Reconditioned monocytes are immunomodulatory and regulate inflammatory environment in sepsis.

Sepsis is caused by dysregulated immune response to severe infection and hyper inflammation plays a central role in worsening the disease. The immunomodulatory properties of mesenchymal stem cells (MSCs) have been evaluated as a therapeutic candidate for sepsis. Reconditioned monocytes (RM), generated from healthy human peripheral blood mononuclear cells (PBMCs) exhibit both macrophage and MSCs-like properties. RM were administered at different stages of sepsis in a mouse model. It reduced serum levels of IL6, MCP-1, IL-10, improved hypothermia, increased survival, and recovery from 0 to 66% when combined with antibiotics in the mouse model. The reduced human leucocyte antigen DR molecules expression on RM enables their co-culture with PBMCs of sepsis patients which resulted in reduced ROS production, and up-regulated TGF-ß while down-regulating IL6, IL8, and IL-10 in-vitro. RM are potentially immunomodulatory, enhance survival in sepsis mouse model and modulate inflammatory behaviour of sepsis patient's PBMCs.

Microdissection of the Rodent Eye.

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, to obtain the anterior and posterior eyecups. With this technique, the sub-parts of the eye, including the corneal tissue, neural tissue, retinal pigment epithelial (RPE) tissue, and lens, can be obtained for wholemounts, cryo-sectioning, and/or single-cell suspensions of a specific ocular tissue. The presence of the third eyelid presents unique and significant advantages, as it benefits the maintenance of the orientation of the eye, which is important for understanding eye physiology following any localized intervention or in studies involving ocular analysis relating to the eye's spatial topography. In this method, we enucleated the eyeball at the socket along with the third eyelid by carefully and slowly cutting through the extraocular muscles and severing the optic nerve. The eyeball was pierced through the corneal limbus using a microblade. The incision was used as the point of entry, allowing for cutting along the corneal-scleral junction by inserting micro-scissors through the incision point. Small and continuous cuts along the circumference were made until the cups separated. These could be further dissected by gently peeling the translucent layer of the neural retina using Colibri suturing forceps to obtain the neural retina and RPE layers. Further, three/four equidistant cuts were made from the periphery perpendicularly to the optic center until the optic nerve was reached. This opened the hemispherical cups into a floret shape so that they fell flat and could be easily mounted. This technique has been used in our lab for corneal wholemounts and retinal sections. The presence of the third eyelid delineates the nasal-temporal orientation, which allows for the study of various cell therapy interventions post-transplantation and, thus, the targeted physiological validation vital for visualization and accurate representation in such studies.

Adjunctive Intravitreal Anti-vascular Endothelial Growth Factor and Moxifloxacin Therapy in Management of Intraocular Tubercular Granulomas.

PURPOSE: To report pre and post treatment levels of VEGF-A in the aqueous humour of patients with intraocular tubercular granulomas and study the effect of a combined intravitreal anti-VEGF bevacizumab and moxifloxacin therapy on their regression. METHODS: Aqueous samples of 10 consecutive patients with intraocular tubercular granulomas obtained before and after initiating treatment were subjected to ELISA for analysing intraocular VEGF-A levels. Intravitreal injections of bevacizumab and moxifloxacin were given weekly till complete regression of these granulomas. All patients received the usual four-drug ATT and oral corticosteroids. RESULTS: Mean baseline VEGF-A level was 1004.27±411.40 pg/ml (401.32-1688.95) that reduced significantly to 27.62±46.86 pg/ml (6.9-131.83) at the last injection. Meannumber of intravitreal injections was 3.1 (2-4). We found significant correlation of decreasing levels of aqueous VEGF-A with the clinical regression of these tubercular granulomas. CONCLUSIONS: Intraocular TB granulomas have high levels of VEGF-A. Weekly intravitreal injections of anti-VEGF bevacizumab with moxifloxacin as an adjunct to the standard care may cause prompt regression of tubercular granulomas. ABBREVIATIONS: TB: Tuberculosis; IOTB: Intraocular tuberculosis; VEGF: Vascular endothelial growth factor; RD: Retinal detachment; Mtb: Mycobacterium tuberculosis; ATT: Antitubercular therapy; AMD: Age-related macular degeneration; SRF: Subretinal fluid; ELISA: Enzyme immunosorbent assay; PCR: Polymerase chain reaction; ONH: Optic nerve head; MDR-TB: Multidrug-resistant tuberculosis; pg/ml: picogram/milliliter; ESR: Erythrocyte sedimentation rate; CECT: Contrast enhanced computed tomography; DNA: Deoxyribonucleic acid; RNA: Ribonucleic acid; BSL: Biosafety level; BCVA: Best corrected visual acuity; HM: Hand movements; KP: Keratic precipitates; PSC: Posterior subcapsular cataract; PS: Posterior synechiae; CRA: Chorio-retinal atrophy; IVMP: Intravenous methyl prednisolone; OCT: Optical coherence tomography; RPE: Retinal pigment epithelium; FFA: Fundus fluorescein angiography; ICG: Indocyanine angiography; RAP: Retinal arterial proliferans.

