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Biomedical subjects

S Dhar

Publications and source records attributed to S Dhar.

At least 19 recordsLinked to original sources
PubMed2002

Vaccination of donkeys against Babesia equi using killed merozoite immunogen.

Protective efficacy of a killed Babesia equi immunogen was assessed in donkeys. The immunogen was prepared from B. equi infected blood so as to contain lysate of 2 x 10(10) parasitised erythrocytes per dose. The immunogen was mixed with an adjuvant Quil A (3mg) and inoculated into four susceptible donkeys (group I). A booster inoculation was given after 21 days of first inoculation followed by challenge with fresh infected blood containing 1x10(11) parasitised erythrocytes 14 days later. Two groups of two donkey each were included as adjuvant only control (group II) and uninoculated control (group III), respectively. After challenge, donkeys were observed for a period of 4 weeks. The immunised donkeys (group I) showed significantly high (P<0.05%) enzyme linked immunosorbant assay (ELISA) antibody titres and significantly high (P<0.05%) stimulation indices (SI) in lymphocyte proliferation assay (LPA) than that of groups II and III donkeys from day 14 PI and day 7 PI onwards, respectively. All the immunised donkeys withstood lethal challenge, whereas, control donkeys died within 10 days post-challenge (PC). Parasitaemia rose to mean maximum 8.0+/-6.0% for 5-7 days in group I donkeys after challenge, whereas, it rose to 55.5% in control groups. The percent rise in rectal temperature, total leucocyte count (TLC), fall in haemoglobin (Hb) was less severe in immunised group as compared to the control groups. Two immunised-challenged donkeys were splenectomised recovery. No parasites appeared in the blood during the observation period following splenectomy 4-week. Three times increase in skin-fold thickness at 24h of intradermal inoculation prior to challenge in group I donkeys was observed, thus, indicating a good in vivo cell mediated immunity. It can be concluded that the B. equi immunogen along with adjuvant Quil A, used in the present study, was optimum to elicit a strong immune response against B. equi in experimental donkeys.

Adjuvants, Immunologic↗
PubMed2001

CpG methylation as a basis for breast tumor-specific loss of NES1/kallikrein 10 expression.

The normal epithelial cell-specific-1 (NES1)/kallikrein 10 gene is expressed in normal mammary epithelial cells, but its expression is dramatically decreased in breast cancer cell lines. Now, we have cloned and characterized the active promoter region of NES1. Using a luciferase reporter system, we demonstrate that most tumor cell lines are able to support full or partial transcription from the NES1 promoter, suggesting a role for promoter-independent cis-acting mechanisms of loss of NES1 expression. We show that hypermethylation of the NES1 gene represents one such mechanism. Using methylation-specific PCR and sequence analysis of sodium bisulfite-treated genomic DNA, we demonstrate a strong correlation between exon 3 hypermethylation and loss of NES1 mRNA expression in a panel of breast cancer cell lines and in primary tumors. Treatment of NES1-nonexpressing cells with a demethylating agent led to reexpression of NES1, suggesting an important role of hypermethylation in the loss of NES1 expression. We suggest that hypermethylation is responsible for tumor-specific loss of NES1 gene expression. Our results also suggest that hypermethylation of the NES1 gene may serve as a potential marker for breast cancer.

Antimetabolites, Antineoplastic↗
PubMed2001

Spontaneously immortalized cell lines obtained from adult Atm null mice retain sensitivity to ionizing radiation and exhibit a mutational pattern suggestive of oxidative stress.

The study of Ataxia-telangiectasia (A-T) has benefited significantly from mouse models with knockout mutations for the Atm (A-T mutation) locus. While these models have proven useful for in vivo studies, cell cultures from Atm null embryos have been reported to grow poorly and then senesce. In this study, we initiated primary cultures from adult ears and kidneys of Atm homozygous mice and found that these cultures immortalized readily without loss of sensitivity to ionizing radiation and other Atm related cell cycle defects. A mutational analysis for loss of expression of an autosomal locus showed that ionizing radiation had a mutagenic effect. Interestingly, some spontaneous mutants exhibited a mutational pattern that is characteristic of oxidative mutagenesis. This result is consistent with chronic oxidative stress in Atm null cells. In total, the results demonstrate that permanent cell lines can be established from the tissues of adult mice homozygous for Atm and that these cell lines will exhibit expected and novel consequences of this deficiency.

