Human amniotic epithelial cells (HAECS); a potential cell type for the hepatic disorders.

OBJECTIVE: To isolate a homogenous population of human amniotic epithelial cells (hAECs) from the amniotic membrane of the human placenta and differentiate them into hepatic-like cells with the help of small molecules. METHODS: hAECs were isolated by using the enzymatic digestion method and characterized for the presence of specific stem cell markers. In-vitro, hepatic differentiation of hAECs was carried out by using a combination of small molecules. Differentiated cells were observed under a live cell imaging microscope for morphological changes followed by gene and protein expression analysis by qPCR and immunocytochemistry respectively. RESULTS: The isolated hAECs attained characteristic cuboid epithelial shape and express stem cells marker. The hepatic differentiation method was optimized based on soluble chemical compounds supplied in the culture medium. The differentiated hAECs phenotypically acquire hepatic-like cell features and expressed hepatic markers as well as hepatic protein albumin at immature levels. CONCLUSIONS: The isolated population of hAECs is highly proliferative. Moreover, hepatic markers expression in the isolated hAECs makes them an exclusive source for the treatment of chronic liver diseases.

Ascorbic acid and salvianolic acid B enhance the valproic acid and 5-azacytidinemediated cardiac differentiation of mesenchymal stem cells.

BACKGROUND: Cardiovascular diseases remain a major cause of death globally. Cardiac cells once damaged, cannot resume the normal functioning of the heart. Bone marrow derived mesenchymal stem cells (BM-MSCs) have shown the potential to differentiate into cardiac cells. Epigenetic modifications determine cell identity during embryo development via regulation of tissue specific gene expression. The major epigenetic mechanisms that control cell fate and biological functions are DNA methylation and histone modifications. However, epigenetic modifiers alone are not sufficient to generate mature cardiac cells. Various small molecules such as ascorbic acid (AA) and salvianolic acid B (SA) are known for their cardiomyogenic potential. Therefore, this study is aimed to examine the synergistic effects of epigenetic modifiers, valproic acid (VPA) and 5-azacytidine (5-aza) with cardiomyogenic molecules, AA and SA in the cardiac differentiation of MSCs. METHODS AND RESULTS: BM-MSCs were isolated, propagated, characterized, and then treated with an optimized dose of VPA or 5-aza for 24 h. MSCs were maintained in a medium containing AA and SA for 21 days. All groups were assessed for the expression of cardiac genes and proteins through q-PCR and immunocytochemistry, respectively. Results show that epigenetic modifiers VPA or 5-aza in combination with AA and SA significantly upregulate the expression of cardiac genes MEF2C, Nkx2.5, cMHC, Tbx20, and GATA-4. In addition, VPA or 5-aza pretreatment along with AA and SA enhanced the expression of the cardiac proteins connexin-43, GATA-4, cTnI, and Nkx2.5. CONCLUSION: These findings suggest that epigenetic modifiers valproic acid and 5-azacytidine in combination with ascorbic acid and salvianolic acid B promote cardiac differentiation of MSCs. This pretreatment strategy can be exploited for designing future stem cell based therapeutic strategies for cardiovascular diseases.

Zinc and hypoxic preconditioning: a strategy to enhance the functionality and therapeutic potential of bone marrow-derived mesenchymal stem cells.

