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Biomedical subjects

H Thakkar

Publications and source records attributed to H Thakkar.

18 recordsLinked to original sources
PubMed2001

Constitutively active Akt is an important regulator of TRAIL sensitivity in prostate cancer.

TRAIL/Apo-2L is a member of the tumor necrosis factor superfamily and has recently been shown to induce apoptosis in cancer cells, but not in normal cells. In nude mice injected with human tumors, TRAIL reduces the size of these tumors without side effects. Akt promotes cell survival and block apoptosis. Some prostate cancer cells express high levels of Akt due to lack of active lipid phosphatase PTEN, a negative regulator of PI-3 kinase pathway, which may be responsible for drug resistance. The objective of this paper is to investigate the intracellular molecules that regulate TRAIL resistance. We have examined caspase-8 activity, BID cleavage, Akt activity, mitochondrial membrane potential (DeltaPsi(m)) and apoptosis in prostate cancer (LNCap, PC-3, PC-3M and DU145) cells treated with or without TRAIL. PC-3, PC-3M and DU145 cells are sensitive to TRAIL, whereas LNCap cells are resistant. LNCap cells express the highest level of constitutively active Akt, which is directly correlated with TRAIL resistance. TRAIL activates caspase-8 in all the cell lines. Downregulation of constitutively active Akt by PI-3 kinase inhibitors (wortmannin and LY-294002), dominant negative Akt or PTEN, renders LNCap cells sensitive to TRAIL. Inhibition of TRAIL sensitivity occurs at the level of BID cleavage. Inhibition of protein synthesis by cycloheximide also causes LNCap cells sensitive to TRAIL. Overexpression of Bcl-2 or Bcl-X(L) inhibits TRAIL-induced DeltaPsi(m) and apoptosis. Overexpression of constitutively active Akt in PC-3M cells (express very low levels of constitutively active Akt) restores TRAIL resistance. These data suggest that elevated Akt activity protects LNCap cells from TRAIL-induced apoptosis, and the PI-3 kinase/Akt pathway may inhibit apoptotic signals by inhibiting processing of BID. Thus, constitutively active Akt is an important regulator of TRAIL sensitivity in prostate cancer.

Androstadienes↗
PubMed2001

Pro-survival function of Akt/protein kinase B in prostate cancer cells. Relationship with TRAIL resistance.

Tumor necrosis factor superfamily member TRAIL/Apo-2L has recently been shown to induce apoptosis in transformed and cancer cells. Some prostate cancer cells express constitutively active Akt/protein kinase B due to a complete loss of lipid phosphatase PTEN gene, a negative regulator of phosphatidylinositol 3-kinase pathway. Constitutively active Akt promotes cellular survival and resistance to chemotherapy and radiation. We have recently noticed that some human prostate cancer cells are resistant to TRAIL. We therefore examined the intracellular mechanisms of cellular resistance to TRAIL. The cell lines expressing the highest level of constitutively active Akt were more resistant to undergo apoptosis by TRAIL than those expressing the lowest level. Down-regulation of constitutively active Akt by phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002, reversed cellular resistance to TRAIL. Treatment of resistant cells with cycloheximide (a protein synthesis inhibitor) rendered cells sensitive to TRAIL. Transfecting dominant negative Akt decreased Akt activity and increased TRAIL-induced apoptosis in cells with high Akt activity. Conversely, transfecting constitutively active Akt into cells with low Akt activity increased Akt activity and attenuated TRAIL-induced apoptosis. Inhibition of TRAIL sensitivity occurs at the level of BID cleavage, as caspase-8 activity was not affected. Enforced expression of anti-apoptotic protein Bcl-2 or Bcl-X(L) inhibited TRAIL-induced mitochondrial dysfunction and apoptosis. We therefore identify Akt as a constitutively active kinase that promotes survival of prostate cancer cells and demonstrate that modulation of Akt activity, by pharmacological or genetic approaches, alters the cellular responsiveness to TRAIL. Thus, TRAIL in combination with agents that down-regulate Akt activity can be used to treat prostate cancer.

Androstadienes↗
PubMed2000

Progressive renal disease: does the quality of the proteinuria matter or only the quantity?

