CDX2 inducible microRNAs sustain colon cancer by targeting multiple DNA damage response pathway factors.

Meta-analysis of transcripts in colon adenocarcinoma patient tissues led to the identification of a DNA damage responsive miR signature called DNA damage sensitive miRs (DDSMs). DDSMs were experimentally validated in the cancerous colon tissues obtained from an independent cohort of colon cancer patients and in multiple cellular systems with high levels of endogenous DNA damage. All the tested DDSMs were transcriptionally upregulated by a common intestine-specific transcription factor, CDX2. Reciprocally, DDSMs were repressed via the recruitment of HDAC1/2-containing complexes onto the CDX2 promoter. These miRs downregulated multiple key targets in the DNA damage response (DDR) pathway, namely BRCA1, ATM, Chk1 (also known as CHEK1) and RNF8. CDX2 directly regulated the DDSMs, which led to increased tumor volume and metastasis in multiple preclinical models. In colon cancer patient tissues, the DDSMs negatively correlated with BRCA1 levels, were associated with decreased probability of survival and thereby could be used as a prognostic biomarker. This article has an associated First Person interview with the first author of the paper.

The Clock-NAD+ -Sirtuin connection in nonalcoholic fatty liver disease.

Nonalcoholic or metabolic associated fatty liver disease (NAFLD/MAFLD) is a hepatic reflection of metabolic derangements characterized by excess fat deposition in the hepatocytes. Identifying metabolic regulatory nodes in fatty liver pathology is essential for effective drug targeting. Fatty liver is often associated with circadian rhythm disturbances accompanied with alterations in physical and feeding activities. In this regard, both sirtuins and clock machinery genes have emerged as critical metabolic regulators in maintaining liver homeostasis. Knockouts of either sirtuins or clock genes result in obesity associated with the fatty liver phenotype. Sirtuins (SIRT1-SIRT7) are a highly conserved family of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, protecting cells from metabolic stress by deacetylating vital proteins associated with lipid metabolism. Circadian rhythm is orchestrated by oscillations in expression of master regulators (BMAL1 and CLOCK), which in turn regulate rhythmic expression of clock-controlled genes involved in lipid metabolism. The circadian metabolite, NAD+ , serves as a crucial link connecting clock genes to sirtuin activity. This is because, NAMPT which is a rate limiting enzyme in NAD+ biosynthesis is transcriptionally regulated by the clock genes and NAD+ in turn is a cofactor regulating the deacetylation activity of sirtuins. Intriguingly, on one hand the core circadian clock regulates the sirtuin activity and on the other hand the activated sirtuins regulate the acetylation status of clock proteins thereby affecting their transcriptional functions. Thus, the Clock-NAD+-Sirtuin connection represents a novel "feedback loop" circuit that regulates the metabolic machinery. The current review underpins the importance of NAD+ on the sirtuin and clock connection in preventing fatty liver disorder.

Selective Estrogen Receptor Modulators Limit Alphavirus Infection by Targeting the Viral Capping Enzyme nsP1.

Alphaviruses cause animal or human diseases that are characterized by febrile illness, debilitating arthralgia, or encephalitis. Selective estrogen receptor modulators (SERMs), a class of FDA-approved drugs, have been shown to possess antiviral activities against multiple viruses, including hepatitis C virus, Ebola virus, dengue virus, and vesicular stomatitis virus. Here, we evaluated three SERM compounds, namely, 4-hydroxytamoxifen, tamoxifen, and clomifene, for plausible antiviral properties against two medically important alphaviruses, chikungunya virus (CHIKV) and Sindbis virus (SINV). In cell culture settings, these SERMs displayed potent activity against CHIKV and SINV at nontoxic concentrations with 50% effective concentration (EC50) values ranging between 400 nM and 3.9 µM. Further studies indicated that these compounds inhibit a postentry step of the alphavirus life cycle, while enzymatic assays involving purified recombinant proteins confirmed that these SERMs target the enzymatic activity of nonstructural protein 1 (nsP1), the capping enzyme of alphaviruses. Finally, tamoxifen treatment restrained CHIKV growth in the infected mice and diminished musculoskeletal pathologies. Combining biochemical analyses, cell culture-based studies, and in vivo analyses, we strongly argue that SERM compounds, or their derivatives, may provide for attractive therapeutic options against alphaviruses.

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.

The microRNAs miR-449a and miR-424 suppress osteosarcoma by targeting cyclin A2 expression.

