Immunological consequences of compromised ocular immune privilege accelerate retinal degeneration in retinitis pigmentosa.

BACKGROUND: Retinitis pigmentosa (RP) is a hereditary retinal disease which leads to visual impairment. The onset and progression of RP has physiological consequences that affects the ocular environment. Some of the key non-genetic factors which hasten the retinal degeneration in RP include oxidative stress, hypoxia and ocular inflammation. In this study, we investigated the status of the ocular immune privilege during retinal degeneration and the effect of ocular immune changes on the peripheral immune system in RP. We assessed the peripheral blood mononuclear cell stimulation by retinal antigens and their immune response status in RP patients. Subsequently, we examined alterations in ocular immune privilege machineries which may contribute to ocular inflammation and disease progression in rd1 mouse model. RESULTS: In RP patients, we observed a suppressed anti-inflammatory response to self-retinal antigens, thereby indicating a deviated response to self-antigens. The ocular milieu in rd1 mouse model indicated a significant decrease in immune suppressive ligands and cytokine TGF-B1, and higher pro-inflammatory ocular protein levels. Further, blood-retinal-barrier breakdown due to decrease in the expression of tight junction proteins was observed. The retinal breach potentiated pro-inflammatory peripheral immune activation against retinal antigens and caused infiltration of the peripheral immune cells into the ocular tissue. CONCLUSIONS: Our studies with RP patients and rd1 mouse model suggest that immunological consequences in RP is a contributing factor in the progression of retinal degeneration. The ocular inflammation in the RP alters the ocular immune privilege mechanisms and peripheral immune response. These aberrations in turn create an auto-reactive immune environment and accelerate retinal degeneration.

Immune function in X-linked retinoschisis subjects in an AAV8-RS1 phase I/IIa gene therapy trial.

This study explored systemic immune changes in 11 subjects with X-linked retinoschisis (XLRS) in a phase I/IIa adeno-associated virus 8 (AAV8)-RS1 gene therapy trial (ClinicalTrials.gov: NCT02317887). Immune cell proportions and serum analytes were compared to 12 healthy male controls. At pre-dosing baseline the mean CD4/CD8 ratio of XLRS subjects was elevated. CD11c+ myeloid dendritic cells (DCs) and the serum epidermal growth factor (EGF) level were decreased, while CD123+ plasmacytoid DCs and serum interferon (IFN)-γ and tumor necrosis factor (TNF)-α were increased, indicating that the XLRS baseline immune status differs from that of controls. XLRS samples 14 days after AAV8-RS1 administration were compared with the XLRS baseline. Frequency of CD11b+CD11c+ DCc was decreased in 8 of 11 XLRS subjects across all vector doses (1e9-3e11 vector genomes [vg]/eye). CD8+human leukocyte antigen-DR isotype (HLA-DR)+ cytotoxic T cells and CD68+CD80+ macrophages were upregulated in 10 of 11 XLRS subjects, along with increased serum granzyme B in 8 of 11 XLRS subjects and elevated IFN-γ in 9 of 11 XLRS subjects. The six XLRS subjects with ocular inflammation after vector application gave a modestly positive correlation of inflammation score to their respective baseline CD4/CD8 ratios. This exploratory study indicates that XLRS subjects may exhibit a proinflammatory, baseline immune phenotype, and that intravitreal dosing with AAV8-RS1 leads to systemic immune activation with an increase of activated lymphocytes, macrophages, and proinflammatory cytokines.

Host Immune Responses after Suprachoroidal Delivery of AAV8 in Nonhuman Primate Eyes.

The suprachoroid is a potential space located between the sclera and choroid of the eye, which provides a novel route for ocular drug or viral vector delivery. Suprachoroidal injection of adeno-associated virus (AAV)8 using transscleral microneedles enables widespread transgene expression in eyes of nonhuman primates, but may cause intraocular inflammation. We characterized the host humoral and cellular immune responses after suprachoroidal delivery of AAV8 expressing green fluorescent protein (GFP) in rhesus macaques, and found that it can induce mild chorioretinitis that resolves after systemic corticosteroid administration, with recovery of photoreceptor morphology, but persistent immune cell infiltration after 3 months, corresponding to a loss of GFP expression from retinal pigment epithelial cells, but persistent expression in scleral fibroblasts. Suprachoroidal AAV8 triggered B cell and T cell responses against GFP, but only mild antibody responses to the viral capsid compared to intravitreal injections of the same vector and dose. Systemic biodistribution studies showed lower AAV8 levels in liver and spleen after suprachoroidal injection compared with intravitreal delivery. Our findings suggest that suprachoroidal AAV8 primarily triggers host immune responses to GFP, likely due to sustained transgene expression in scleral fibroblasts outside the blood-retinal barrier, but elicits less humoral immune reactivity to the viral capsid than intravitreal delivery due to lower egress into systemic circulation. As GFP is not native to primates and not a clinically relevant transgene, suprachoroidal AAV delivery of human transgenes may have significant translational potential for retinal gene therapy.

Peripheral blood-derived monocytes show neuronal properties and integration in immune-deficient rd1 mouse model upon phenotypic differentiation and induction with retinal growth factors.