Animals↗
PubMed2001

Characterization of ataxia telangiectasia fibroblasts with extended life-span through telomerase expression.

Ataxia-telangiectasia (A-T) is an autosomal recessive disease characterized by progressive cerebellar degeneration, immunodeficiencies, genomic instability and gonadal atrophy. A-T patients are hypersensitive to ionizing radiation and have an elevated cancer risk. Cells derived from A-T patients require higher levels of serum factors, exhibit cytoskeletal defects and undergo premature senescence in culture. We show here that expression of the catalytic subunit of telomerase (hTERT) in primary A-T patient fibroblasts can rescue the premature senescence phenotype. Ectopic expression of hTERT does not rescue the radiosensitivity or the telomere fusions in A-T fibroblasts. The hTERT+AT cells also retain the characteristic defects in cell-cycle checkpoints, and show increased chromosome damage before and after ionizing radiation. Although A-T patients have an increased susceptibility to cancer, the expression of hTERT in A-T fibroblasts does not stimulate malignant transformation. These immortalized A-T cells provide a more stable cell system to investigate the molecular mechanisms underlying the cellular phenotypes of Ataxia-telangiectasia.

Animals↗
PubMed2001

Intracytoplasmic and intranuclear Reinke's crystals in a testicular Leydig-cell tumor diagnosed by fine-needle aspiration cytology: a case report with review of the literature.

We report on the cytopathologic findings of a Leydig-cell tumor of the testis in a young adult male with no evidence of endocrine dysfunction. The preoperative diagnosis was based on fine-needle aspiration cytology (FNAC) alone, which was subsequently confirmed on histopathology. The present case was of interest on account of the paucity of literature regarding the cytodiagnosis of this lesion. In addition, the finding of intracytoplasmic lipofuscin pigment and several intracytoplasmic as well as intranuclear Reinke's crystals served to clinch the diagnosis on FNA. Therefore, the use of FNAC, especially in the presence of diagnostic Reinke's crystals, may vitiate the need for more invasive biopsy procedures in the preoperative diagnosis of testicular Leydig-cell tumors.

Adult↗
PubMed2001

Temporal effects of the novel antitumour pyridyl cyanoguanidine (CHS 828) on human lymphoma cells.

CHS 828, a novel pyridyl cyanoguanidine, has shown potent antitumour activity both in vitro and in vivo and is currently undergoing phase I evaluation in humans in collaboration with the European Organization for Research and Treatment of Cancer (EORTC). Here we study the temporal effects of CHS 828 on cytotoxicity, protein and DNA synthesis, cellular morphology and ultra structure using the lymphoma cell line U-937 GTB as the primary tumour model. In vitro analysis of tumour cell survival in response to CHS 828 revealed a cytotoxic effect progressively increased as a function of exposure time with maximum efficacy observed after 72 h. Activity of CHS 828 on U-937 GTB cells grown in vivo was also found. CHS 828 induced-cell death was dependent on intact protein synthesis and most cells appeared to lose their membrane integrity in the presence of a relatively well preserved nuclear structure. The results indicate that CHS 828 induced active and delayed cell death with a non-apoptotic morphology.

Antineoplastic Agents↗
PubMed2001

Juvenile hormone III-dependent conformational changes of the nuclear receptor ultraspiracle.

