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Biomedical subjects

Sanjeev Gupta

Publications and source records attributed to Sanjeev Gupta.

At least 19 recordsLinked to original sources
PubMed2003

Inhibitor of apoptosis proteins are substrates for the mitochondrial serine protease Omi/HtrA2.

The mature serine protease Omi/HtrA2 is released from the mitochondria into the cytosol during apoptosis. Suppression of Omi/HtrA2 by RNA interference in human cell lines reduces cell death in response to TRAIL and etoposide. In contrast, ectopic expression of mature wildtype Omi/HtrA2, but not an active site mutant, induces potent caspase activation and apoptosis. In vitro assays demonstrated that Omi/HtrA2 could degrade inhibitor of apoptosis proteins (IAPs). Consistent with this observation, increased expression of Omi/HtrA2 in cells increases degradation of XIAP, while suppression of Omi/HtrA2 by RNA interference has an opposite effect. Combined, our data demonstrate that IAPs are substrates for Omi/HtrA2, and their degradation could be a mechanism by which the mitochondrially released Omi/HtrA2 activates caspases during apoptosis.

Apoptosis↗
PubMed2003

Heterotopic auxiliary liver in an isolated and vascularized segment of the small intestine in rats.

BACKGROUND: Development of an auxiliary liver is of interest for treating several conditions. To examine whether an isolated intestinal segment will support development of a heterotopic auxiliary liver, we studied the fate of liver microfragments in rats. METHODS: Small intestinal segments with intact circulation were created, and the small intestinal mucosa was removed. The intestinal segments were filled with autologous liver microfragments, and animals were studied for various periods. RESULTS: Initially, liver microfragments were engulfed by a serosanguineous exudate enriched in polymorphonuclear leukocytes, suggesting an early granulation-type response. Transplanted liver fragments were subsequently reorganized and showed morphologic integrity with typical hepatic lobular organization. Transplanted tissue contained healthy hepatocytes with abundant glycogen content. Transplanted liver remained intact in the small intestine for up to 40 days, although at later times portal fibrosis and bile ductular proliferation were apparent, despite the absence of cholestasis or hepatocellular abnormalities. In contrast, instillation of liver microfragments in the peritoneal cavity led to rapid loss of tissue integrity and phagocytotic clearance of transplanted tissue. CONCLUSIONS: Small intestinal segments denuded of the mucosal layer can support heterotopically transplanted liver. Further development of this auxiliary liver system will provide insights into mechanisms concerning neo-organogenesis and into potential therapeutic applications of heterotopic liver in specific diseases.

Animals↗
PubMed2003

Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells.

Beta-cell replacement is considered to be the most promising approach for treatment of type 1 diabetes. Its application on a large scale is hindered by a shortage of cells for transplantation. Activation of insulin expression, storage, and regulated secretion in stem/progenitor cells offers novel ways to overcome this shortage. We explored whether fetal human progenitor liver cells (FH) could be induced to differentiate into insulin-producing cells after expression of the pancreatic duodenal homeobox 1 (Pdx1) gene, which is a key regulator of pancreatic development and insulin expression in beta cells. FH cells possess a considerable replication capacity, and this was further extended by introduction of the gene for the catalytic subunit of human telomerase. Immortalized FH cells expressing Pdx1 activated multiple beta-cell genes, produced and stored considerable amounts of insulin, and released insulin in a regulated manner in response to glucose. When transplanted into hyperglycemic immunodeficient mice, the cells restored and maintained euglycemia for prolonged periods. Quantitation of human C-peptide in the mouse serum confirmed that the glycemia was normalized by the transplanted human cells. This approach offers the potential of a novel source of cells for transplantation into patients with type 1 diabetes.

Animals↗
PubMed2003

Induction of Mad expression leads to augmentation of insulin gene transcription.

