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T G Rosano

Publications and source records attributed to T G Rosano.

At least 19 recordsLinked to original sources
PubMed1995

Fucosyltransferase and alpha-L-fucosidase activities and fucose levels in normal and malignant endometrial tissue.

Previous immuno- and lectin-histochemical studies using mAbs and Ulex europaeus lectin I, which recognize various fucose-containing blood group antigens, have shown an increased expression of Lewis and H blood group antigens in endometrial carcinoma. We investigated the biochemical basis of aberrant fucose-containing antigen expression by comparing the activity of fucosyltransferases (FTase) and alpha-L-fucosidase in tissue biopsies from normal (n = 18) and malignant (n = 20) endometrium. Alteration of FTase activity in tumor tissue homogenates was evaluated by using a panel of FTase substrates including N-acetyllactosamine (type 2), lacto-N-biose I (type 1), and phenyl-beta-D-galactoside. Based on histological subtyping, the endometrioid group (n = 14) showed a significant (P < 0.05) increase in tumor FTase activity with all three substrates, while no significant increase was detected for the papillary serous group (n = 4). Matched pair analysis of normal and tumor tissue from a subgroup (n = 5) of the patients with increased tumor enzyme activity also showed higher FTase activity (P < 0.05) in the tumor tissue when the type 1 substrate was used. Regression analysis showed a correlation between the FTase activities acting on type 2 or type 1 substrates (r = 0.821 and r = 0.722, respectively) and the endogenous fucose levels in tumor homogenates. Spectrophotometric analysis of alpha-L-fucosidase activity using p-nitrophenyl-alpha-L-fucoside revealed a higher activity in tumor homogenates than in normal homogenates (P < 0.05) and, therefore, could not account for the enhanced expression of fucose-containing antigens. The current study suggests that aberrant expression of fucose-containing antigens, such as the H and the Lewis blood-group antigens, in endometrial carcinoma is consequential to the change in FTase rather than in alpha-L-fucosidase activity. In addition, the investigation suggests that different glycosylation mechanisms are operative in different subtypes of endometrial cancer.

ABO Blood-Group System↗
PubMed1995

Clinical and analytical evaluation of an immunoradiometric assay for corticotropin.

We evaluated a new IRMA developed commercially for the measurement of corticotropin (ACTH) in human plasma. The assay involves purified polyclonal goat capture antibodies specific for ACTH 26-39 and an 125I-labeled monoclonal signal antibody specific for ACTH 1-17. CVs for intraassay and total precision at ACTH concentrations between 9 and 801 ng/L ranged from 2.5% to 4.7% and from 3.3% to 9.3%, respectively, with an assay detection limit of 1.7 ng/L. The reference interval determined for adults with the new method (16-52 ng/L) differed significantly (P < 0.05) from that for an established ACTH IRMA (9-54 ng/L). Method comparison with clinical samples (n = 179) revealed a correlation coefficient of 0.970 and a best-fit equation of y (new IRMA) = (1.011 +/- 0.019)x + (4.17 +/- 3.31) with Sylx = 40.2. Both methods showed equivalent clinical sensitivity in evaluating Cushing disease, adrenal tumors, ectopic ACTH-producing tumors, hypopituitarism, steroid suppression, surgical adrenalectomy, Nelson syndrome, Addison disease, and corticotropin-releasing hormone stimulation. We conclude that the new IRMA is technically simple to perform and provides a specific and sensitive method for evaluating of adrenocortical function.

Adrenal Cortex Diseases↗
PubMed1994

Inhibition of calcineurin phosphatase activity in adult bone marrow transplant patients treated with cyclosporine A.

In vitro studies have demonstrated that cyclosporine A (CsA) acts by inhibiting the phosphatase activity of calcineurin, an important mediator of T-cell activation. The relationship of CsA administration in vivo, calcineurin activity, and graft-versus-host disease (GVHD) has yet to be studied. The calcineurin activities of mononuclear cells isolated from 62 bone marrow transplant recipients and 12 normal volunteers were determined and analyzed with respect to administration of CsA, presence or absence of CsA in plasma, and presence or absence of GVHD. Of 62 patients, 33 were taking CsA and 29 were not. Early posttransplant (< 100 days), the calcineurin activity of patients on CsA was significantly lower than that of patients not on CsA (P = .0004) and than that of normal volunteers (P < .0001). Similarly, late posttransplant (> 100 days), the calcineurin activity of patients taking CsA was inhibited compared with normal volunteers (P < .05). The calcineurin activity of patients with acute GVHD who were taking CsA was lower than that of patients on CsA without acute GVHD matched for time posttransplant (P = .02). Calcineurin activity in patients on CsA with chronic GVHD was similar to those without chronic GVHD on drug. In conclusion, calcineurin activity is significantly suppressed by in vivo administration of CsA. The lower calcineurin activity of patients on CsA with acute GVHD suggests that CsA-resistant GVHD is not the result of inadequate suppression of calcineurin activity. These data suggest that if inhibition of calcineurin is the only physiologic target of CsA administration, simply increasing doses of CsA or treatment with other inhibitors of calcineurin, such as FK506, would not be expected to ameliorate GVHD.