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.

Prevalence of Obesity in Inguinal Hernia Repair Patients in a Tertiary Care Center.

INTRODUCTION: Inguinal hernia is a common surgical problem, with a lifetime risk of 27% in men and 3% in women. Its cumulative incidence is 17.2% and 12.3% in body mass index of <25 kg/m2 and 25-30 kg/m2 respectively. Obesity had been regarded as the risk factor for the development of an inguinal hernia. However, recent epidemiologic studies have suggested the decreased prevalence of inguinal hernia in increased weight and body mass index individuals. The aim of this study is to find out the prevalence of obesity in inguinal hernia repair patients in a tertiary care center. METHODS: A descriptive cross-sectional observational study was performed in Bir Hospital from May 2018 to December 2019 after taking ethical approval from the institutional review board of NAMS. Convenient sampling was done with a sample size of 219. Statistical analysis was done using SPSS version 23 and Microsoft Excel software by descriptive statistics. RESULTS: The mean body mass index was 22.10±3.07 kg/m2. Body mass index category 18.5-22.9 kg/m2 had 133 (61%) male and seven (3.2%) female patients, category ≥30 kg/m2 had four (1.8%) male. Most of inguinal hernia repair patients were farmers 158 (72.5%). Common risk factors noted were smoking 142 (65.1%), heavy work 112 (51.4%), chronic cough 65 (29.8%). Most of the complications occurred in the normal body mass index category and the prevalence of complications decreased as the body mass index increased. The recurrence was found in 3 (1.4%) inguinal hernia repairs. CONCLUSIONS: The majority of inguinal hernia repair patients were non-obese, and complications were less in obese patients.

The safety and efficacy of BCG encapsulated alginate particle (BEAP) against M.tb H37Rv infection in Macaca mulatta : A pilot study.

Due to the limited utility of Bacillus Calmette-Guérin (BCG), the only approved vaccine available for tuberculosis, there is a need to develop a more effective and safe vaccine. We evaluated the safety and efficacy of a dry powder aerosol (DPA) formulation of BCG encapsulated alginate particle (BEAP) and the conventional intradermal BCG immunization in infant rhesus macaques (Macaca mulatta). The infant macaques were immunized intratracheally with DPA of BEAP into the lungs. Animals were monitored for their growth, behaviour, any adverse and allergic response. The protective efficacy of BEAP was estimated by the ex-vivo H37Rv infection method. Post-immunization with BEAP, granulocytes count, weight gain, chest radiography, levels of liver secreted enzymes, cytokines associated with inflammation like TNF and IL-6 established that BEAP is non-toxic and it does not elicit an allergic response. The T cells isolated from BEAP immunized animals' blood, upon stimulation with M.tb antigen, secreted high levels of IFN-γ, TNF, IL-6 and IL-2. The activated T cells from BEAP group, when co-cultured with M.tb infected macrophages, eliminated largest number of infected macrophages compared to the BCG and control group. This study suggests the safety and efficacy of BEAP in Non-human primate model.