The identification of potential endogenous or synthetic ligands for orphan receptors in the steroid receptor superfamily is important both for discerning endogenous regulatory pathways and for designing receptor inhibitors. The insect nuclear receptor Ultraspiracle (USP), an ortholog of vertebrate RXR, has long been treated as an orphan receptor. We have tested here the fit of terpenoid ligands to the JH III-binding site of monomeric and homo-oligomeric USP from Drosophila melanogaster (dUSP). dUSP specifically bound juvenile hormone III (JH III), but not control farnesol or JH III acid, and also specifically changed in conformation upon binding of JH III in a fluorescence binding assay. Juvenile hormone III binding caused intramolecular changes in receptor conformation, and stabilized the receptor's dimeric/oligomeric quaternary structure. In both a radiometric competition assay and the fluorescence binding assay the synthetic JH III agonist methoprene specifically competed with JH III for binding to dUSP, the first demonstration of specific binding of a biologically active JH III analog to an insect nuclear receptor. The recombinant dUSP bound with specificity to a DR12 hormone response element in a gel shift assay. The same DR12 element conferred enhanced transcriptional responsiveness of a transfected juvenile hormone esterase core promoter to treatment of transfected cells with JH III, but not to treatment with retinoic acid or T3. The activity of JH III or JH III-like structures, but not structures without JH III biological activity, to bind specifically to dUSP and activate its conformational change, provide evidence of a terpenoid endogenous ligand for Ultraspiracle, and offer the prospect that synthetic, terpenoid structures may be discovered that can agonize or antagonize USP function in vivo.

Animals↗
PubMed2001

Analysis of normal epithelial cell specific-1 (NES1)/kallikrein 10 mRNA expression by in situ hybridization, a novel marker for breast cancer.

PURPOSE: Normal epithelial cell specific-1 (NES1)/kallikrein 10 gene is expressed in normal mammary and prostate epithelial cells, but the expression of NES1 mRNA and protein is markedly reduced in established breast and prostate cancer cell lines although the NES1 gene is intact. Here, we wished to assess whether NES1 expression is down-regulated in primary breast cancers. EXPERIMENTAL DESIGN: We developed and used an in situ hybridization technique with an antisense NES1 probe to detect NES1 mRNA in sections of normal breast specimens, typical and atypical ductal hyperplasia, ductal carcinoma in situ, and infiltrating ductal carcinoma. RESULTS: All of the 30 normal breast specimens showed high NES1 expression. Notably, 18 (75%) of 24 typical and atypical breast hyperplasia specimens showed high NES1 expression, with weak-to-moderate expression in 6 (25%). Significantly, 13 (46%) of 28 ductal carcinoma in situ specimens lacked NES1 expression, and the remaining 15 (54%) showed weak-to-moderate expression. Finally, 29 of 30 (97%) infiltrating ductal carcinoma grades I-III samples lacked NES1 mRNA, with weak expression in the remaining one sample. CONCLUSIONS: Our results demonstrate that NES1 mRNA is expressed in normal breast tissue and benign lesions, with loss of NES1 expression during tumor progression. We suggest that NES1 expression may serve as a molecular tool in the study of breast cancer progression. Studies with larger series of specimens should help assess whether NES1 expression can be a diagnostic and/or prognostic marker in breast and other cancers.

Biomarkers, Tumor↗
PubMed2000

Identification of severe and extensive coronary artery disease by postexercise regional wall motion abnormalities in Tc-99m sestamibi gated single-photon emission computed tomography.

Postexercise wall motion abnormality (WMA) in patients with normal resting myocardial perfusion may represent prolonged postischemic stunning, and may be related to the presence of severe angiographic coronary artery disease (CAD). This study assesses the diagnostic value of postexercise WMA by technetium-99m (Tc-99m) sestamibi gated single-photon emission computed tomography (SPECT) in patients with normal resting perfusion. Ninety-nine patients underwent exercise gated Tc-99m sestamibi/resting thallium-201 SPECT and coronary angiography within 90 days of nuclear testing. All patients had normal perfusion at rest. Multivariate logistic regression analysis demonstrated an incremental value of wall motion and perfusion over perfusion data alone in identifying severe and extensive CAD. Sensitivity for identifying any severely stenosed coronary artery by WMA was significantly higher than by severe perfusion defect (78% vs 49%, p <0.0001). Overall specificities of severe perfusion defect and WMA were 91% and 85%, respectively (p = NS). Thus, postexercise WMA detected by gated Tc-99m sestamibi SPECT in patients with normal resting perfusion is a sensitive marker of severe and extensive CAD.

Aged↗
PubMed2000

Regulation of the hTERT telomerase catalytic subunit by the c-Abl tyrosine kinase.