The therapeutic use of bone marrow mesenchymal stem cells (BM-MSCs) requires a large number of cells (1-100 × 106 cells/kg of body weight). Extensive in vitro growth is limited due to the aging of cultured BM-MSCs which leads to abnormal morphology and senescence. Hypoxia increases BM-MSC proliferation, but the question of whether hypoxia preconditioning is safe for clinical application of BM-MSCs remains to be answered. Zinc is essential for cell proliferation and differentiation, especially for the regulation of DNA synthesis and mitosis. It is a structural constituent of numerous proteins on a molecular level, including transcription factors and enzymes of cellular signaling machinery. All the tissues, fluids, and organs of the human body contain zinc. More than 95% of zinc is intracellular, of which 44% is involved in the transcription of DNA. We investigated the effects of ZnCl2 on proliferation, morphology, migration, population doubling time (PDT), and gene expression of BM-MSCs under hypoxic (1% O2) and normoxic (21% O2) environments. BM-MSCs were preconditioned with optimized concentrations of ZnCl2 under normoxic and hypoxic environments and further examined for morphology by the phase-contrast inverted microscope, cell proliferation by MTT assay, PDT, cell migration ability, and gene expression analysis. Zinc significantly enhanced the proliferation of BM-MSCs, and it decreases PDT under hypoxic and normoxic environments as compared to control cells. Migration of BM-MSCs toward the site of injury increased and expression of HIF1-α significantly decreased under hypoxic conditions as compared to non-treated hypoxic cells and control. At late passages (P9), the morphology of normoxic BM-MSCs was transformed into large, wide, and flat cells, and they became polygonal and lost their communication with other cells. Conversely, zinc-preconditioned BM-MSCs retained their spindle-shaped, fibroblast-like morphology at P9. The expression of proliferative genes was found significantly upregulated, while downregulation of genes OCT4 and CCNA2 was observed in zinc-treated BM-MSCs under both normoxic and hypoxic conditions. ZnCl2 treatment can be used for extensive expansion of BM-MSCs in aged populations to obtain a large number of cells required for systemic administration to produce therapeutic efficacy.

Pre-weaning fluoxetine exposure caused anti-depressant like behavior at adulthood via perturbing tryptophan metabolism in rats.

The perinatal depression exposes the child to antidepressants during vulnerable window of development, which can chronically impact the mental wellbeing of new born. Active pharmaceuticals are not tested for this long term neurobehavioral aspect of toxicity during drug development process. Keeping this in view, the current study was designed to study the effect of pre-weaning fluoxetine exposure on depression-like behavior of the offspring upon attaining adulthood using FST (Forced swim test). Additionally, the brain tryptophan, 5-HT (5-hydroxytryptamine) and its metabolite 5-HIAA (5-hydroxyindoleacetic acid) levels were quantified using Enzyme linked Immunosorbent Assay (ELISA), while expression of SERT (serotonin receptor), 5-HT1A receptor, TPH (tryptophan hydroxylase) genes were monitored using qPCR. Our data showed that pre-weaning fluoxetine (10, 50 or 100 mg/kg) exposure decreased depression-like behavior. The 5-HT and 5-HIAA levels showed declining trend. However, the 5-HT synthetic precursor i.e. tryptophan levels were found to be significantly elevated in both brain and plasma as compared to control rats. The gene expression study did not reveal any significant alterations as compared to control. In conclusion, the present study demonstrate that pre-weaning fluoxetine exposure decreased depression-like behavior upon adulthood via perturbing tryptophan metabolism.

Pre-weaning fluoxetine exposure perturbs social behavior at adulthood via altering hippocampal morphometry and PSD-95 expression in rats.

The depression during and after pregnancy cause significant exposure of fluoxetine to the child at early life through mother. This exposure to the child, during the vulnerable window of development, can have a long lasting impact on overall mental wellbeing. Long term neurobehavioral aspect of developmental toxicity is neglected as the part of testing requirements in the process of drug developmental. In this context, the present study was designed to study the possible effect of pre-weaning fluoxetine exposure on the social behavior of rats upon adulthood followed by assessing hippocampal morphometry (hematoxylin-eosin and silver staining) and post-synaptic density protein 95 (PSD-95) expression (using qPCR). Our data showed that the fluoxetine exposure (10, 50 and 100mg/kg) caused predominant increase in the social behavior of rats; the effect more pronounced in female rats. The morphometric analysis revealed significant increase in cell population and count of dentate gyrus (DG) region of hippocampus along with enhanced dendritic arborization. Furthermore, the PSD-95 expression was found to be down regulated in the fluoxetine treated group as compared to control. In conclusion, the present study demonstrate that the early post-natal exposure to fluoxetine cause hypersociability upon attaining adulthood, which may be attributed to enhanced neuronal proliferation and decrease PSD-95 expression in the hippocampus.

Phloroglucinol ameliorated methylglyoxal induced harmful effects in rats.

Glycation is among the underlying mechanisms attributed to ageing and associated morbidities. There is no drug available to combat this deleterious phenomenon. The present study aimed to explore phloroglucinol (PHL) for its anti-glycation potential at preclinical level. The rats were treated with methylglyoxal (MGO, 17.25 mg/kg, i.p. for 14 days) to induce glvcative stress. The treatment groups received additional administration of test drug (PHL; 0.25mg/kg, 0.5mg/kg, and 1mg/kg) or standard aminoguanidine (AG, 50 mg/kg) or saline (control, 5ml/kg). During 14 days, the weight and food intake was noted. Afterwards, the cognitive function was evaluated using Morris Water Maze (MWM) while hepatic and renal functions were assessed through liver function test (bilirubin, alkaline phosphatase, SGPT, and SGOT) and creatinine respectively, using chemical analyzer. The carboxymethyllysine (CML) levels were quantified in the blood using ELISA technique. Histopathological study was performed on the brain, liver, and kidney using H&E staining. Additionally, the qPCR was used to quantify the expression of TNF-α, RAGE and BACE-1 (brain), RAGE, TNF-α, and glyoxalase-I (liver) and RAGE, TNF-α, and VEGF (kidney), while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference housekeeping gene. The data regarding weight and food intake did not reveal significant alterations. In MWM, the MGO treatment caused significant increase in the time to reach target quadrant, while decrease in the time spent in target quadrant and number of crossings through platform position. All these effects were inhibited by both AG and PHL. The navigation maps also exhibit that the retention of spatial memory. Additionally, the MGO-induced alteration in hepatic and renal function indicators was ameliorated by both AG and PHL treatments. The plasma CML levels were found to be elevated following MGO treatment, while the concomitant administration of AG and PHL has resisted this raise. Histopathological assessment revealed no specific pathology in liver kidney and brain tissues. The qPCR data revealed enhanced expression of all genes, especially TNF-α and BACE, which were found to be reduced following both AG and PHL treatments. PHL prevented the brain, hepatic, and renal impairments caused by MGO induced glycative stress. Hence, the PHL, a clinically used anti-spasmodic drug, presents itself as a potential candidate to be repurposed as anti-glycation drug.

Phloroglucinol inhibited glycation via entrapping carbonyl intermediates.

Advanced glycation end products (AGEs) play an important role in the pathogenesis of age-linked disorders and diabetes mellitus. The aim of this study was to assess the repurposing potential of Phloroglucinol (PHL the antispasmodic drug), as an anti-glycation agent using Fructose-BSA model. The ability of PHL to inhibit AGE formation was evaluated using AGEs formation (Intrinsic fluorescence), fructosamine adduct (NBT) and free lysine availability (TNBSA) assays. The BSA protein conformation was assessed through Thioflavin-T, Congo-Red and Circular Dichroism assays. The lysine blockade and carbonyl entrapment were explored as possible mode of action. Our data showed that PHL significantly decreased the formation of AGEs with an IC50 value of 0.3mM. The fructosamine adducts and free lysine load was found to be reduced. Additionally, the BSA conformation was preserved by PHL. Mechanistic assays did not reveal involvement of lysine blockade as underlying reason for reduction in AGEs load. This was also supported by computational data whereby PHL failed to engage any catalytic residue involved in early fructose-BSA interaction. However, it was found to entrap the carbonyl moieties. In conclusion, the PHL demonstrated anti-glycation potential, which can be attributed to its ability to entrap carbonyl intermediates. Hence, the clinically available antispasmodic drug, presents itself as a promising candidate to be repurposed as anti-glycation agent.