Proteinuria is now accepted to be not just a sign of renal disease but also a contributory factor to the development of progressive tubulointerstitial fibrosis. Excellent correlations between the degree of proteinuria and rate of decline of glomerular filtration rate have been demonstrated. What has been investigated less is whether the type of protein found in the urine is important. Using transformed and primary human proximal tubular epithelial cells, we have investigated the binding of albumin and retinol binding protein to plasma membrane preparations and studied the response of the intact cells to increasing concentrations of these same proteins. We have preliminary evidence for differences in the pattern of binding of these two proteins to the plasma membrane receptors and also for differential release of pro-inflammatory cytokines from intact cells. These in vitro results, along with those of other groups, and some recent clinical findings suggest that the quality of proteinuria may play a role in the early development of interstitial fibrosis. Furthermore, the use of such in vitro model systems based on human proximal epithelial cell culture can provide a means of evaluating the potential significance of different markers of tubular damage.

Cell Line↗
PubMed2000

Reference ranges for plasma cystatin C and creatinine measurements in premature infants, neonates, and older children.

AIM: To establish a reference range in the paediatric population for the new glomerular filtration rate (GFR) marker, cystatin C, and to compare it with that of creatinine. METHODS: Cystatin C and creatinine were measured by particle enhanced nephelometric immunoassay (PENIA) and fixed interval Jaff¿e methods, respectively, in 291 children aged 1 day to 17 years, including 30 premature infants with gestational ages ranging from 24 to 36 weeks. RESULTS: In the premature infants, concentrations of both cystatin C and creatinine were significantly raised compared with term infants, with cystatin C concentrations being between 1.10 and 2.06 mg/litre and creatinine between 32 and 135 micromol/litre. In premature infants, there was no significant relation between gestational age and cystatin C or creatinine concentration. Creatinine concentrations fell to a nadir at 4 months of age, rising gradually to adult values by about 15-17 years of age, in contrast to cystatin C, which fell to a mean concentration of 0.80 mg/litre by the 1st year of life, and remained constant throughout adulthood up to the age of 50 years. Neither analyte showed any influence of sex. CONCLUSION: The measurement of cystatin C, rather than creatinine, is more practical for monitoring GFR changes in the paediatric population.

Adolescent↗
PubMed1998

Measurement of the kinetics of protein uptake by proximal tubular cells using an optical biosensor.

BACKGROUND: The affinity and specificity of protein reabsorption by proximal tubular cells have been investigated using techniques for monitoring endocytosis, demonstrating a high capacity but low affinity process. It is not known whether uptake is through binding to a single binding site/receptor with differing affinities, or if there are several classes of binding sites receptors, each specific for differing proteins or groups, such as, high or low molecular weight proteins. METHODS: We have developed a novel technique for analyzing the kinetics of protein binding to tubular cells using a optical biosensor system. We have studied the binding of cultured LLCPK cells to albumin and RBP immobilized onto the sensor. By adding increasing concentrations of competing proteins [varying in molecular weight from 66,000 to 11,800 D and pI from 4.6 to 9.2 as represented by albumin, alpha1-microglobulin (alpha1M), retinol binding protein (RBP), cystatin C and beta2-microglobulin (beta2m)], specific and inhibitable cell binding was demonstrated. RESULTS: Equilibrium constants, KA, could be calculated from the reciprocal of the protein concentration causing 50% inhibition in binding rate. These were: albumin = 8.0 x 10(4) M(-1), alpha1M = 2.0 x 10(5) M(-1), RBP = 2.7 x 10(4) M(-1), cystatin C = 2.0 x 10(4) M(-1), beta2m = 4.2 x 10(3) M(-1). There were no significant differences between the measured KA's whether RBP or albumin were immobilized on the surface. CONCLUSIONS: All the proteins gave similar shaped inhibition profiles, suggesting that there is one binding site/receptor for all proteins studied, regardless of molecular weight or charge, but there are differing affinities for each protein.

Alpha-Globulins↗
PubMed1998

Development and validation of an automated latex-enhanced immunoassay for prealbumin.