Transplantation of retinal pigment epithelium and photoreceptors generated concomitantly via small molecule-mediated differentiation rescues visual function in rodent models of retinal degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is a result of degeneration/damage of the retinal pigment epithelium (RPE) while retinitis pigmentosa (RP), an inherited early-onset disease, results from premature loss of photoreceptors. A promising therapeutic approach for both is the replacement of lost/damaged cells with human induced pluripotent stem cell (hiPSC)-derived retinal cells. METHODS: The aim of this study was to investigate the in vivo functionality of RPE and photoreceptor progenitor (PRP) cells derived from a clinical-grade hiPSC line through a unified protocol. De novo-generated RPE and PRP were characterized extensively to validate their identity, purity, and potency. RESULTS: RPE expressed tight junction proteins, showed pigmentation and ciliation, and secreted polarization-related factors vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). PRP expressed neural retina proteins and cone and rod markers, and responded to KCl-induced polarization. Transcriptomic analysis demonstrated an increase in the expression of mature retinal tissue-specific genes coupled with concomitant downregulation of genes from undesired lineages. RPE transplantation rescued visual function in RCS rats shown via optokinetic tracking and photoreceptor rescue. PRP transplantation improved light perception in NOD.SCID-rd1 mice, and positive electroretinography signals indicated functional photoreceptor activity in the host's outer nuclear layer. Graft survival and integration were confirmed using immunohistochemistry, and no animals showed teratoma formation or any kind of ectopic growth in the eye. CONCLUSIONS: To our knowledge, this is the first demonstration of a unified, scalable, and GMP-adaptable protocol indicating strong animal efficacy and safety data with hiPSC-derived RPE and PRP cells. These findings provide robust proof-of-principle results for IND-enabling studies to test these potential regenerative cell therapies in patients.

Apoptosis-inducing factor deficient mice fail to develop hepatic steatosis under high fat high fructose diet or bile duct ligation.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein involved in redox signalling and programmed cell death. The role of AIF has been well recognized in diabetes and obesity. However, the aspect of AIF deficiency in the development of hepatic steatosis and liver injury is unknown. Therefore, in the current study, Harlequin (Hq mutant) mouse with markedly reduced content of AIF was investigated to explore the role of AIF on the initiation of liver injury. The wild type (WT) developed physiological and pathological features of non-alcoholic fatty liver disease (NAFLD) that were not seen in the Hq mice with AIF deficiency, when fed on high fat high fructose (HFHF) diet. Following bile duct ligation (BDL), the liver associated pathological changes were less conspicuous in Hq mice as compared to WT mice. The expression of AIF protein and apoptosis was markedly lesser as compared to their respective control in Hq mice on HFHF diet. Furthermore, the genes involved in fatty acid metabolism were also altered in the group of treated Hq mice. In conclusion, Hq mice failed to develop diet induced hepatic steatosis, suggestive of a role of AIF mediated pathway in the initiation and progression of liver inflammation. Thus, partial loss of AIF appears to be hepatoprotective. SIGNIFICANCE OF THE STUDY: AIF deficiency has multiple roles in altered pathology processes and cellular metabolism, thereby compromising the cellular homeostasis. Considering the molecular functions of AIF in other organ pathology little is known about its role in diet induced liver injury. Hence, the aim of the current study was to investigate the role of AIF deficiency in liver injury and diseases with focus on NAFLD. The study will help to deliniate the mechanisms of NAFLD using Harliquin Mice.

Mean core to peripheral temperature difference and mean lactate levels in first 6 hours of hospitalisation as two indicators of prognosis: an observational cohort study.

BACKGROUND: To study mean core to peripheral temperature difference (CPTD) and the mean lactate levels over the first 6 h of admission to hospital, as indicators of prognosis in critically ill children. METHODS: A prospective observational study in a tertiary level Pediatrics ICU in Delhi, India. Seventy eight paediatric patients from 1 month to 12 years were studied. Children with physical trauma, post-surgical patients and patients with peripheral vascular disease were excluded. Core temperature (skin over temporal artery) to peripheral temperature (big toe) difference was measured repeatedly every minute over 6 h and mean of temperature difference was calculated. Pediatric Risk of Mortality (PRISM) II, lactate clearance and mean lactate levels during that time were also studied. In-hospital mortality was used as the outcome measure. RESULTS: Mean temperature difference During the first 6 h after admission the mean temperature difference was 9.37 ± 2 °C in those who died and 3.71 ± 2.27 °C in those who survived (p < 0.0001). The area under the receiver operating curve (AUROC) was 0.953 (p < 0.0001). The comparable AUROC of PRISM II was 0.999 (p < 0.0001). Mean Lactate Mean lactate level in the first 6 h was 7.1 ± 2.02 mg/dl in those who died compared to 2.86 ± 0.87 mg/dl in those who survived (p < 0.0001). The AUROC curve for mean lactate was 0.989 (95% CI = 0.933 to 0.999; p < 0.0001). AUROC for the lactate clearance was 0.682 (p = 0.0214). CONCLUSIONS: The mean core to peripheral temperature difference over the first 6 h is an easy-to-use and non-invasive method that is useful to predict mortality in children admitted to the Pediatric ICU. The mean lactate during the first 6 h of Pediatric ICU admission is a better index of prognosis than the lactate clearance over the same time period. They may be used as components of a scoring system to predict mortality.

