intrasplenic-transplantation-hepatocytes-after-partial-hepatectomy

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological ...

generation-rat-model-acute-liver-failure-combining-70-partial

Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and ...

National Institute of Immunology

Researchers had developed and characterized transgenic green/red fluorescent protein (GFP/RFP) nude mouse with ubiquitous RFP or GFP expression, but none has evaluated the level of immune cells and expression levels of GFP in this model.

Development and Evaluation of Transgenic Nude Mice Expressing Ubiquitous Green Fluorescent Protein.

The role of Invariant chain (CD74 or Ii) in antigen presentation via Antigen Presenting Cells (APC), macrophage recruitment as well as survival, T cell activation and B cell differentiation has been well recognized. However, the aspect of CD74 which is involved in the development of hepatic steatosis and the pathways through which it acts remain to be studied. In this study, we investigated the role of CD74 in the inflammatory pathway and its contribution to development of hepatic steatosis. For this, wild type C57BL/6J and CD74 deficient mice (Ii(-/-) mice) were fed with high fat high fructose (HFHF) diet for 12 weeks. Chronic consumption of this feed did not develop hepatic steatosis, glucose intolerance or change in the level of immune cells in Ii(-/-) mice. Moreover, there was relatively delayed expression of genes involved in development of non alcoholic fatty liver disease (NAFLD) in HFHF fed Ii(-/-) mice as compared to that of C57BL/6J phenotype. Taken together, the data suggest that HFHF diet fed Ii(-/-) mice fail to develop hepatic steatosis, suggesting that Ii mediated pathways play a vital role in the initiation and propagation of liver inflammation.

Role of antigen presenting cell invariant chain in the development of hepatic steatosis in mouse model.

In today's context when liver diseases have spread across countries and people of all ages, it is of high importance to consider novel methods of non-toxic and long lived therapeutics. Among various therapies, immunotherapy for acute and chronic liver diseases is rapidly moving to the forefront among treatment options in hepatology medicine. Liver has a unique immuno- biological advantage which is utilized to maintain a balance between immunity and tolerance. This intricate balance of hepatic immune cells can be modulated to effect treatments in various liver diseases. The present review covers the recent advances in immunotherapy of hepatocellular carcinoma (HCC), viral hepatitis, cirrhosis, obesity induced inflammation, non alcoholic fatty liver diseases (NAFLD), drug induced liver injury (DILI) and disease pertaining to autoimmunity in liver.

Immunotherapy in Liver Diseases: A Balance Between Immunity and Tolerance.

In view of the escalating need for autologous cell-based therapy for treatment of liver diseases, a novel candidate has been explored in the present study. The monocytes isolated from hepatitis B surface antigen (HBsAg) nucleic acid test (NAT)-positive (HNP) blood were differentiated to hepatocyte-like cells (NeoHep) in vitro by a two-step culture procedure. The excess neutrophils present in HNP blood were removed before setting up the culture. In the first step of culture, apoptotic cells were depleted and genes involved in hypoxia were induced, which was followed by the upregulation of genes involved in the c-MET signaling pathway in the second step. The NeoHep were void of hepatitis B virus and showed expression of albumin, connexin 32, hepatocyte nuclear factor 4-α, and functions such as albumin secretion and cytochrome P450 enzyme-mediated detoxification of xenobiotics. The engraftment of NeoHep derived from HBsAg-NAT-positive blood monocytes in partially hepatectomized NOD.CB17-Prkdcscid /J mice liver and the subsequent secretion of human albumin and clotting factor VII activity in serum make NeoHep a promising candidate for cell-based therapy. Stem Cells Translational Medicine 2017;6:174-186.

Autologous NeoHep Derived from Chronic Hepatitis B Virus Patients' Blood Monocytes by Upregulation of c-MET Signaling.

