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Biomedical subjects

M Bhatia

Publications and source records attributed to M Bhatia.

At least 91 records · Page 5Linked to original sources
PubMed1997

Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice.

The purification of primitive human hematopoietic stem cells has been impaired by the absence of repopulation assays. By using a stringent two-step strategy involving depletion of lineage-positive cells followed by fluorescence-activated cell sorting, we have purified a cell population that is highly enriched for cells capable of multilineage repopulation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) recipients. These SCID-repopulating cells (SRCs) were exclusively found in a cell fraction that expressed high levels of CD34 and no CD38. Through limiting dilution analysis using Poisson statistics, we calculated a frequency of 1 SRC in 617 CD34(+) CD38(-) cells. The highly purified SRC were capable of extensive proliferation in NOD/SCID mice. Mice transplanted with 1 SRC (at limiting cell doses) were able to produce approximately 400, 000 progeny 6 weeks after the transplant. Detailed flow cytometric analysis of the marrow of highly engrafted mice demonstrated both lymphoid and myeloid differentiation, as well as the retention of a significant fraction of CD34(+) CD38(-) cells. These highly purified fractions should be useful for identification of the cellular and molecular mechanisms that regulate primitive human hematopoietic cells. Moreover, the ability to detect and purify primitive cells provides a means to develop conditions for maintaining and/or expanding these cells during in vitro culture.

ADP-ribosyl Cyclase↗
PubMed1997

A cholecystokinin-releasing factor mediates ethanol-induced stimulation of rat pancreatic secretion.

The mechanisms by which short-term ethanol administration alters pancreatic exocrine function are unknown. We have evaluated the effects of ethanol administration on pancreatic secretion of digestive enzymes. In our studies, anesthetized as well as conscious rats were given ethanol at a rate sufficient to cause the blood ethanol concentration to reach levels associated with clinical intoxication. Ethanol was administered over a 2-h period during which blood ethanol levels remained stably elevated. We report that intravenous administration of ethanol results in a transient increase in pancreatic amylase output and plasma cholecystokinin (CCK) levels. The ethanol-induced increase in amylase output can be completely inhibited by the CCK-A receptor antagonist L-364,718 and partially inhibited by the muscarinic cholinergic antagonist atropine. The ethanol-induced rise in amylase output can be completely prevented by instillation of trypsin into the duodenum or by lavage of the duodenum with saline during ethanol administration. Furthermore, the intraduodenal activity of a CCK-releasing factor is increased by infusion of ethanol. These studies indicate that administration of ethanol causes rat pancreatic exocrine secretion to increase. This phenomenon is mediated by a trypsin-sensitive CCK-releasing factor which is present within the duodenal lumen. These observations lead us to speculate that repeated CCK-mediated ethanol-induced stimulation of pancreatic digestive enzyme secretion may play a role in the events which link ethanol abuse to the development of pancreatic injury.

Alcoholism↗
PubMed1997

Assay of human stem cells by repopulation of NOD/SCID mice.

The only conclusive method to assay stem cells is to follow their ability to repopulate conditioned recipients, making it difficult to study human stem cells. The development of systems to transplant human hematopoietic cells into immune-deficient mice lays the foundation for such an experimental repopulation assay for primitive human cells. Cell purification and gene marking studies have shown that the repopulating cells, termed severe-combined immunodeficiency (SCID) mouse-repopulating cells (SRC), are primitive and distinct from most of the progenitors that are detected using short and long-term in vitro culture assays. The SRC are exclusively CD34+CD38- and poorly infected with retrovirus vectors. These gene marking data are reminiscent of the human clinical trials establishing that the SRC assay is a good surrogate to develop improved transduction methods. Limiting dilution analysis has been used to establish a quantitative assay for SRC that can be used to precisely determine the effect of various cytokine cocktails on the proliferation and differentiation of SRC during in vitro culture.