MicroRNAs of the miR-16 and miR-34 families have been reported to inhibit cell cycle progression, and their loss has been linked to oncogenic transformation. Utilizing a high-throughput, genome-wide screen for miRNAs and mRNAs that are differentially regulated in osteosarcoma (OS) cell lines, we report that miR-449a and miR-424, belonging to the miR-34 and miR-16 families, respectively, target the major S/G2 phase cyclin, cyclin A2 (CCNA2), in a bipartite manner. We found that the 3'-UTR of CCNA2 is recognized by miR-449a, whereas the CCNA2 coding region is targeted by miR-424. Of note, we observed loss of both miR-449a and miR-424 in OS, resulting in derepression of CCNA2 and appearance of aggressive cancer phenotypes. Ectopic expression of miR-449a and miR-424 significantly decreased cyclin A2 levels and inhibited proliferation rate, migratory potential, and colony-forming ability of OS cells. To further probe the roles of miR-449a and miR-424 in OS, we developed an OS mouse model by intraosseous injection of U2OS cells into the tibia bone of NOD-scid mice, which indicated that miR-449a and miR-424 co-expression suppresses tumor growth. On the basis of this discovery, we analyzed the gene expression of human OS biopsy samples, revealing that miR-449a and miR-424 are both down-regulated, whereas cyclin A2 is significantly up-regulated in these OS samples. In summary, the findings in our study highlight that cyclin A2 repression by miRNAs of the miR-16 and miR-34 families is lost in aggressive OS.

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.

Bone marrow stem cell therapy partially ameliorates pathological consequences in livers of mice expressing mutant human α1-antitrypsin.

Alpha-1-antitrypsin (AAT) deficiency (AATD) is a genetic disease, caused by mutation of the AAT gene. Accumulation of mutated AAT protein aggregates in hepatocytes leads to endoplasmic reticulum stress, resulting in impairment of liver functions and, in some cases, hepatocellular carcinoma, whereas decline of AAT levels in sera is responsible for pulmonary emphysema. In advanced liver disease, the only option for treatment is liver transplantation, whereas AAT replacement therapy is therapeutic for emphysema. Given that hepatocytes are the primary affected cells in AATD, we investigated whether transplantation of bone marrow (BM)-derived stem cells in transgenic mice expressing human AATZ (the Z variant of AAT) confers any competitive advantages compared to host cells that could lead to pathological improvement. Mouse BM progenitors and human mesenchymal stem cells (MSCs) appeared to contribute in replacement of 40% and 13% host hepatocytes, respectively. Transplantation of cells resulted in decline of globule-containing hepatocytes, improvement in proliferation of globule-devoid hepatocytes from the host-derived hepatocytes, and apparently, donor-derived cells. Further analyses revealed that transplantation partially improves liver pathology as reflected by inflammatory response, fibrosis, and apoptotic death of hepatocytes. Cell therapy was also found to improve liver glycogen storage and sera glucose level in mice expressing human AATZ mice. These overall improvements in liver pathology were not restricted to transplantation of mouse BM cells. Preliminary results also showed that following transplantation of human BM-derived MSCs, globule-containing hepatocytes declined and donor-derived cells expressed human AAT protein. CONCLUSION: These results suggest that BM stem cell transplantation may be a promising therapy for AATD-related liver disease. (Hepatology 2017;65:1319-1335).

Carbohydrate-Neuroactive Hybrid Strategy for Metabolic Glycan Engineering of the Central Nervous System in Vivo.

Sialic acids are abundant in the central nervous system (CNS) and are essential for brain development, learning, and memory. Dysregulation in biosynthesis of sialo-glycoconjugates is known to be associated with neurological disorders, CNS injury, and brain cancer. Metabolic glycan engineering (MGE) and bioorthogonal ligation have enabled study of biological roles of glycans in vivo; however, direct investigations of sialoglycans in brain have been intractable. We report a simple strategy utilizing carbohydrate-neuroactive hybrid (CNH) molecules, which exploit carrier-mediated transport systems available at the blood-brain barrier, to access brain via tail vein injection in mice. Peracetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz) conjugated with neuroactive carriers, namely, nicotinic acid, valproic acid, theophylline-7-acetic acid, and choline, were synthesized and evaluated in SH-SY5Y (human neuroblastoma) cells for MGE. Intravenous administration of CNH molecules in mice (C57BL/6J and BALB/cByJ) resulted in robust expression of N-azidoacetyl-neuraminic acid (NeuAz)-carrying glycoproteins in both brain and heart, while the nonhybrid molecule Ac4ManNAz showed NeuAz expression in heart but not in brain. Successful neuroactive carriers were then conjugated with N-butanoyl-d-mannosamine (ManNBut) with a goal to achieve modulation of polysialic acid (polySia) on neural cell adhesion molecules (NCAM). PolySia levels on NCAM in adult mice were reduced significantly upon administration of Ac3ManNBut-nicotinate hybrid, but not with Ac4ManNBut. This novel application of MGE not only offers a noninvasive tool for investigating brain glycosylation, which could be developed in to brain mapping applications, but also serves as a potential drug by which modulation of neural glycan biosynthesis and thus function can be achieved in vivo.