Insulin gene transcription is critical for the maintenance of pancreatic beta-cell differentiation and insulin production. In this study, we found that the basic helix-loop-helix transcription factor Mad, which usually acts as a repressor to c-Myc, enhances insulin gene transcription. In isolated rat islets adenoviral overexpression of Mad augmented insulin mRNA expression and insulin protein content, as well as glucokinase and GLUT2 mRNA expression. Also, Mad overexpression upregulated insulin promoter activity in beta-cell-derived cell lines, MIN6 and betaTC1, as well as in non-insulin producing liver cell line, HepG2. Mad overexpression in rat islets enhanced PDX-1 expression and its DNA binding activity. We found that Mad mediated increased PDX-1 expression by an E-box dependent transcriptional regulation of the PDX-1 gene. That the effects of Mad on insulin expression were mediated through PDX-1 was further substantiated by studies showing inhibition of insulin promoter activation by Mad in the presence of mutated PDX-1 binding site. Although Mad functions as a negative regulatory factor for multiple target genes, these studies establish the fact that Mad can also function as a positive regulatory factor for insulin gene transcription. Such regulation of insulin expression by Mad with modulation of PDX-1 expression and DNA binding activity could offer useful therapeutic and/or experimental tools to promote insulin production in appropriate cell types.

Adenoviridae↗
PubMed2003

Sestamibi is a substrate for MDR1 and MDR2 P-glycoprotein genes.

Technetium-99m sestamibi has attracted interest for assessment of the function of P-glycoproteins, which are well expressed in the liver and have roles in biliary transport and the removal of chemotherapeutic drugs. To further examine the cross-reactivity of (99m)Tc-sestamibi for P-glycoprotein family members, we conducted studies in animals. Hepatobiliary secretion of (99m)Tc-sestamibi was determined in normal FVB/N mice, mutant mice with specific P-glycoprotein deficiencies in the FVB/N background, normal Long-Evans Agouti (LEA) rats, and Long-Evans Cinnamon (LEC) rats with abnormal copper transport and liver disease but intact P-glycoprotein expression. After intrasplenic injection, (99m)Tc-sestamibi was rapidly incorporated in the mouse and rat liver, with maximal accumulation after 102+/-31 and 109+/-16 s, respectively ( P=NS). In normal mice and rats, 55%+/-11% and 55%+/-6%, respectively, of the maximal sestamibi activity was retained in the liver after 1 h ( P=NS). In double knockout mice lacking both mdr1a and mdr1b homologs of the human MDR1 ( ABCB1) gene, 88%+/-11% of maximal sestamibi activity was retained in the liver after 1 h ( P<0.001). In knockout mice deficient in either mdr1a gene or mdr2 ( ABCB4) gene, biliary sestamibi excretion was also impaired, although this impairment was relatively less pronounced in ABCB4-deficient mice than in double knockout mice lacking both ABCB1 gene homologs ( P<0.03). Hepatobiliary sestamibi excretion in LEC rats was not different from that in control normal rats, despite the presence of significant liver disease in the former. Hepatobiliary sestamibi excretion requires P-glycoproteins and is unperturbed in chronic liver disease. Sestamibi appears to be a substrate for both ABCB1 and ABCB4 genes, although the former utilizes it far more efficiently. Assessment of P-glycoprotein activity with sestamibi should consider how regulation of ABCB1 and related family members might modulate sestamibi incorporation.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2003

Public spending on health care and the poor.

This paper uses new cross-country data to assess the relationship between public spending on health care and the health status of the poor. Data are drawn from two sources: (i) existing data on health status by income quintile tabulated from demographic health surveys in 44 countries; and (ii) our estimates of the health status of the poor in over 70 countries drawn from a new technique in decomposing social indicators. Our estimates confirm that the poor have significantly worse health status than the nonpoor and the regression results provide new evidence that public spending on health care matters more to them. However, the results suggest that increased public spending alone will not be sufficient to significantly improve health status.

Health Expenditures↗
PubMed2003

Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential.