Animals↗
PubMed1997

Usefulness of short-term video EEG recording with saline induction in pseudoseizures.

OBJECTIVES: To study the usefulness of short-term recording of video electroencephalography (VEEG) as an outpatient procedure with placebo induction (PLIN) and intravenous saline in cases of pseudoseizures (Psz). MATERIAL AND METHODS: Fifty cases of suspected Psz were enrolled. They were divided into 2 groups: Group 1 consisted of patients with frank Psz, Group 2 those where diagnosis was uncertain. VEEG recording was done and 10 ml of saline used for placebo-induction. RESULTS: Of 50 patients, 24 (48%) were in Group 1 and 26 (52%) in Group 2. Fifteen (30%) had a spontaneous event during VEEG and 33% had an event only on PLIN. The diagnosis was confirmed in 60 %. In 24% of patients anti-epileptic drugs were discontinued. CONCLUSION: Short-term monitoring with VEEG using PLIN is a useful initial screening procedure and in patients where it is inconclusive, long term recordings may be done.

Adolescent↗
PubMed1997

Lipid in renal clear cell carcinoma: detection on opposed-phase gradient-echo MR images.

PURPOSE: To determine if comparison of in-phase and opposed-phase gradient-echo magnetic resonance (MR) images enables detection of lipid in renal clear cell carcinoma. MATERIALS AND METHODS: A retrospective search of MR and pathologic records identified 43 patients with biopsy-proved renal masses who underwent in-phase and opposed-phase MR imaging. Thirty-three patients had renal cell carcinoma (27 with clear cell carcinoma), and 10 patients had other renal tumors. With MR images, a region-of-interest measurement of signal intensity of the renal mass was divided by that of reference tissue. In each patient, a ratio of these region-of-interest measurements on the opposed-phase images to those on the in-phase images was calculated and termed the opposed-phase/in-phase signal intensity ratio (OIR). RESULTS: The mean OIR of clear cell carcinomas was significantly different from that of other renal masses (P < .0002); in 16 (59%) of 27 patients with clear cell carcinoma, the OIR was less than 2 standard deviations below the mean OIR of other masses. In cases of clear cell carcinoma, focal signal intensity on opposed-phase images was less than that on in-phase images. CONCLUSION: On opposed-phase images, some clear cell carcinomas show relative focal and diffuse loss of signal intensity. In renal masses, this signal intensity loss-which is consistent with lipid-does not necessarily indicate angiomyolipoma.

Adenocarcinoma, Clear Cell↗
PubMed1996

1,25-Dihydroxyvitamin D3 stimulates expression and translocation of protein kinase Calpha and Cdelta via a nongenomic mechanism and rapidly induces phosphorylation of a 33-kDa protein in acute promyelocytic NB4 cells.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) primes NB4 cells for 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiation in a dose- and sequence-dependent fashion. Experiments utilizing 1,25-(OH)2D3 analogues and kinase/phosphatase inhibitors suggested that tyrosine kinase and serine/threonine phosphorylation cascades, rather than vitamin D3 receptor-mediated signals, were involved in 1,25-(OH)2D3 action. Here we show that NB4 cells express the alpha and delta (but not the beta, epsilon, and theta) isoforms of protein kinase C (PKC). Both authentic 1, 25-(OH)2D3 and the nongenomic analogue 1alpha,25-dihydroxyprevitamin D3 (HF) increased expression of PKCalpha and PKCdelta. PKCalpha and PKCdelta were translocated to the nucleus of the cell in response to 1,25-(OH)2D3 or HF. The effects of HF were attenuated by the nongenomic antagonist 1beta,25-dihydroxyvitamin D3, suggesting that changes in PKC expression are mediated by a nongenomic signaling pathway. Consistent with the involvement of serine, threonine, and tyrosine phosphorylation cascades mediating 1,25-(OH)2D3 action, enhanced phosphorylation of a variety of cellular proteins at serine and threonine residues and the specific enhanced phosphotyrosyl content of a 33-kDa protein (vdrp33) were observed immediately after 1,25-(OH)2D3 addition. We propose that 1,25-(OH)2D3 primes NB4 cells for 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiation by increasing the expression of specific PKC isoforms and inducing the specific phosphorylation of key protein signaling intermediates.