Retinitis pigmentosa (RP) is a common retinal degeneration disease caused by mutation in any gene of the photo transduction cascade and results in photoreceptor dystrophy. Over decades, several animal models have been used to address the need for the elucidation of effective therapeutics and factors regulating retinal degeneration to prohibit or renew the damaged retina. However, controversies over the immune privilege of retina during cell transplantation and the role of immune modulation during RP still remain largely uninvestigated because of the lack of suitable animal models. Here, we have developed an immunocompromised mouse model, NOD.SCID-rd1, for retinitis pigmentosa (RP) by crossing CBA/J and NOD SCID mice and selecting homozygous double mutant animals for further breeding. Characterization of the newly developed RP model indicates a similar retinal degeneration pattern as CBA/J, with a decreased apoptosis rate and rhodopsin loss. It also exhibits loss of T cells, B cells and NK cells. The NOD.SCID-rd1 model is extremely useful for allogenic and xenogenic cell-based therapeutics, as indicated by the higher cell integration capacity post transplantation. We dissect the underlying role of the immune system in the progression of RP and the effect of immune deficiency on immune privilege of the eye using comparative qPCR studies of this model and the immune-competent RP model.

A novel immunodeficient NOD.SCID-rd1 mouse model of retinitis pigmentosa to investigate potential therapeutics and pathogenesis of retinal degeneration.

: Magnetic sorting of cells, based on  microbead conjugated antibodies (Abs), employs positive as well as negative immunomagnetic separation methods, for isolation of a specific cell population. These microbeads are suggested to be nontoxic, biodegradable carriers conjugated to various antibodies. Isolation of cells through positive selection involves the attachment of antibody conjugated microbeads to the cells of interest, followed by their isolation in the presence of a strong magnetic field to obtain higher purity. Negative selection involves attachment of microbead conjugated antibodies to all other cell populations except the cells of interest, which remain untagged. In the present study, we compared the two methods for their effect on functional and immunophenotypic behavior of isolated CD14+ monocytes. : Peripheral blood mononuclear cells (PBMCs) were isolated from blood collected from healthy volunteers by density gradient centrifugation. Human blood derived monocytes were isolated through positive selection and negative selection, making use of the appropriate monocyte isolation kit. Monocytes were then stimulated with lipopolysaccharide (LPS) and their activation and proliferation capacity were examined. The degradation or dissociation of cell-bound microbeads was also investigated. : We observed an impaired LPS sensitivity as well as poor activation and proliferation capacity upon stimulation by LPS in positively sorted CD14+ monocytes as compared to negatively sorted CD14+ monocytes. The attached microbeads did not degrade and remained attached to the cells even after 6 days of culture. : Our results suggest that positively sorted CD14+ cells exhibit hampered functionality and may result in inaccurate analysis and observations in downstream applications. However, these cells can be used for immediate analytical procedures.

Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour.

Non-alcoholic fatty liver disease (NAFLD)-like conditions enhance the production and action of clotting factors in humans. However, studies examining the effect of NAFLD due to high-fat high-fructose (HFHF) diet in factor VIII-deficient (haemophilia A) animals or patients have not been reported previously. In this study, we investigated the individual role of factor VIII in the progression of diet-induced NAFLD in the factor 8 (F8 ) mouse model system and its consequences on the haemophilic status of the mice. The F8 mice were fed with HFHF diet for 14 weeks. Physiological, biochemical, haematological, molecular, pathological, and immune histochemical analyses were performed to evaluate the effect of this diet. The F8 mice developed hepatic steatosis after 14 weeks HFHF diet and displayed lower energy metabolism, higher myeloid cell infiltration in the liver, decreased platelet count, upregulated de novo fatty acid synthesis, lipid accumulation, and collagen deposition. This study helps to understand the role of factor VIII in NAFLD pathogenesis and to analyse the severity and consequences of steatosis in haemophilic patients as compared to normal population. This study suggests that haemophilic animals (F8 mice) are highly prone to hepatic steatosis and thrombocytopenia.

Evaluation of high-fat high-fructose diet treatment in factor VIII (coagulation factor)-deficient mouse model.

The cultivation of mycobacteria often requires the use of several antibiotics to limit the growth of other rapidly growing micro-flora present in the growth medium. This antibiotic cocktail is one of the most expensive reagents required for mycobacterium culture. Here we present a customized antibiotics mix that is easy to prepare at a fraction of the cost of the commercially available antibiotic mixture that protects against transient flora, which are normally present in lungs, without affecting mycobacterial colony number.

Alaknanda Mishra

National Institute of Immunology

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen