Modeling Liver Development and Disease in a Dish.

Historically, biological research has relied primarily on animal models. While this led to the understanding of numerous human biological processes, inherent species-specific differences make it difficult to answer certain liver-related developmental and disease-specific questions. The advent of 3D organoid models that are either derived from pluripotent stem cells or generated from healthy or diseased tissue-derived stem cells have made it possible to recapitulate the biological aspects of human organs. Organoid technology has been instrumental in understanding the disease mechanism and complements animal models. This review underscores the advances in organoid technology and specifically how liver organoids are used to better understand human-specific biological processes in development and disease. We also discuss advances made in the application of organoid models in drug screening and personalized medicine.

Direct Differentiation of Human Embryonic Stem Cells to 3D Functional Hepatocyte-like Cells in Alginate Microencapsulation Sphere.

BACKGROUND: The lack of a stable source of hepatocytes is one of major limitations in hepatocyte transplantation and clinical applications of a bioartificial liver. Human embryonic stem cells (hESCs) with a high degree of self-renewal and totipotency are a potentially limitless source of a variety of cell lineages, including hepatocytes. Many techniques have been developed for effective differentiation of hESCs into functional hepatocyte-like cells. However, the application of hESC-derived hepatocyte-like cells (hESC-Heps) in the clinic has been constrained by the low yield of fully differentiated cells, small-scale culture, difficulties in harvesting, and immunologic graft rejection. To resolve these shortcomings, we developed a novel 3D differentiation system involving alginate-microencapsulated spheres to improve current hepatic differentiation, providing ready-to-use hESC-Heps. METHODS: In this study, we used alginate microencapsulation technology to differentiate human embryonic stem cells into hepatocyte-like cells (hESC-Heps). Hepatic markers of hESC-Heps were examined by qPCR and Western blotting, and hepatic functions of hESC-Heps were evaluated by indocyanine-green uptake and release, and ammonia removal. RESULTS: The maturity and hepatic functions of the hESC-Heps derived from this 3D system were better than those derived from 2D culture. Hepatocyte-enriched genes, such as HNF4α, AFP, and ALB, were expressed at higher levels in 3D hESC-Heps than in 2D hESC-Heps. 3D hESC-Heps could metabolize indocyanine green and had better capacity to scavenge ammonia. In addition, the 3D sodium alginate hydrogel microspheres could block viral entry into the microspheres, and thus protect hESC-Heps in 3D microspheres from viral infection. CONCLUSION: We developed a novel 3D differentiation system for differentiating hESCs into hepatocyte-like cells by using alginate microcapsules.

Prognostic potential of CK-19 managing disseminated oral squamous cell carcinomas in Pakistan: A descriptive cross-sectional study.

A cross-sectional study was conducted to assess the adoptability of CK-19 as a routine diagnostic assay and potential prognostic marker following disseminated oral squamous cell carcinoma in Pakistani population. The current descriptive study was conducted at Isra Dental College Hospital, Isra University, Hyderabad, Pakistan. Suspected patients of oral squamous cell carcinomas (OSCC), who visited the Isra Dental College Hospital's outpatient department from January 2014 up to January 2015 with four year follow up (from January 2015 up to December 2019), were included after ethical approval of the Institutional board. SPSS version 21.0 was used for data analysis. Sixty cases of oral squamous cell carcinoma (OSCC) were selected for CK-19 quantification by using PCR before and after incisional biopsy. Of the 60 included subjects, fifty-two (87 %) were male, whereas only 8 were female. The mean age of females was 43.2±21.5years and the mean age of males was 36.14±14.1years. Of the 12 CK-19 positive cases, only seven cases of OSCCs were found positive following four year follow up duration. Our study shows that CK-19 has a positive (20%) prognostic potential for diagnosing disseminated carcinomas (p=0.0001). Before adopting CK-19 as a routine laboratory assay for diagnosing disseminated carcinomas, proper research is required to fulfil existing knowledge gap and standardising clinical and histopathological criteria for disseminating OSCCs in parallel to CK-19 concentration.

Incidental Findings Of Polypoid Cystitis In A 6-Year Old Child Without Any History Of Lower Urinary Tract Symptoms And Previous Urinary Catheterization.

A 6-year-old boy presented to ER with acute pain in right iliac fossa without any history of lower urinary tract symptoms, haematuria and urinary catheterization. Ultrasound showed loculated cystic area in pelvis with non-visualized appendix. A CT scan abdomen and pelvis revealed loculated, multi-septated cystic lesion in right hemipelvis thought to be a collection from possible appendicular inflammation / perforation. The laboratory findings revealed raised CRP and normal urine routine examination and culture. Exploratory laparotomy revealed cystic urinary bladder growth involving dome with normal appendix, partial cystectomy was done. Histopathology confirmed polypoid cystitis with no evidence of malignancy. This is a very rare presentation of polypoidal cystitis, not previously reported in literature.

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells.

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T overexpressing human NTCP, HNF4α, RXRα and PPARα) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines. HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.

Small-cell neuroendocrine carcinoma of the urinary bladder: A case report.