BACKGROUND: Cell therapy is one of the most promising therapeutic interventions for retinitis pigmentosa. In the current study, we aimed to assess if peripheral blood-derived monocytes which are highly abundant and accessible could be utilized as a potential candidate for phenotypic differentiation into neuron-like cells. METHODS: The peripheral blood-derived monocytes were reconditioned phenotypically using extrinsic growth factors to induce pluripotency and proliferation. The reconditioned monocytes (RM) were further incubated with a cocktail of growth factors involved in retinal development and growth to induce retinal neuron-like properties. These cells, termed as retinal neuron-like cells (RNLCs) were characterized for their morphological, molecular and functional behaviour in vitro and in vivo. RESULTS: The monocytes de-differentiated in vitro and acquired pluripotency with the expression of prominent stem cell markers. Treatment of RM with retinal growth factors led to an upregulation of neuronal and retinal lineage markers and downregulation of myeloid markers. These cells show morphological alterations resembling retinal neuron-like cells and expressed photoreceptor (PR) markers. The induced RNLCs also exhibited relative membrane potential change upon light exposure suggesting that they have gained some neuronal characteristics. Further studies showed that RNLCs could also integrate in an immune-deficient retinitis pigmentosa mouse model NOD.SCID-rd1 upon sub-retinal transplantation. The RNLCs engrafted in the inner nuclear layer (INL) and ganglion cell layer (GCL) of the RP afflicted retina. Mice transplanted with RNLCs showed improvement in depth perception, exploratory behaviour and the optokinetic response. CONCLUSIONS: This proof-of-concept study demonstrates that reconditioned monocytes can be induced to acquire retinal neuron-like properties through differentiation using a defined growth media and can be a potential candidate for cell therapy-based interventions and disease modelling for ocular diseases.

Mother-to-Child Transfer of Reactivated Varicella-Zoster Virus DNA and Varicella-Zoster IgG in Pregnancy.

Stress-induced subclinical reactivation of varicella-zoster virus (VZV) has been studied previously. However, subclinical reactivation of VZV induced by the stress of pregnancy has not been investigated. The objective was to study varicella DNA and varicella antibody levels in mothers and their newborn babies. VZV immunoglobulin G (IgG) levels in 350 mother-newborn dyads were studied using indirect enzyme-linked immunosorbent assay testing. A subset of 73 dyads was selected, DNA was isolated from the serum samples, and quantitative polymerase chain reaction (qPCR) was performed. Nearly 15% (14.6%) mothers tested were positive for varicella antibodies (>100 mIU/dL) and 16% were borderline (<100 and >50 mIU/dL). Approximately 16.9% of the babies were positive, and 18% were in borderline. Among those tested for VZV-DNA, 70% of mothers with low VZ-IgG (<100 mIU/dL) and 11.32% of those with high VZ-IgG (>100 mIU/dL) were positive for DNA. Among the newborns, 60% of those with low VZ-IgG and 15% of those with high VZ-IgG were positive for DNA. Mothers who have had VZV infection in the past can transmit VZV DNA to their babies.

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 detoxifying functions of the liver. In most irreversible liver damage cases, orthotropic liver transplant (OLT) remains the only available treatment. To study the therapeutic potential of a treatment for ALF, its prior testing in an animal model of ALF is essential. In the current study, an ALF model in rats was developed by combining 70% partial hepatectomy (PHx) and injections of acetaminophen (APAP) that provides a therapeutic window of 48 h. The median and left lateral lobes of the liver were removed to excise 70% of the liver mass and APAP was given 24 h postsurgically for 2 days. Survival in ALF-induced animals was found to be severely decreased. The development of ALF was confirmed by altered serum levels of the enzymes alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP); changes in prothrombin time (PT); and assessment of the international normalized ratio (INR). Study of the gene expression profile by qPCR revealed an increase in expression levels of genes involved in apoptosis, inflammation, and in the progression of liver injury. Diffused degeneration of hepatocytes and infiltration of immune cells was observed by histological evaluation. The reversibility of ALF was confirmed by the restoration of survival and serum levels of ALT, AST, and ALP after intrasplenic transplantation of syngeneic healthy rat hepatocytes. This model presents a reliable alternative to the available ALF animal models to study the pathophysiology of ALF as well as to evaluate the potential of a novel therapy for ALF. The use of two different approaches also makes it possible to study the combined effect of physical and drug-induced liver injury. The reproducibility and feasibility of current procedure is an added benefit of the model.

An inexpensive and easy-to-make customized antibiotics mix for mycobacterium culture.

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.

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

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.

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 conditions. Partial hepatectomy also facilitates the increased engraftment and proliferation of transplanted cells by accelerating neovascularization and cell migration towards the liver. Here, we describe a simple protocol for performing 30% hepatectomy and transplantation of cells in the spleen of a non-obese diabetic/severe combined immunodeficient NOD.SCID (NOD.CB17-Prkdcscid/J) mouse. In this procedure, two small incisions are made. The first incision is to expose and resect the left lobe of the liver, and another small incision is made to expose the spleen for the intrasplenic transplantation of cells. This procedure does not require any specialized surgical skills, and it can be completed in 5-7 minutes with less stress and pain, faster recovery, and better survival. We have demonstrated the transplantation of hepatocytes isolated from a green fluorescent protein (GFP) expressing mouse (Transgenic C57BL/6-Tg (UBC-GFP) 30Scha/J), as well as hepatocyte like cells of human origin (NeoHep) in partially hepatectomized NOD.SCID mice.

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

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.

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

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.

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

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

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

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.

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

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.

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

PURPOSE: 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. PROCEDURE: The nude GFP mice were evaluated by imaging, hematological indices, and flow cytometry to compare the proportion of immune T cells. Quantitative real-time PCR (qRT-PCR) was done for evaluating the relative expression of GFP transcripts in few organs of the nude GFP mice. RESULTS: The hematological and immune cells of nude GFP were within the range of nude mice. However, the gene expression levels were relatively less in various tissues compared with B6 GFP mice. CONCLUSIONS: These findings suggest that nude GFP is an ideal model resembling normal nude mice; however, GFP expression in various tissues by fluorescence should be considered, as the expression of GFP differs in various organs.