BACKGROUND: Telomeres consist of repetitive (TTAGGG) DNA sequences that are maintained by the multisubunit telomerase ribonucleoprotein. Telomerase consists of an RNA, which serves as template for the sequence tracts, and a catalytic subunit that functions in reverse transcription of the RNA template. Cloning and characterization of the human catalytic subunit of telomerase (hTERT) has supported a role in cell transformation. How telomerase activity is regulated, however, is largely unknown. RESULTS: We show here that hTERT associates directly with the c-Abl protein tyrosine kinase. We also found that c-Abl phosphorylates hTERT and inhibits hTERT activity. Moreover, our findings demonstrate that exposure of cells to ionizing radiation induces tyrosine phosphorylation of hTERT by a c-Abl-dependent mechanism. The functional significance of the c-Abl-hTERT interaction is supported by the demonstration that cells deficient in c-Abl show telomere lengthening. CONCLUSIONS: The ubiquitously expressed c-Abl tyrosine kinase is activated by DNA double-strand breaks. Our finding of telomere lengthening in c-Abl-deficient cells and the functional interactions between c-Abl and hTERT support a role for c-Abl in the regulation of telomerase function.

Catalytic Domain↗
PubMed2000

Herpes simplex virus 1 late gene expression is preferentially inhibited during infection of the TAF250 mutant ts13 cell line.

A key component of the polymerase II transcription machinery is the transcription factor TFIID, a complex that contains the TATA-box binding protein and several (10-12) associated factors designated as TAFs (TBP-associated factors). ts13 cells, which contain a temperature-sensitive mutant in TAF250, the largest subunit of TFIID, exhibit promoter-specific defects in gene expression at the nonpermissive temperature, suggesting that individual TAFs are required for transcription of specific subsets of eukaryotic genes. Herpes simplex virus 1 (HSV-1) does not replicate in ts13 cells at the nonpermissive temperature, but the point at which the replicative process is blocked is not known. We used the TAF250 defect in ts13 cells to investigate the role of TAF250 in the expression of HSV-1 genes of each temporal class. At a low m.o.i., expression of most immediate-early mRNAs is reduced at the nonpermissive temperature, and consequently, there is little expression of early genes and no viral DNA replication. In contrast, at high m.o.i., expression of immediate-early genes is unaffected by the TAF250 defect and is not dependent on de novo viral protein synthesis. Early genes and early proteins are produced under these conditions, and viral DNA replication ensues, albeit at somewhat reduced levels. In contrast, late gene expression and late protein synthesis are severely restricted, even in the presence of appreciable viral DNA replication. Thus the lack of late protein synthesis is responsible for the inability of HSV-1 to replicate in ts13 cells at the nonpermissive temperature. Further, it appears that late viral gene expression may be preferentially inhibited by the TAF250 mutation in ts13 cells.

Animals↗
PubMed2000

Ionizing radiation activates the ATM kinase throughout the cell cycle.

The ATM protein kinase is a critical intermediate in a number of cellular responses to ionizing irradiation (IR) and possibly other stresses. ATM dysfunction results in abnormal checkpoint responses in multiple phases of the cell cycle, including G1, S and G2. Though downstream targets of the ATM kinase are still being elucidated, it has been demonstrated that ATM acts upstream of p53 in a signal transduction pathway initiated by IR and can phosphorylate p53 at serine 15. The cell cycle stage-specificity of ATM activation and p53Ser15 phosphorylation was investigated in normal lymphoblastoid cell line (GM536). Ionizing radiation was found to enhance the kinase activity of ATM in all phases of the cell cycle. This enhanced activity was apparent immediately after treatment of cells with IR, but was not accompanied by a change in the abundance of the ATM protein. Since IR activates the ATM kinase in all phases of the cell cycle, DNA replication-dependent strand breaks are not required for this activation. Further, since p53 protein is not directly required for IR-induced S and G2-phase checkpoints, the ATM kinase likely has different functional targets in different phases of the cell cycle. These observations indicate that the ATM kinase is necessary primarily for the immediate response to DNA damage incurred in all phases of the cell cycle.

Ataxia Telangiectasia↗
PubMed2000

Primary diagnosis of ameloblastoma by fine-needle aspiration: a report of two cases.

Ameloblastoma is the most common epithelial odontogenic tumor, comprising 1% of tumors and cysts arising in the jaws. We describe two cases of ameloblastoma of the jaw diagnosed by fine-needle aspiration cytology. The patients presented with swelling in the parotid region. Cytological examination of the particulate and sticky bloodstained fluid obtained on aspiration showed tightly packed groups of basaloid cells arranged in nests with rounded edges. Palisading epithelial cells and squamous cells with spherical keratinized bodies were the distinctive cytological features. Histologic examination confirmed the presence of ameloblastoma.

Adult↗
PubMed2000

Recurrent arterial embolization from a metastatic germ cell tumor invading the left atrium.

Arterial embolization of a malignant tumor is extremely rare. We report an unusual case of a young adult man who presented with acute lower limb ischemia and a mass in the right lung and left atrium. These clinical manifestations were the result of metastases and embolization from a germ cell tumor and were the first indication of malignancy in this patient. The importance of appropriate investigations in the subsequent treatment is stressed.

Adult↗
PubMed2000

Metastatic pancreatic adenocarcinoma to the heart diagnosed antemortem.

Pancreatic tumors frequently metastasize widely, though it is rare to diagnose pancreatic cardiac metastases in the antemortem state. We report an unusual case of metastatic pancreatic adenocarcinoma to the right atrium. Transesophageal echocardiography showed that the tumor was attached to the superior aspect of the right atrium, prolapsing through and obstructing the tricuspid valve in diastole and retracting back into the right atrium during systole. The tumor was excised, and histologic examination confirmed the presence of moderately differentiated adenocarcinoma with a papillary architectural pattern and with desmoplastic stroma, features comparable to the original primary pancreatic neoplasm.

Adenocarcinoma↗
PubMed2000

Antitumour activity of suramin analogues in human tumour cell lines and primary cultures of tumour cells from patients.

Suramin has shown promising antitumour activity against several tumour types, both in vitro and in vivo, but the clinical utility of this compound is hampered by its unfavourable toxicity profile. In the present study, the semi-automated fluorometric microculture cytotoxicity assay (FMCA) was employed for evaluation of the cytotoxicity of seven suramin analogues in vitro in a panel of human tumour cell lines and in primary cultures of tumour cells from patients. Like suramin, the analogues showed little sensitivity to resistance mechanisms involving P-glycoprotein, topoisomerase II, multidrug resistance associated protein and glutathione-mediated drug resistance. In the cell line panel, NF067 and FCE 26644 showed activity comparable with suramin. All analogues were less potent than suramin in patient cells except for FCE 26644. Correlation to suramin activity patterns in the cell line panel was highest for NF037 and low to moderate for the remaining analogues. In patient cells, high correlation coefficients were obtained for FCE 26644, NF110, NF031 and NF037. The results indicate that the cytotoxic activity of suramin on patient tumour cells is shared by the analogues with FCE 26644 being the most active. The pharmacophore for cytotoxicity in patient cells may be different from that observed in the cell lines.

Antineoplastic Agents↗
PubMed2000

Differential activity of topotecan, irinotecan and SN-38 in fresh human tumour cells but not in cell lines.

The topoisomerase I inhibitors topotecan irinotecan (CPT-11) and its metabolite SN-38 were studied in a panel of cell lines and in primary tumour cells from patients, using a non-clonogenic cytotoxicity assay. All three substances showed similar activity patterns in the panel of cell lines established to classify the drugs mechanistically. In the patient tumour cells the drugs had different effects. In haematological and ovarian cancer samples, SN-38 was much more potent than topotecan, followed by irinotecan, while in colorectal cancer samples only irinotecan showed substantial activity. This in vitro activity pattern seems to agree with clinical experiences to date. The inactivity of SN-38 in colorectal cancer suggests irinotecan may also have some other role in addition to being a prodrug to SN-38. This study raises questions as to the role and relevance of early preclinical model systems in anticancer drug development, and suggests that important information can be obtained from studies using primary cultures of human tumour cells.

Antineoplastic Agents↗