Primary neuroendocrine carcinomas of the urinary bladder are rare. A 60-year-old male presented with gross hematuria for the past 3 months. Diagnostic flexible cystoscopy revealed a papillary lesion above the right ureteric orifice. Transurethral resection of bladder tumor was performed and resected tissue was sent for histopathology that revealed high-grade urothelial carcinoma with small-cell neuroendocrine differentiation. Lamina propria, muscularis propria, and perineural invasion was seen which was later also confirmed by immunohistochemistry. The patient received neoadjuvant four cycles of chemotherapy and then underwent radical cystoprostatectomy with ileal conduit. The patient's recovery was uneventful and he is on regular follow-up from the past 12 months without any disease recurrence. Early detection and aggressive management can improve the survival and prognosis of these patients.

Rhazyaminine from Rhazya stricta Inhibits Metastasis and Induces Apoptosis by Downregulating Bcl-2 Gene in MCF7 Cell Line.

BACKGROUND: The role of alkaloids isolated from Rhazya stricta Decne (Apocynaceae family) (RS) in targeting genes involved in cancer and metastasis remains to be elucidated. OBJECTIVE: Identify and characterize new compounds from RS, which inhibit gene(s) involved in the survival, invasion, self-renewal, and metastatic processes of cancer cells. METHODS: Bioinformatics study was performed using HISAT2, stringtie, and ballgown pipeline to understand expressional differences between a normal epithelial cell line-MCF10A and MCF7. NMR and ATR-FTIR were performed to elucidate the structure of rhazyaminine (R.A), isolated from R stricta. Cell viability assay was performed using 0, 25, and 50 µg/mL of total extract of R stricta (TERS) and R.A, respectively, for 0, 24, and 48 hours, followed by scratch assay. In addition, total RNA was isolated for RNA- seq analysis of MCF7 cell line treated with R.A followed by qRT-PCR analysis of Bcl-2 gene. RESULTS: Deptor, which is upregulated in MCF7 compared with MCF10A as found in our bioinformatics study was downregulated by R.A. Furthermore, R.A effectively reduced cell viability to around 50% ( P < .05) and restricted cell migration in scratch assay. Thirteen genes, related to metastasis and cancer stem cells, were downregulated by R.A according to RNA- seq analysis. Additionally, qRT-PCR validated the downregulation of Bcl-2 gene in R.A-treated cells by less than 0.5 folds ( P < .05). CONCLUSION: R.A successfully downregulated key genes involved in apoptosis, cell survival, epithelial-mesenchymal transition, cancer stem cell proliferation, and Wnt signal transduction pathway making it an excellent "lead candidate" molecule for in vivo proof-of-concept studies.

Targeting signal transduction pathways of cancer stem cells for therapeutic opportunities of metastasis.

Tumor comprises of heterogeneous population of cells where not all the disseminated cancer cells have the prerogative and "in-build genetic cues" to form secondary tumors. Cells with stem like properties complemented by key signaling molecules clearly have shown to exhibit selective growth advantage to form tumors at distant metastatic sites. Thus, defining the role of cancer stem cells (CSC) in tumorigenesis and metastasis is emerging as a major thrust area for therapeutic intervention. Precise relationship and regulatory mechanisms operating in various signal transduction pathways during cancer dissemination, extravasation and angiogenesis still remain largely enigmatic. How the crosstalk amongst circulating tumor cells (CTC), epithelial mesenchymal transition (EMT) process and CSC is coordinated for initiating the metastasis at secondary tissues, and during cancer relapse could be of great therapeutic interest. The signal transduction mechanisms facilitating the dissemination, infiltration of CSC into blood stream, extravasations, progression of metastasis phenotype and angiogenesis, at distant organs, are the key pathologically important vulnerabilities being elucidated. Therefore, current new drug discovery focus has shifted towards finding "key driver genes" operating in parallel signaling pathways, during quiescence, survival and maintenance of stemness in CSC. Understanding these mechanisms could open new horizons for tackling the issue of cancer recurrence and metastasis-the cause of ~90% cancer associated mortality. To design futuristic & targeted therapies, we propose a multi-pronged strategy involving small molecules, RNA interference, vaccines, antibodies and other biotechnological modalities against CSC and the metastatic signal transduction cascade.

Hepatitis C virus and neurological damage.

Chronic hepatitis C virus (HCV) infection exhibits a wide range of extrahepatic complications, affecting various organs in the human body. Numerous HCV patients suffer neurological manifestations, ranging from cognitive impairment to peripheral neuropathy. Overexpression of the host immune response leads to the production of immune complexes, cryoglobulins, as well as autoantibodies, which is a major pathogenic mechanism responsible for nervous system dysfunction. Alternatively circulating inflammatory cytokines and chemokines and HCV replication in neurons is another factor that severely affects the nervous system. Furthermore, HCV infection causes both sensory and motor peripheral neuropathy in the mixed cryoglobulinemia as well as known as an important risk aspect for stroke. These extrahepatic manifestations are the reason behind underlying hepatic encephalopathy and chronic liver disease. The brain is an apt location for HCV replication, where the HCV virus may directly wield neurotoxicity. Other mechanisms that takes place by chronic HCV infection due the pathogenesis of neuropsychiatric disorders includes derangement of metabolic pathways of infected cells, autoimmune disorders, systemic or cerebral inflammation and alterations in neurotransmitter circuits. HCV and its pathogenic role is suggested by enhancement of psychiatric and neurological symptoms in patients attaining a sustained virologic response followed by treatment with interferon; however, further studies are required to fully assess the impact of HCV infection and its specific antiviral targets associated with neuropsychiatric disorders.