Primary ileal volvulus: a rare twist in an elderly patient-case report.

BACKGROUND: Small bowel volvulus is a rare entity and it is even rarer for the ileum to undergo torsion without any known predisposing factors. It presents as acute abdomen with features of intestinal obstruction. As it is a life-threatening condition, it should be kept as a differential for small bowel obstruction despite its rarity. Therefore, we report this case. CASE REPORT: A 60-year-old gentleman presented to our emergency department with a 2-day history of worsening abdominal pain, vomiting, abdominal distension and obstipation. Exploratory laparotomy was done which revealed ileal volvulus with no predisposing factors. Derotation of the segment was done. The postoperative period was uneventful and on follow up after a month, he had a satisfying recovery. CONCLUSION: Though primary ileal volvulus is a rare diagnosis, it should be kept in mind in any patient with small bowel obstruction with pain out of proportion and resistant to opioid management. Early diagnosis and urgent surgical intervention is the key to prevent bowel necrosis and associated morbidity and mortality.

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.

Correlation of vascular endothelial growth factor with the clinical regression of tubercular granuloma.

Tubercular granulomas are a common manifestation of intraocular tuberculosis. These are said to be hypoxic granulomas with increased expression of vascular endothelial growth factor (VEGF). Management of these granulomas includes a combination of antitubercular therapy (ATT) and oral corticosteroids. We report a case of tubercular granuloma with exudative retinal detachment which was treated with weekly intravitreal anti-VEGF and antibiotic injections along with ATT and corticosteroids. The VEGF levels measured paralleled with the clinical regression of the granuloma.

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.

Aerosol immunization by alginate coated mycobacterium (BCG/MIP) particles provide enhanced immune response and protective efficacy than aerosol of plain mycobacterium against M.tb. H37Rv infection in mice.

BACKGROUND: With the aim of preparing a more effective, safe and economical vaccine for tuberculosis, inhalable live mycobacterium formulations were evaluated. METHODS: Alginate particles in the size range of 2-4 µm were prepared by encapsulating live Bacille Calmette-Guérin (BCG) and "Mycobacterium indicus pranii" (MIP). These particles were characterized for their size, stability and release profile. Mice were immunized with liquid aerosol or dry powder aerosol (DPA) alginate encapsulated mycobacterium particles and their in-vitro recall response and infection with mycobacterium H37Rv were investigated. RESULTS: It was found that the DPA of alginate encapsulated mycobacterium particles invoked superior immune response and provided higher protection in mice than the liquid aerosol. The BCG encapsulated in alginate particles (BEAP) and MIP encapsulated in alginate particles (MEAP) were engulfed by bone marrow dendritic cells (BMDCs) and co-localized with lysosome. The MEAP/BEAP activated BMDCs exhibited higher chemotaxis movement and had enhanced ability of antigen presentation to T cells. The in-vitro recall response of BEAP/MEAP immunized mice when compared in terms of proliferation index and Interferon gamma (IFN-gamma) released by splenocytes and mediastinal lymph node cells was found to be higher than mice immunized by liquid aerosol of BCG/MIP. Finally, different groups of immunized mice were infected with M. tb H37Rv and after 16 weeks the Colony forming units (CFUs) in lung and spleen estimated. The bacilli burden in the BEAP/MEAP immunized mice was significantly less than the respective liquid aerosol immunized mice and the histopathology of BEAP/MEAP immunized mice lungs showed very little damage. CONCLUSIONS: These inhale-able vaccines formulation of alginate coated live mycobacterium are more immunogenic as compared to the aerosol of bacilli and they provide better protection in mice when infected with H37Rv.

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.