BACKGROUND & AIMS: The availability of in vitro expandable human hepatocytes would greatly advance liver-directed cell therapies. Therefore, we examined whether human fetal hepatocytes are amenable to telomerase-mediated immortalization without inducing a transformed phenotype and disrupting their differentiation potential. Telomerase is a ribonucleoprotein that plays a pivotal role in maintaining telomere length and chromosome stability. Human somatic cells, including hepatocytes, exhibit no telomerase activity. Consequently, their telomeres progressively shorten with each cell cycle until critically short telomeres trigger replicative senescence. METHODS: The catalytic subunit, telomerase reverse transcriptase, was expressed in human fetal hepatocytes. Transduced cells were characterized for telomerase activity, telomere length, proliferative capacity, hepatocellular functions, oncogenicity, and their in vivo maturation potential. RESULTS: The expression of human telomerase reverse transcriptase restored telomerase activity in human fetal hepatocytes. Telomerase-reconstituted cells were capable of preserving elongated telomeres, propagated in culture beyond replicative senescence for more than 300 cell doublings (to date), and maintained their liver-specific nature, as analyzed by a panel of hepatic growth factors, growth factor receptors, and transcription factors as well as albumin, glucose-6-phosphatase, glycogen synthesis, cytochrome P450 (CYP) expression profiles, and urea production. Moreover, the immortalized cells exhibited no oncogenicity, and no up-regulation of c-Myc was detected. The cells engrafted and survived in the liver of immunodeficient mice with hepatocellular gene expression. CONCLUSIONS: Reconstitution of telomerase activity induces indefinite replication in human fetal hepatocytes and offers unique opportunities for examining basic biologic mechanisms and for considering development of stable cell lines for liver-directed therapies.

Animals↗
PubMed2003

Klippel-Trenaunay syndrome with a life-threatening thromboembolic event.

Klippel-Trenaunay syndrome is a congenital disorder characterised by the triad of cutaneous vascular nevi, soft tissue or bony hypertrophy, and varicose veins or venous malformations involving one or more extremities. An incidence of venous thromboembolism of up to 22% has been reported in this disorder. Also reported but rare is the development of trophic changes. Herein, we report the case of a male with Klippel-Trenaunay syndrome, deep vein thrombosis, venous ulceration, and death due to recurrent pulmonary embolism.

Anticoagulants↗
PubMed2003

Essential hypertension: antecedents in children.

A prospective study was conducted to determine the incidence of essential hypertension (EH) and identify markers, if any, in children of essential hypertension families. The study group included 90 children (2-18 years) with a parent or grandparent with EH while the control group had 25 age matched children from non-hypertensive families. Around 30% children (n=27) from these families had a diastolic blood pressure of >95th centile and an additional 27% (n=24) had borderline hypertension. The serum cholesterol, serum triglycerides and 24 hour urinary sodium excretion were significantly higher in the study group (p < 0.05) as compared to controls. The children from the study group also had a significant high salt (p < 0.001) and fat intake (P < 0.05).

Adolescent↗
PubMed2002

A nuclear protein tyrosine phosphatase activates p53 and induces caspase-1-dependent apoptosis.

PTP-S2/TC45 is a nuclear protein tyrosine phosphatase, which induces p53-dependent apoptosis. Here we show that the p53 protein level increased in MCF-7 cells in response to PTP-S2 overexpression. PTP-S2-induced p53 protein was transcriptionally active and it could activate caspase-1 gene expression from endogenous as well as ectopic promoter. Coexpression of an active site mutant of procaspase-1 strongly inhibited PTP-S2-induced apoptosis. Mutant procaspase-1 also inhibited apoptosis induced by p53 overexpression or doxorubicin treatment, which induce caspase-1 gene expression. In contrast, apoptosis induced by staurosporine or cycloheximide, which do not increase caspase-1 gene expression, was not affected by mutant procaspase-1. These results suggest that caspase-1 may be one of the mediators of p53-dependent apoptosis in human cells.

Apoptosis↗
PubMed2002

Cell transplantation after oxidative hepatic preconditioning with radiation and ischemia-reperfusion leads to extensive liver repopulation.