Costimulatory Molecules OX40 and OX40L Upregulation in Oral Squamous Cell Carcinoma: A Blood-Based Study.

OBJECTIVES: This research aimed to determine OX40 and OX40L mRNA expression in blood samples of naive oral squamous cell carcinoma (OSCC) patients in different histological grades and clinical stages. The in silico analysis was performed using the STRING database for functional association and a better understanding of the interactions of OX40 and its ligand with other proteins. MATERIALS AND METHODS: In this study, we recruited 141 newly diagnosed patients of OSCC. Levels of OX40 and OX40L mRNA expression were explored using real-time quantitative polymerase chain reaction. An in silico tool was also utilized to evaluate the OX40/OX40L interactome. RESULTS: The results showed higher OX40 expressional levels in the late stage (23-fold) compared with the early stage (8.5-fold) (p = < 0.001). A similar trend was seen in OX40L mRNA expression, revealing a fold change of 5.8 in the early stage in comparison to 9.9-fold change in the late stage (p = < 0.001). Overexpression of OX40 and OX40L was found in different histological grades (p = 0.005 and p = < 0.001, respectively). Overexpression of OX40 and OX40L was detected in habits such as smoking and paan intake, whereas statistically significant upregulation was observed in the cheek, lip, and alveolus tumors. However, there was no substantial difference in OX40 and OX40L expression based on age or gender. The functional interactions, that is, interactomes of OX40 and OX40L with other proteins have been determined by in silico analysis. CONCLUSION: Based on current study findings, despite OX40 and OX40L upregulation in newly diagnosed OSCC patients, it is speculated that the physiological function of these molecules is altered due to immune system exhaustion.

Applications of Metformin in Dentistry-A review.

Metformin is a versatile drug with numerous medical uses. It is known primarily as an anti-hyperglycemic drug that has become the main oral blood-glucose-lowering medication for managing type 2 diabetes mellitus globally. Its use has been reported in a variety of oral conditions and dentistry in general. Recent clinical trials have indicated the effectiveness of adjunct topical application of metformin in improving the periodontal parameters of patients with diabetes and periodontitis. Additionally, studies have suggested that metformin stimulates odontogenic differentiation and mineral synthesis of stem cells in the tooth pulp. Metformin also stimulates osteoblast proliferation, decreases osteoclast activity and exerts regenerative effects on periodontal bone, thus making it a viable candidate for periodontal regeneration. Metformin monotherapy significantly enhances osseointegration of endosseous implants and has been reported to have anti-cancer effects on oral squamous cell carcinoma by impeding tumor progression. Animal studies have indicated that metformin improves orthodontic tooth movement and resists orthodontic appliance corrosion. This narrative review aims to provide a current summary of research highlighting the prospective uses of metformin in dentistry.

Serum Levels of OX40 in Early and Late-Stage Oral Squamous Cell Carcinoma.

Background The tumor necrosis factor receptor superfamily, member 4 (OX40) and its ligand (OX40L) are members of the tumor necrosis factor superfamily and play roles as costimulatory immunomodulators to combat infectious diseases as well as cancers. Presently, many therapeutic agents focused on OX40 and OX40L are in trials for antitumor efficacy. In Pakistan, oral squamous cell carcinoma (OSCC) is the second most prevalent cancer with a mortality of 50% despite the availability of various therapeutic modalities. Data regarding serum levels of OX40 in patients with OSCC is lacking. Therefore, the study aimed to assess the OX40 levels in serum and their association with the clinicopathological features of the tumor. Methodology A cross-sectional study was conducted and serum samples of 78 biopsy-confirmed OSCC patients were collected prior to any treatment along with 10 healthy persons after informed consent. Serum levels of OX40 were measured via sandwich enzyme-linked immunosorbent assay (ELISA). Results The mean serum levels of OX40 were 1.65 ± 0.64 ng/ml and 2.39 ± 0.58 ng/ml in early and late-stage disease patients of OSCC, respectively (p =<0.005). However, based on gender and tumor site, male gender and buccal mucosa tumors in late-stage OSCC patients showed higher mean levels of OX40, 2.42± 0.58 ng/ml and 2.41 ± 0.58 ng/ml (p =<0.05), respectively. Patients with well-differentiated tumors demonstrated mean serum levels of 2.28 ng/ml, and in moderately differentiated tumors, the mean levels were 2.19 ng/ml (p =0.47). Conclusions A high OX40 level is associated with advanced-stage disease and a poor prognosis, possibly reflecting the immune-exhausted status against OSCC.

