encouraging response.

Name of the Course

Name of the Course No. of seats
Ph.D ( Biomedical Sciences) 6
Short term and long training for MSc and MTech candidates for thesis 7
Summer training programme As per applications

Department's Achievements

  • The department was awarded the prestigious DST infrastructural grant to establish a facility for alternative methods for liver regeneration and from department of Ayush for testing drugs on animal models of Fatty liver.
  • A Center of Excellence grant, from Ministry of AYUSH, Govt. of India, has been awarded wherein dietary composition for establishing a 'healthy Indian diet' is being studied.
  • The department has received extramural projects and fundings from several national, international and private agencies for conducting focused research in areas of molecular medicine as follows: DBT (05, 1.3cr), DST (03, 1cr), SERB (06, 95lakhs), Ayush (02, 4.4Cr), ICMR (14, 6.2Cr), BIRAC (01, 3Lakhs), CCRAS (01, 21 Lakhs), CCRUM (01, 14lakhs), GRIFOLS (01, 46 Lakhs), Lady TATA memorial (01, 9Lakhs), MHFW (01, 70 lakhs), Mylan (02, 2.1Crore), SRIC Roorkee (01, 9.2 lakhs).
  • Students have presented their ongoing work in the prestigious national and international conferences like AASLD, EASL, ISGCON etc.
  • The students have bagged travel grants, bursaries, and best presentation awards in many reputed National and International meetings.
  • Three students have been awarded PhD Degree in Biomedical Sciences.
  • The department also successfully conducted several workshops and training programmes
 

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