Blotting, Western↗
PubMed1996

1,25-dihydroxyvitamin D3 primes acute promyelocytic cells for TPA-induced monocytic differentiation through both PKC and tyrosine phosphorylation cascades.

NB4 cells are the only in vitro model of differentiation in acute promyelocytic leukemia (APL). Although these cells respond to all-trans-retinoic acid to form neutrophils, our group has recently shown that these cells are capable of terminal monocytic differentiation in response to combined treatment with 1,25-dihydroxyvitamin D3 (1,25 D3) and 12-O-tetradecanoylphorbol-13-acetate (TPA). We show here that the agents need not be present simultaneously, but may be added sequentially. TPA treatment prior to 1,25 D3 led to the appearance of adherent cells; however, when 1,25 D3 treatment preceded TPA treatment cells expressed all differentiation markers reflective of terminal differentiation. This priming effect of 1,25 D3 was both dose and time dependent. Increasing the interval between 1,25 D3 and TPA treatment caused a decrease in this priming potential indicative of limited commitment inducing capacity of 1,25 D3. In order to characterize the mechanism of action of 1,25 D3 and TPA, chemical inhibitors of phosphorylation were used. Staurosporine and bisindolymaleimide GF 109203X treatment prior to and during 1,25 D3 treatment or TPA treatment caused attenuation of the differentiation response. Experiments utilizing tyrosine kinase and phosphatase inhibitors supported the hypothesis that 1,25 D3 signaling was mediated by both serine/threonine and tyrosine phosphorylation cascades. Results from this study provide evidence to support the hypothesis that 1,25 D3 signaling occurs via nongenomic mechanisms which when combined with the signaling effects of TPA, allow for the terminal differentiation of APL cells. This model should be used to develop new differentiation therapies for APL and other leukemias.

Alkaloids↗
PubMed1996

Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy.

The development of stem-cell gene therapy is hindered by the absence of repopulation assays for primitive human hematopoietic cells. Current methods of gene transfer rely on in vitro colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, as well as inference from other mammalian species. We have identified a novel human hematopoietic cell, the SCID-repopulating cell (SRC), a cell more primitive than most LTC-ICs and CFCs. The SRC, exclusively present in the CD4+CD8- fraction, is capable of multilineage repopulation of the bone marrow of nonobese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice). SRCs were rarely transduced with retroviruses, distinguishing them from most CFCs and LTC-ICs. This observation is consistent with the low level of gene marking seen in human gene therapy trials. An SRC assay may aid in the characterization of hematopoiesis, as well as the improvement of transduction methods.

ADP-ribosyl Cyclase↗
PubMed1996

Overexpression of poly(ADP-ribose) polymerase promotes cell cycle arrest and inhibits neutrophilic differentiation of NB4 acute promyelocytic leukemia cells.

The t(15;17) translocation causes a disruption of the retinoic acid receptor alpha (RAR-alpha) and allows for the expression of the PML-RAR alpha fusion protein considered to be responsible for the differentiation block in acute promyelocytic leukemia (APL). Patients being treated with all-trans retinoic acid (ATRA) undergo remission due to the differentiation of leukemic cells to functional neutrophils but relapse due to subsequent ATRA resistance. Our group has shown recently that NB4 cells, the only in vitro model of APL, are capable of monocytic differentiation in response to 1,25-dihydroxyvitamin D3 and 12-O-tetradecanoylphorbol-13-acetate in addition to the neutrophilic differentiation response that occurs with ATRA treatment. Poly(ADP-ribose) polymerase (PARP) is a ubiquitous protein that plays a role in DNA metabolism and repair. We have shown that, unlike HL-60 cells, NB4 cells completely down-regulate PARP in the neutrophilic lineage and up-regulate PARP 90-fold in the monocytic lineage. To ascertain whether PARP is an active participant in the bipotent differentiation of APL cells, NB4 cells were transiently transfected by lipid-mediated gene transfer with the human PARP gene under the control of the human metallothionein promoter. A 4-fold overexpression of PARP, in response to 8 microM CdCl2, promoted arrest of NB4 cells in the S phase of the cell cycle. Overexpression of PARP alone had no effect on cell viability or induction of phenotypic markers in the monocytic or neutrophilic lineages. However, increased PARP expression did result in an increase in the number of cells in the subdiploid population likely to include apoptotic cells. Overexpression of PARP, alone with 12-O-tetradecanoylphorbol-13-acetate (200 nM), 1,25-dihydroxyvitamin D3 (200 nM), or a suboptimal dose of the combined agents, did not alter the expected monocytic differentiation marker profile over cells transfected with control plasmid (pSV2Neo). In contrast, PARP overexpression blocked the appearance of phenotypic markers of terminally differentiated neutrophils in 85% of the transfected population in response to 1 microM ATRA. Comparable to wild-type NB4 cells, 90% of cells transfected with pSV2Neo developed neutrophilic differentiation markers (nitroblue tetrazolium-positive and multi-lobed nuclei) in response to 1 microM ATRA. These data suggest that overexpression of PARP arrests APL cells and blocks ATRA-induced terminal neutrophilic differentiation. We propose that normal down-regulation of PARP in NB4 cells is a requirement for neutrophilic maturation.

Cell Cycle↗
PubMed1996

Injection induced nerve injury : an iatrogenic tragedy.

11 patients with injection induced nerve injury were evaluated. There were 9 adults and 2 children, 8 had radial nerve injury and 3 sciatic nerve injury. 6 had evidence of severe involvement with active denervation. Nature of the drug was not known in 81%, thus faulty site of injection was the most important factor responsible. The need to discourage indiscriminate use of intramuscular injections and choice of a proper site of selection is stressed.

Adolescent↗
PubMed1996

Multiple sclerosis in India: A.I.I.M.S. experience.

Fifty patients of multiple sclerosis were reviewed. The commonest initial neurologic symptom was motor weakness (30%) followed by visual impairment (26%). Absence of internuclear opthalmoplegia was noted in addition to the infrequent occurrence of optic nerve involvement and Devic's disease. Evoked potential studies and radiological investigations were in agreement with others series reported.

Adolescent↗
PubMed1996

Video--EEG monitoring in intractable attacks of uncertain etiology.

Video-EEG monitoring was done in 26 patients with paroxysmal intractable attacks of uncertain etiology to determine the nature and cause. Two broad categories were identified: Those with prominent motor activity and those with attacks of collapse/limpness. Events were recorded in 16 patients of which 11 showed no abnormality on the simultaneously recorded EEG, thus helping to confirm their diagnosis of pseudoseizures.

Adolescent↗
PubMed1995

Monocytic differentiation of acute promyelocytic leukemia cells in response to 1,25-dihydroxyvitamin D3 is independent of nuclear receptor binding.

We have shown that 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) primes NB4 cells, the only available acute promyelocytic leukemia cell line, for 12-O-tetradecanoyl-phorbol-13-acetate-induced monocytic differentiation. Here, we have used isomers of 1,25(OH)2D3 to investigate the role of 1,25(OH)2D3 and its putative nuclear receptor (VDR) in NB4 cell monocytic differentiation. 1 beta,25-dihydroxyvitamin D3 (HL), a specific antagonist of only the nongenomic signals of 1,25(OH)2D3, attenuated the priming effect of 1,25(OH)2D3. The 6-cis conformer of 1,25(OH)2D3 (HF), which is unable to bind to VDR, was 20 times more potent than 1,25(OH)2D3 as a priming agent for monocytic differentiation. This response was also blocked by the HL antagonist. Unlike myelocytic HL-60 cells, which respond to 1,25(OH)2D3 with increases in VDR expression and monocytic differentiation, neither HF nor 1,25(OH)2D3 regulated VDR expression in NB4 cells. In the monocytic differentiation of acute promyelocytic leukemia cells, 1,25(OH)2D3 appears to signal through a pathway independent of VDR/VDRE action.