Hypoxic Preconditioning Improves the Therapeutic Potential of Aging Bone Marrow Mesenchymal Stem Cells in Streptozotocin-Induced Type-1 Diabetic Mice.

Insulin replacement is the current therapeutic option for type-1 diabetes. However, exogenous insulin cannot precisely represent the normal pattern of insulin secretion. Another therapeutic strategy is transplantation of pancreatic islets, but this is limited by immune rejection, intrinsic complications, and lack of donor availability. Stem cell therapy that results in the regeneration of insulin-producing cells represents an attractive choice. However, with advancing age, stem cells also undergo senescence, which leads to changes in the function of various cellular processes that result in a decrease in the regeneration potential of these aging stem cells. In this study, the effect of young and aging mesenchymal stem cells (MSCs) on the regeneration of pancreatic beta cells in streptozotocin (STZ)-induced type-1 diabetic mice was observed after hypoxic preconditioning. Hypoxia was chemically induced by 2, 4-dinitrophenol (DNP). Plasma insulin and glucose levels were measured at various time intervals, and pancreatic sections were analyzed histochemically. The effect of DNP was also analyzed on apoptosis of MSCs by flow cytometry and on gene expression of certain growth factors by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). We observed that hypoxic preconditioning caused changes in the gene expression levels of growth factors in both young and aging MSCs. Young MSCs showed significant regeneration potential compared with the aging cells in vivo. However, hypoxic preconditioning was able to improve the regeneration potential of aging MSCs. It is concluded from the present study that the regeneration potential of aging MSCs into pancreatic ß-cells can be enhanced by hypoxic preconditioning, which causes changes in the gene expression of certain growth factors.

Conditioned media trans-differentiate mature fibroblasts into pancreatic beta-like cells.

AIM: The study was carried out to evaluate the role of preconditioning strategies on the trans-differentiation of mature fibroblasts (NIH3T3 cells) into insulin producing ß-cells. METHODS: The NIH3T3 cells were treated with dexamethasone (5µM) and pancreatic extract (0.05 and 0.4mg/mL) separately or in combination. The treated cells were analyzed for the morphological changes, and expression of pancreatic genes and proteins by phase contrast microscopy, RT-PCR and flow cytometry/immunocytochemistry, respectively. RESULTS: Treatment of mature fibroblasts with different combinations of dexamethasone and pancreatic extract in the form of conditioned media resulted in comparable morphological changes and expression of certain pancreatic genes and proteins; however, their expression varied with each treatment. Most prominent effect was observed in case of combined treatment which resulted in significant increase (p<0.001) in gene expression levels of insulin, MafA, and Ngn3. Variable pattern was observed in insulin, MafA, Ngn3 and Sca1 expressions at the protein level. CONCLUSION: It is concluded from this study that preconditioning of NIH3T3 cells with conditioned media containing different combinations of dexamethasone and pancreatic extract can induce trans-differentiation of these cells into pancreatic ß-like cells. The conditioned media however, need to be optimized. The study may offer the possibility of improved regeneration of mature cell type that could serve as a future therapeutic option for diabetes.