Acute Disease↗
PubMed1994

Benztropine identification and quantitation in a suicidal overdose.

For a human fatality involving suspected overdose with the anticholinergic agent benztropine, GC-MS analysis was utilized for identification, quantitation, and investigation of metabolism. Organic extracts of blood and urine were analyzed by a capillary-column gas chromatograph interfaced with an ion-trap mass spectrometer, which was programmed for wide-spectrum data acquisition. Electron impact and chemical ionization were used for benztropine detection. The chemical structures of the ion fragments are proposed. Benztropine-d3 was synthesized and used as an internal standard. Quantitative determinations of benztropine revealed 0.183 mg/L in blood and 7.12 mg/L in urine from the decedent. Drug concentrations were interpreted relative to the case findings, published data, and a limited evaluation of the therapeutic concentrations in psychiatric patients. In addition, the possible metabolic conversion to norbenztropine was investigated by the synthesis of the norbenztropine derivative. Chromatographic evaluation of samples from the case study did not reveal significant bioconversion via the N-desmethylation pathway.

Adult↗
PubMed1994

Comparative quantitative immunohistochemical and immunoradiometric determinations of cathepsin D in endometrial adenocarcinoma: predictors of tumor aggressiveness.

Previous comparisons of results of biochemical and immunohistochemical assays for Cathepsin D protease (CD) have conflicted in breast tumors and have not been applied to endometrial adenocarcinomas. We studied CD levels in 31 endometrial adenocarcinomas comparing immunoradiometric assay (IRA) of tumor cytosol with quantitative immunohistochemistry (QIH). Corresponding formalin-fixed paraffin-embedded sections were stained with a polyclonal antibody and measured with the CAS 200 Image Analyzer using a modified cytoplasmic antigen quantification program. Significant and trended increases in CD levels by IRA and QIH were similarly seen in high-grade tumors (IRA p < 0.001; QIH p < ns), papillary serous carcinoma subtype, and cases with deep myometrial invasion (IRA p < 0.005; QIH p < 0.04). Higher mean CD levels by QIH correlated significantly with positive lymph node status (LN+ 0.18 U/cell; LN- 0.09 U/cell; p < 0.03). No correlation of CD by IRA or QIH with estrogen- and progesterone-receptor tumor status was seen. We conclude that image quantification immunohistochemistry is an objective and sensitive method for determining tumor CD levels, CD by QIH directly correlates with standard prognostic indicators such as myometrial invasion and lymph node metastasis, and CD by QIH may prove clinically useful as a predictive adjuvant study for endometrial adenocarcinoma biopsy specimens.

Adenocarcinoma↗
PubMed1992

Correlation of tumor cytosol cathepsin D with differentiation and invasiveness of endometrial adenocarcinoma.

The lysosomal acidic protease cathepsin D, a recognized independent predictor of prognosis in human breast cancer, has not been studied widely in patients with endometrial adenocarcinoma. Cathepsin D levels (52-kD precursor plus 48-kD intermediate and 34/14-kD mature form) were measured in tumor cytosols from 26 hysterectomy specimens by immunoradiometric assay. Significant correlation between cathepsin D levels and tumor differentiation was noted with linear increase in cathepsin D from 8 pmol/mg (standard error of the mean [SEM], 1.73 pmol/mg) for Grade I tumors to 28 pmol/mg (SEM, 3.91 pmol/mg) for Grade III tumors. A group of four papillary serous carcinomas showed relatively high cathepsin D levels reaching 39 pmol/mg. A significant stepwise increase in cathepsin D levels was associated with increased depth of myometrial invasion. Noninvasive tumors averaged 7 pmol/mg (SEM, 4.0 pmol/mg); intramural tumors averaged 15 pmol/mg (SEM, 2.45 pmol/mg); and transmural invasive tumors averaged 30 pmol/mg (SEM, 3.72 pmol/mg). There was no significant correlation of cathepsin D levels with age, estrogen/progesterone receptor hormone status, clinical stage, and lymph node metastasis. Cathepsin D levels correlate significantly with tumor differentiation and myometrial invasiveness and may show promise as a clinically useful adjunct to prognosis assessment and the planning of therapy for patients with endometrial adenocarcinoma.