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.

High-fat diet- and angiotensin II-induced aneurysm concurrently elicits splenic hypertrophy.

BACKGROUND: Angiotensin II (Ang II) and high-fat diet are implicated in causing pathological changes in the vascular endothelium, brain, kidney and liver. The association of aneurysm leading to histopathological changes in the splenic compartment remains elusive. Further, the salubrious credentials of antioxidants, especially α-tocopherol and ß-carotene in the resolution of splenic pathology have not been investigated. METHODS: Four-month-old Apoe(-/-) mice were used in the induction of aneurysm by infusing Ang II, and subsequently were orally administered with α-tocopherol and ß-carotene-enriched diet for 60 days. RESULTS: We observed splenomegaly in Ang II-infused aneurysm and high-fat diet-supplemented mice as compared to normal mice. These observations were further confirmed through histopathological investigations, demonstrating splenic follicular hypertrophy. We observed a remarkable decrease in the size of spleen in α-tocopherol and ß-carotene-treated Apoe(-/-) mice as compared with Ang II-treated animals. Furthermore, no marked changes in the histopathological splenic sections were seen in the ß-carotene-treated group. However, hyperplasia and proliferation of immature lymphocytes in the follicles were observed in the α-tocopherol-treated animals. We found that CD4+ T-cell levels were increased in the high-fat diet group relative to the control group and were decreased in the ß-carotene-treated animals. CONCLUSIONS: Our study provides evidence that Ang II infusion and high-fat supplementation induces abdominal aortic aneurysm that has pathological implications to the spleen. The use of ß-carotene but not α-tocopherol as an antioxidant markedly ameliorates the pathological changes in spleen.

Pigmented skin exhibits accelerated wound healing compared to the nonpigmented skin in Guinea pig model.

This study investigated and compared the wound healing kinetics of pigmented (PG) and non-pigmented (NP) skin in guinea pigs, focusing on histological and transcriptional changes. Full-thickness wounds created on PG and NP skin were evaluated at various time points post-injury. Fontana-Masson staining and ultrastructural analysis suggested the presence of melanin and melanosomes in PG skin, which coincided with an upregulation of melanogenic genes cKIT, TYR, and DCT. On day 9 post-wound, PG skin exhibited a rapid transition from the inflammatory to proliferative phase, which correlated with the reappearance of epidermal pigmentation whereas the NP skin exhibited a delayed neo-epidermis formation. Furthermore, the study revealed that melanocyte-derived growth factors (conditioned media) positively regulated keratinocyte migration while inhibiting fibroblast differentiation. These effects were more prominent in tyrosine-treated (hyperpigmented) melanocyte-CM as was TGF- ß expression. These findings provide valuable insights into the mechanisms underlying skin repair and pigmentation.

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.

Protective Effects of Rifampicin and Its Analog Rifampicin Quinone in a Mouse Model of Obesity-Induced Type 2 Diabetes.

Advanced glycation end-products (AGEs) form when glucose reacts non-enzymatically with proteins, leading to abnormal protein function, oxidative stress, and inflammation. AGEs are associated with aging and age-related diseases; their formation is aggravated during diabetes. Therefore, drugs preventing AGE formation can potentially treat diabetic complications, positively affecting health. Earlier, we demonstrated that rifampicin and its analogs have potent anti-glycating activities and increase the life span of Caenorhabditis elegans. This study aimed to investigate the effects of rifampicin during hyperglycemia in C. elegans and in a mouse model of obesity-induced type 2 diabetes. The effects of rifampicin were assessed by determining the life span of C. elegans cultured in the presence of glucose and by measuring HbA1c, AGE levels, and glucose excursions in the diabetic mouse model. Our results show that rifampicin protects C. elegans from glucose-induced toxicity and increases life span. In mice, rifampicin reduces HbA1c and AGEs, improves insulin sensitivity, and reduces indications of diabetic nephropathy without inducing hepatotoxicity. Rifampicin quinone, an analog with lower anti-microbial activity, also reduces HbA1c levels, improves glucose homeostasis and insulin sensitivity, and lowers indications of diabetic nephropathy, without adversely affecting the liver of the diabetic mice. Altogether, our results indicate that rifampicin and its analog have protective roles during diabetes without inflicting hepatic damage and may potentially be considered for repositioning to treat hyperglycemia-related complications in patients.