Proteomic analysis of mice fed methionine and choline deficient diet reveals marker proteins associated with steatohepatitis.

The mechanisms underlying the progression of simple steatosis to steatohepatitis are yet to be elucidated. To identify the proteins involved in the development of liver tissue inflammation, we performed comparative proteomic analysis of non-alcoholic steatohepatitis (NASH). Mice fed a methionine and choline deficient diet (MCD) developed hepatic steatosis characterized by increased free fatty acid (FFA) and triglyceride levels as well as alpha-SMA. Two-dimensional proteomic analysis revealed that the change from the normal diet to the MCD diet affected the expressions of 50 proteins. The most-pronounced changes were observed in the expression of proteins involved in Met metabolism and oxidative stress, most of which were significantly downregulated in NASH model animals. Peroxiredoxin (Prx) is the most interesting among the modulated proteins identified in this study. In particular, cross-regulated Prx1 and Prx6 are likely to participate in cellular defense against the development of hepatitis. Thus, these Prx isoforms may be a useful new marker for early stage steatohepatitis. Moreover, curcumin treatment results in alleviation of the severity of hepatic inflammation in steatohepatitis. Notably, curcumin administration in MCD-fed mice dramatically reduced CYP2E1 as well as Prx1 expression, while upregulating Prx6 expression. These findings suggest that curcumin may have a protective role against MCD fed-induced oxidative stress.

EFFICACY OF ENDOSCOPIC TREATMENT FOR PRIMARY VESICOURETERIC REFLUX IN CHILDREN.

BACKGROUND: Vesicoureteral reflux (VUR) is a common anomaly affecting 1-3% of all children and 30-50% of those with urinary tract infection (UTI). In the past febrile vesicoureteric reflux on chronic antibiotic prophylaxis were treated by open surgery. Now a day's endoscopic injection of a bulking material has replaced open surgical procedure in cases of primary VUR. Our objective was to assess the efficacy of endoscopic treatment for primary vesico-ureteric reflux in children. METHODS: This was a descriptive case series. One hundred and five patients with either unilateral or bilateral VUR (181 ureters) underwent endoscopic treatment for primary VUR between January 2011 and January 2014. Children from 1 to 12 years of age with grade-II to IV reflux on preoperative voiding cystourethrogram (VCUG) were enrolled through consecutive non-probability sampling. Efficacy of treatment was evaluated at three months post injection by a standard VCUG. Ureters with no or grade-I reflux were considered successful treatment. RESULTS: Out of 105 patients 76 had bilateral while 29 had unilateral reflux. Mean age was 5.7 years (SD ± .7). Among 181 refluxing ureters, 116 (64%) were free of reflux, while 49 (27%) showed down gradation and 16 (8.8%) showed no response to treatment on postoperative VCUG. CONCLUSION: Endoscopic treatment for VUR is a viable option for patients with primary VUR and may be considered in management of such cases.

Adiponectin: regulation of its production and its role in human diseases.

Adiponectin is a white and brown adipose tissue hormone, also known as gelatin-binding protein-28 (GBP28), AdipoQ, adipocyte complement-related protein (ACRP30), or apM1. Adiponectin circulates in the bloodstream in trimeric, hexameric, and high-molecular-mass species, while different forms of adiponectin have been found to play distinct roles in the balance of energy homoeostasis. Adiponectin is an insulin sensitizing hormone that exerts its action through its receptors AdipoR1, AdipoR2, and T-cadherin. AdipoR1 is expressed abundantly in muscle, whereas AdipoR2 is predominantly expressed in the liver. Adiponectin is inversely proportional to obesity, diabetes, and other insulin-resistant states. In this review we present the current findings regarding the regulation of its production and several new findings pertaining to its biological effects. Adiponectin enhances AMPK and the PPARα pathway in the liver and skeletal muscle. Adiponectin increases fatty acids oxidation, which lowers circulating free fatty acids and prevents insulin resistance. Adiponectin has been reported to exert an antiatherosclerotic effect. It inhibits macrophage activation and foam cell accumulation, while it also augments endothelial nitrous oxide production and protects the vasculature by reducing platelet aggregation and vasodilation. Apart from causing metabolic dysfunction, adiponectin deficiency may also contribute to coronary heart disease, steatohepatitis, insulin resistance, nonalcoholic fatty liver disease, and a wide array of cancers. In this study, we present ample evidence that adiponectin mediates multiple molecular pathways. We therefore support the concept that it shows distinct potential for being of therapeutic value in the treatment of obesity related diseases, ranging from metabolic syndrome to malignancies.