The inability of transplanted cells to proliferate in the normal liver hampers cell therapy. We considered that oxidative hepatic DNA damage would impair the survival of native cells and promote proliferation in transplanted cells. Dipeptidyl peptidase-deficient F344 rats were preconditioned with whole liver radiation and warm ischemia-reperfusion followed by intrasplenic transplantation of syngeneic F344 rat hepatocytes. The preconditioning was well tolerated, although serum aminotransferase levels rose transiently and hepatic injury was observed histologically, along with decreased catalase activity and 8-hydroxy adducts of guanine, indicating oxidative DNA damage. Transplanted cells did not proliferate in the liver over 3 months in control animals and animals preconditioned with ischemia-reperfusion alone. Animals treated with radiation alone showed some transplanted cell proliferation. In contrast, the liver of animals preconditioned with radiation plus ischemia-reperfusion was replaced virtually completely over 3 months. Transplanted cells integrated in the liver parenchyma and liver architecture were preserved normally. These findings offer a paradigm for repopulating the liver with transplanted cells. Progressive loss of cells experiencing oxidative DNA damage after radiation and ischemia-reperfusion injury could be of significance for epithelial renewal in additional organs.

Animals↗
PubMed2002

Enzymatically labeled chromosomal probes for in situ identification of human cells in xenogeneic transplant models.

Analysis of the viability, differentiation, clonogenicity and function of human stem/progenitor cells requires suitable xenograft models. However, the identification of transplanted cells has been generally difficult. Fluorescence in situ hybridization is a tedious method for analyzing tissues, and localization of transplanted cells with X or Y chromosome probes is limited by the sparse signals produced. Therefore, we examined the possibility of generating either pan-nuclear signals with a total human DNA probe or multiple nuclear signals with a pan-centromeric human DNA probe. The probes were labeled with digoxigenin to make reaction products visible by light microscopy and to allow the use of immunohistochemistry methods incorporating various color schemes to demonstrate specific properties of transplanted cells. The ability to localize all types of nucleated human cells with such probes will facilitate studies of stem cell biology and cell and gene therapy, as well as the development of new animal models.

Animals↗
PubMed2002

Isolation of human progenitor liver epithelial cells with extensive replication capacity and differentiation into mature hepatocytes.

The liver can regenerate itself through the progenitor cells it harbors. Here we demonstrate isolation of epithelial progenitor/stem cells from the fetal human liver, which contains a large number of hepatoblasts. Progenitor liver cells displayed clonogenic capacity, expressed genes observed in hepatocytes, bile duct cells and oval cells, and incorporated genes transferred by adenoviral or lentiviral vectors. Under culture conditions, progenitor cells proliferated for several months, with each cell undergoing more than forty divisions, but they retained normal karyotypes. Progenitor cells differentiated into mature hepatocytes in mice with severe combined immunodeficiency, both when in an ectopic location and when in the liver itself. Cells integrated in the liver parenchyma and proliferated following liver injury. An abundance of progenitor cells in the fetal human liver is consistent with models indicating depletion of progenitor/stem cells during aging and maturation of organs. The studies indicate that isolation of progenitor cells from fetal organs will be appropriate for establishing novel systems to investigate basic mechanisms and for cell and gene therapy.

Animals↗
PubMed2002

Therapeutic potential of hepatocyte transplantation.

Liver repopulation with transplanted cells offers unique opportunities for treating a variety of diseases and for studies of fundamental mechanisms in cell biology. Our understanding of the basis of liver repopulation has come from studies of transplanted cells in animal models. A variety of studies established that transplanted hepatocytes as well as stem/progenitor cells survive, engraft, and function in the liver. Transplanted cells survive life-long, although cells do not proliferate in the normal liver. On the other hand, the liver is repopulated extensively when diseases or other injuries afflict native hepatocytes but spare transplanted cells. The identification of ways to repopulate the liver with transplanted cells has greatly reinvigorated the field of liver cell therapy. The confluence of insights in stem/progenitor cells, transplantation immunology, cryobiology, and liver repopulation in specific models of human diseases indicates that the field of liver cell therapy will begin to reap the promised fruit in the near future.

Animals↗
PubMed2002

Hepatocyte transplantation.