Taurine deficiency as a driver of aging.

Aging is associated with changes in circulating levels of various molecules, some of which remain undefined. We find that concentrations of circulating taurine decline with aging in mice, monkeys, and humans. A reversal of this decline through taurine supplementation increased the health span (the period of healthy living) and life span in mice and health span in monkeys. Mechanistically, taurine reduced cellular senescence, protected against telomerase deficiency, suppressed mitochondrial dysfunction, decreased DNA damage, and attenuated inflammaging. In humans, lower taurine concentrations correlated with several age-related diseases and taurine concentrations increased after acute endurance exercise. Thus, taurine deficiency may be a driver of aging because its reversal increases health span in worms, rodents, and primates and life span in worms and rodents. Clinical trials in humans seem warranted to test whether taurine deficiency might drive aging in humans.

Embryonic osteocalcin signaling determines lifelong adrenal steroidogenesis and homeostasis in the mouse.

Through their ability to regulate gene expression in most organs, glucocorticoid (GC) hormones influence numerous physiological processes and are therefore key regulators of organismal homeostasis. In bone, GC hormones inhibit expression of the hormone Osteocalcin for poorly understood reasons. Here, we show that in a classical endocrine feedback loop, osteocalcin in return enhanced the biosynthesis of GC as well as mineralocorticoid hormones (adrenal steroidogenesis) in rodents and primates. Conversely, inactivation of osteocalcin signaling in adrenal glands significantly impaired adrenal growth and steroidogenesis in mice. Embryo-made osteocalcin was necessary for normal Sf1 expression in fetal adrenal cells and adrenal cell steroidogenic differentiation and therefore determined the number of steroidogenic cells present in the adrenal glands of adult animals. Embryonic, not postnatal, osteocalcin also governed adrenal growth, adrenal steroidogenesis, blood pressure, electrolyte equilibrium, and the rise in circulating corticosterone levels during the acute stress response in adult offspring. This osteocalcin-dependent regulation of adrenal development and steroidogenesis occurred even in the absence of a functional hypothalamus/pituitary/adrenal axis and explains why osteocalcin administration during pregnancy promoted adrenal growth and steroidogenesis and improved the survival of adrenocorticotropic hormone signaling-deficient animals. This study reveals that a bone-derived embryonic hormone influences lifelong adrenal functions and organismal homeostasis in the mouse.

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.

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.

iCa2+ Flux, ROS and IL-10 Determines Cytotoxic, and Suppressor T Cell Functions in Chronic Human Viral Infections.

Exhaustion of CD8+ T cells and increased IL-10 production is well-known in chronic viral infections but mechanisms leading to loss of their cytotoxic capabilities and consequent exhaustion remain unclear. Exhausted CD8+T cells also called T suppressors are highly immune suppressive with altered T cell receptor signaling characteristics that mark it exclusively from their cytotoxic counterparts. Our study found that iCa2+ flux is reduced following T cell receptor activation in T suppressor cells when compared to their effector counterpart. Importantly chronic activation of murine cytotoxic CD8+ T cells lead to reduced iCa2+ influx, decreased IFN-γ and enhanced IL-10 production and this profile is mimicked in Tc1 cells upon reduction of iCa2+ flux by extracellular calcium channel inhibitors. Further reduced iCa2+ flux induced ROS which lead to IFN-γ reduction and increased IL-10 producing T suppressors through the STAT3-STAT5 axis. The above findings were substantiated by our human data where reduced iCa2+ flux in chronic Hepatitis infections displayed CD8+ T cells with low IFN-γ and increased IL-10 production. Importantly treatment with an antioxidant led to increased IFN-γ and reduced IL-10 production in human chronic Hep-B/C samples suggesting overall a proximal regulatory role for iCa2+ influx, ROS, and IL-10 in determining the effector/ suppressive axis of CD8+ T cells.

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.