Biomarkers↗
PubMed1995

Modulation of poly(ADP-ribose) polymerase during neutrophilic and monocytic differentiation of promyelocytic (NB4) and myelocytic (HL-60) leukaemia cells.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which has been shown to play a role in the differentiation of haematopoietic cells. We report here that neutrophils are the first nucleated mammalian cell type demonstrated to be devoid of immunoreactive PARP. Both NB4 acute promyelocytic leukaemia and HL-60 (acute myelocytic leukaemia) cells were differentiated into non-malignant neutrophils with all-trans-retinoic acid (ATRA). Western blot analysis demonstrated that ATRA had no effect on PARP expression in HL-60 cells. However, PARP was completely down-regulated in NB4 cells within 36 h of treatment initiation. This decrease in PARP polypeptide coincided with growth arrest and preceded the appearance of neutrophilic differentiation features. NB4 cells require a combination of 1,25-dihydroxyvitamin D3 (1,25-D3) and phorbol 12-myristate 13-acetate (PMA) to differentiate completely into monocyte/macrophages, whereas HL-60 cells can be made to differentiate by combined or single agents. PARP expression was up-regulated 90-fold when NB4 cells were treated with PMA and 1,25-D3 together, and this increase accompanied expression of the monocyte/macrophage phenotype. Only modest changes in PARP expression were observed when each agent was used alone in NB4 cells or when HL-60 cells were differentiated along the monocyte/macrophage pathway. In addition, PARP activity was modulated in a pattern similar to protein levels when NB4 cells were induced to differentiate along the neutrophilic and monocyte/macrophage pathways. This suggests that the activity of PARP may be controlled through regulation of protein levels during NB4 cell differentiation. We conclude that PARP levels are dramatically modulated during monocyte/macrophage and neutrophilic differentiation. On the basis of the tremendous changes in PARP polypeptide and total activity during myeloid differentiation, we propose that modulation of PARP gene expression is required for cellular maturation in both lineages.

Calcitriol↗
PubMed1995

Alternative splicing of the dopamine D2 receptor directs specificity of coupling to G-proteins.

Two isoforms of the dopamine D2 receptor have been characterized, D2L (long) and D2S (short), generated by alternative splicing from the same gene. They differ by an in-frame insert of 29 amino acids specific to D2L within the putative third intracytoplasmic loop of the receptor. We have previously demonstrated (Montmayeur, J.-P., Guiramand, J., and Borelli, E. (1993) Mol. Endocrinol. 7, 161-170) that D2S and D2L, although presenting very similar pharmacological profiles, couple differently to the alpha-subunit of guanine nucleotide-binding regulatory proteins (G-proteins). In particular, D2L, but not D2S, requires the presence of the alpha-subunit of the inhibitory G-protein (G alpha i2) to elicit greater inhibition of adenylyl cyclase activity. The insert present in D2L must therefore confer the specificity of interaction with G alpha i2. Thus, we introduced substitution mutations within the D2L insert. These mutant receptors were expressed in JEG3 cells, a G alpha i2-deficient cell line, scoring for those presenting an increased inhibition of adenylyl cyclase by dopamine. Our analysis identified two mutants, S259/262A and D249V, with these properties. These results clearly show that the insert present in D2L plays a critical role in the selectivity for the G-proteins interacting with the receptor.

Adenylyl Cyclase Inhibitors↗