Repopulation of the liver with transplanted cells holds significant promise for developing novel therapies. The liver is a most suitable target for treating a variety of genetic, metabolic and acquired diseases. Liver disease, such as chronic viral hepatitis, constitutes an enormous burden worldwide. Advancing liver cell therapy requires insights into mechanisms of cell engraftment and proliferation, as well as unique requirements of specific diseases for correction by cell transplantation. This review highlights recent developments in the area of hepatocyte transplantation. Aspects concerning modulation of cell engraftment, regulation of gene expression and proliferation of transplanted cells are discussed. Other issues concern the current status of clinical applications of hepatocyte transplantation, as well as novel sources of cells that could benefit cell therapy in the future. The general conclusion is that cell therapy has become more practical in recent years and insights into how the normal liver and the diseased liver can be repopulated will offer effective ways to treat many disorders in the near future.

Cell Transplantation↗
PubMed2002

Early cell transplantation in LEC rats modeling Wilson's disease eliminates hepatic copper with reversal of liver disease.

BACKGROUND & AIMS: The Long-Evans Cinnamon (LEC) rat is an excellent model of Wilson's disease with impaired copper excretion, hypoceruloplasminemia, and copper toxicosis. We hypothesized that early hepatocyte transplantation would improve copper excretion and liver disease in Wilson's disease. METHODS: Normal syngeneic Long-Evans Agouti rat hepatocytes were transplanted intrasplenically into 2-week-old LEC rats. Untreated LEC pups were controls. Liver repopulation was shown by changes in serum ceruloplasmin, hepatic atp7b messenger RNA, and bile and liver copper levels. Histologic analysis of the liver was performed. RESULTS: Significant copper accumulation and liver disease were observed in 5-month-old LEC rats, with occasional treated rats showing increased bile copper excretion. The liver was repopulated extensively in 10 of 14 treated LEC rats (71%) 20 months after cell transplantation. In these 10 rats, hepatic copper content was virtually normal in 6 rats (53 +/- 12 microg/g liver) and substantially less in 4 others (270 +/- 35 microg/g) compared with elevated liver copper levels in untreated LEC rats (1090 +/- 253 microg/g) (P < 0.001). Changes in serum ceruloplasmin levels, bile copper excretion capacity, and liver histology were in concordance with decreases in liver copper levels. CONCLUSIONS: Transplanted cells proliferated subsequent to the onset of liver injury, and the liver was repopulated over an extended period. Cell transplantation eventually restored copper homeostasis and reversed liver disease without hepatic preconditioning in LEC rats.

Animals↗
PubMed2002

Kupffer cells participate in early clearance of syngeneic hepatocytes transplanted in the rat liver.

BACKGROUND & AIMS: Kupffer cells are activated shortly after deposition of hepatocytes in liver sinusoids, with clearance of a significant fraction of transplanted cells, especially when cells are entrapped in portal spaces. We determined whether perturbation of Kupffer cells would improve transplanted cell engraftment. METHODS: Dipeptidyl peptidase IV-deficient rats were used as recipients of syngeneic Fischer 344 rat hepatocytes. Kupffer cell function was analyzed by measuring phagocytic activity with carbon particle or (99m)Tc-sulfur colloid incorporation. Transplanted cell survival and integration in the liver parenchyma was determined by histochemical analysis of tissues. Transplanted cell proliferation was analyzed in rats conditioned with retrorsine and partial hepatectomy. RESULTS: Gadolinium chloride significantly impaired Kupffer cell function, especially in periportal areas, where transplanted cells were localized. Transplanted cell survival increased by approximately 2-fold in animals treated with gadolinium chloride 24 hours before cell transplantation. In gadolinium-treated rats, more transplanted cells were observed in portal vein radicles, as well as in liver sinusoids, albeit integration of cells in the liver parenchyma was slower in gadolinium-treated rats and cells separated from other hepatocytes in portal vein radicles that failed to exhibit bile canalicular reconstitution. Finally, hepatocyte transplantation in rats primed with retrorsine and partial hepatectomy showed accelerated kinetics of liver repopulation in animals pretreated with gadolinium chloride. CONCLUSIONS: Perturbation of Kupffer cell activity will benefit liver repopulation with cells and further analysis of clinically suitable approaches to exploit this mechanism will be appropriate.

Animals↗