Adenocarcinoma↗
PubMed1992

Assessment of the in vivo immunosuppressive activity of the major cyclosporine metabolite by leukemia allograft rejection.

AM1 (M17) is the major metabolite of cyclosporine found in the blood of human transplant recipients, and trough levels of this derivative exceed those of the parent compound approximately two-fold. Studies performed in vitro indicate that AM1 retains only 10-20% of the biological activity of the parent compound, but very little is known about its in vivo immunosuppressive effects. We therefore developed a rapid and sensitive method, based on the rejection of allogeneic L1210 (H-2d) leukemia cells by C57BL/6 (H-2b) mice, to assess the immunosuppressive activity of AM1 in vivo. Rejection of the leukemia allograft was determined by analyzing the spleens from mice injected intravenously with 10(5) L1210 cells for the presence of H-2Kd-positive cells by flow cytometry using an FITC-conjugated monoclonal anti-H-2Kd antibody. Nonimmunosuppressed mice rejected the allogeneic cells and survived indefinitely. Spleens from these mice were virtually free of H-2Kd-positive cells (0.51 +/- 0.21%) by day 7. In contrast, C57BL/6 mice treated with 10 mg/kg/day s.c. of CsA all died from the L1210 challenge (mean survival time of 9 +/- 1 days). Spleens from mice treated in this manner contained 11.02 +/- 3.31% H-2Kd-positive cells on day 7. There was a direct correlation between the dose of CsA administered (7.5-50 mg/kg/day) and the percentage of H-2Kd-positive cells in the spleen. We then compared the immunosuppressive activity of AM1 and CsA in this model. AM1 was purified from the urine of CsA-treated renal allograft recipients by a combination of preparative adsorption-desorption chromatography and preparative elution high-performance liquid chromatography. AM1 at a dose of 10 mg/kg/day exhibited no demonstrable immunosuppressive effect, and trough levels of AM1 on day 7 were only 36 +/- 4 ng/ml. Increasing the dose of AM1 to 50 mg/kg/day resulted in only 1.05 +/- 0.16% H-2Kd-positive cells in the spleens (P = NS) and a mean trough level of 221 +/- 27 ng/ml. In contrast, mice treated with 50 mg/kg/day of CsA exhibited 17.7 +/- 2.9% H-2Kd-positive cells in their spleens and a mean trough CsA level of 3036 +/- 277 ng/ml. The half-life of a single subcutaneous dose of 10 mg/kg of AM1 (4.6 hr) was significantly shorter than that of CsA (9.7 hr) in mice. Compared with CsA, the lack of immunosuppressive effect of AM1 in vivo therefore appears to be due to a combination of decreased immunosuppressive activity and increased rate of clearance in mice.

Animals↗
PubMed1991

The clinical significance of cyclosporine metabolites.

Cyclosporine (CsA) is extensively metabolized, with over 14 metabolites having been characterized to date. The confirmation of structure and purity is a prerequisite for studies involving CsA metabolites. Analytical techniques such as fast atom bombardment/mass spectroscopy (FAB/MS), tandem mass spectrometry (MS), 1H- and 13C-nuclear magnetic resonance (NMR) can be used for such purposes. In vitro experiments indicate that metabolites are considerably less immunosuppressive and toxic than CsA. In vivo studies have been hampered by sufficient quantities of metabolites and a suitable animal model. Preliminary results in the rat suggest that CsA metabolites are less immunosuppressive and toxic than CsA, although these results must be confirmed using a more suitable animal model. Present data indicate that the routine monitoring of metabolites is not warranted in transplant patients, although additional information is required to confirm these findings.

Amino Acid Sequence↗
PubMed1991

Advances in catecholamine and metabolite measurements for diagnosis of pheochromocytoma.

Assessment of catecholamine production and excretion is important in the laboratory detection of pheochromocytoma, a rare but curable cause of hypertension. Advances in catecholamine and metabolite methodologies have enhanced the diagnostic acumen by increasing analytical sensitivity and eliminating many of the interferences observed with earlier methods. Estimation of urinary catecholamines metanephrine and vanillylmandelic acid is routinely used in the biochemical detection of pheochromocytoma and in monitoring the completeness of tumor excision as well as the possibility of recurrence. Traditional spectrophotometric and fluorometric methods for urinary catecholamines and their metabolites are being replaced by highly sensitive and selective chromatographic methods. The ability to quantify individual catecholamines and metanephrines by high-performance liquid chromatography is of particular value for detecting familial forms of the tumor that may secrete epinephrine. Plasma norepinephrine and epinephrine measurements are of additional diagnostic value in determining recent catecholamine release and response to clonidine suppression. For either urine or plasma measurements, appropriate patient preparation, sample collection, and method validation along with an understanding of the variable pattern of catecholamine secretion and metabolism in pheochromocytoma are essential. Advances in laboratory methodology and reference intervals for catecholamines for clinical interpretation are reviewed.

Adrenal Gland Neoplasms↗
PubMed1990

Candidate reference method for determining creatinine in serum: method development and interlaboratory validation.

We describe a "high-performance" liquid chromatographic (HPLC) method for accurately determining creatinine in serum. After prechromatographic precipitation of protein, we performed isocratic ion-exchange chromatography with ultraviolet detection (234 nm). Analytical results showed linearity up to 1770 mumol/L, a detection limit of 22 mumol/L, an average analytical recovery of 101%, and a CV ranging from 3% to 11%. We used certified human serum (National Institute of Standards and Technology), and additional lyophilized serum pools also assayed by definitive isotope-dilution mass spectrometry, to validate the accuracy of the HPLC method. In addition, the isocratic HPLC results showed close agreement with those obtained with a step-gradient HPLC method. We also compared the isocratic HPLC method with alkaline picrate and enzymatic methods. Our findings with samples from nonuremic, uremic, and diabetic ketoacidotic patients confirmed the positive bias previously reported with the alkaline picrate method. Interlaboratory transferability of the method was demonstrated with various commercial instruments and analytical columns. We evaluated column stability and possible interference from endogenous or exogenous compounds. On the basis of our analytical findings, we recommend the isocratic HPLC method as a candidate Reference Method for determining creatinine in serum.

Chemistry, Clinical↗
PubMed1989

Assessment of renal function by inulin clearance: comparison with creatinine clearance as determined by enzymatic methods.

We compared creatinine clearances determined by enzymatic (Kodak Ektachem 700 single-slide, Boehringer Mannheim creatinine PAP) and nonenzymatic (Jaffé, HPLC) methods with glomerular filtration rate measured by inulin clearance in patients with varying degrees of renal function. The Kodak enzymatic assay gave values for creatinine 2 to 3 mg/L higher than the other methods. This resulted in significantly lower creatinine clearances than inulin clearances and creatinine clearances determined by the other methods. However, correlations between all methods for serum and urinary creatinine values and clearances were good. To avoid between assay (enzymatic vs nonenzymatic) discrepancies, manufacturers should agree to an acceptable standard of calibration under the usual conditions used with patients.

Chromatography, High Pressure Liquid↗
PubMed1988

Monoclonal antibodies for radioimmunoassay of cyclosporine: a multicenter comparison of their performance with the Sandoz polyclonal radioimmunoassay kit.

The performance of a radioimmunoassay kit containing monoclonal specific and nonspecific antibodies to cyclosporine (Sandimmun-Kit; Sandoz Ltd., Basle, Switzerland) was compared with that of the original Sandoz polyclonal radioimmunoassay kit (Ciclosporin RIA-Kit). A total of 1320 blood and plasma samples from patients receiving cyclosporine after kidney, heart, liver, and bone-marrow transplantation were analyzed at six centers. For blood samples the median result on using the specific assay was about 50% of the polyclonal assay result after kidney and bone-marrow transplantation, about 33% after heart and liver transplantation; comparable figures for plasma samples were 70 and 40%. The monoclonal nonspecific-antibody assay produced results 10% to 140% higher than polyclonal-assay results, depending on sample matrix and transplant indication; the largest difference was seen in samples from heart- and liver-transplant recipients. Evidently the specific-antibody assay provides a convenient alternative to high-performance liquid chromatography for specific measurement of the drug, but the role of the new nonspecific antibody, possessing an even broader spectrum of cross-reactivity with cyclosporine metabolites than the original polyclonal antiserum, has yet to be defined.

Antibodies↗