The measurement of circulating prealbumin has been shown to be clinically useful in the assessment of nutritional status, both as an initial screen and in the monitoring of nutritional recovery. We describe a fully automated, noncompetitive, homogeneous, light-scattering immunoassay that has been developed for this analyte on a Dimension (Dade) analyzer. A sheep anti-prealbumin IgG fraction was covalently coupled to 40-nm chloromethyl styrene particles and, after the addition of sample, polyethylene glycol-assisted immunoagglutination was monitored by turbidimetry. The prealbumin working assay range was 8-550 mg/L at a sample volume of 2 microL and a reaction time of 6.5 min. When data were analyzed using ANOVA, total and within-run assay imprecision values (CVs) were 1-5%, and calibration and reagent stabilities were in excess of 40 days. Mean analytical recoveries were 102% +/- 4% (SD), and there was no lack of parallelism. Hemolysis, lipemia, and bilirubin did not interfere. Both plasma anticoagulated with heparin or EDTA and serum from plain or serum-separation tubes were acceptable as sample matrices. Comparison with the Beckman Array method gave a Passing and Bablok regression of: Dimension analyzer = 1.01Beckman + 7.1 (n = 103), using a common calibrator. We conclude that the prealbumin method is appropriate for clinical use according to the analytical criteria used in this study.

Agglutination↗
PubMed1997

A simple, automated, nonextraction assay for serum digoxin using particle-enhanced immunoassay.

We have adapted a latex particle-enhanced immunoassay for serum digoxin to a centrifugal analyser. A 4-microliter serum sample (without pretreatment) inhibits the monoclonal antibody induced aggregation of digoxin-coated latex particles. The total assay time is 10 min and mean analytical recoveries were 98.1%. Intra and interassay precision were < 4.2 and < 15.0%, respectively. Method comparison with an established fluorescence polarisation immunoassay (FPIA) gave R = 0.97, and a Deming regression analysis of particle-enhanced turbidimetric inhibition immunoassay (PETINIA) = FPIA x 0.78 + 0.007 microgram/L (n = 91). There was no evidence of significant interference from digoxin-like immunoreactive compounds in patients with chronic renal failure. This assay can be adapted to most photometric analysers used in routine laboratories and is a significant advance in the sensitivity of latex particle-enhanced immunoassays in a serum matrix.

Cardiotonic Agents↗
PubMed1997

Measurement of albumin and low molecular weight proteins in the urine of newborn infants using a cotton wool ball collection method.

OBJECTIVE: The aim of this study was to investigate the inter-relationship between urinary excretion of alpha-1-microglobulin (A1M), retinol-binding protein (RBP) and albumin in term and premature neonates, with urine collected into cotton wool balls and extracted by a novel method. SUBJECTS AND METHODS: Sixty-four infants were studied on the first day of life; 26 had been born at term (37-42 weeks gestation) and 38 prematurely (24-28 weeks n = 16, 29-36 weeks n = 22). Urine collected into cotton wool balls was analysed following a new detergent extraction method, which resulted in a recovery rate of 94-107% for albumin. A1M, RBP and creatinine. RESULTS: Urinary protein excretion, expressed as a ratio to urinary creatinine, decreased significantly with increasing gestational age (24-28 weeks, 29-36 weeks, 37-42 weeks: albumin:creatinine ratio mg/mmol mean 96.9, 31.7, 19.3; A1M:creatinine ratio mg/mmol mean 99.3, 37.0, 7.8; RBP:creatinine ratio mg/mmol mean 16.2, 3.8, and < 0.01, below the limit of detection, respectively). When results were corrected for birthweight, this gestation-associated effect was still present for A1M and RBP, but not for albumin. In premature infants there was a significant positive correlation between A1M:creatinine ratio and RBP:creatinine ratio (r = 0.85), and also between albumin and both A1M and RBP (r = 0.82 and 0.77). CONCLUSION: Increased excretion of A1M, RBP and albumin at earlier gestational ages is probably due to proximal tubular immaturity, although tubular damage and also glomerular dysfunction cannot be excluded as possible explanations.

Albuminuria↗
PubMed1997

Development and validation of a particle-enhanced turbidimetric inhibition assay for urine albumin on the Dade aca analyzer.

The measurement of urine albumin now has a well-established role in the monitoring of patients with diabetes mellitus. We have developed a particle-enhanced immunoturbidimetric inhibition assay for urine albumin on the Dade aca analyzer. The inhibition approach removes any of the potential antigen excess difficulties that could be expected from the wide clinical range of urine albumin, but retains the sensitivity advantages of latex-enhanced immunoturbidimetry. Human serum albumin (HSA) is covalently attached to 40-nm poly(chloromethyl)styrene-modified latex particles. This reagent, along with monoclonal antibody to HSA, is aliquoted into the aca reagent pack along with polyethylene glycol 8000 in a tablet form (giving a final reaction concentration of 15 g/L). A 150 mmol/L phosphate buffer, pH 7.8, is used to fill the reagent pack in the instrument and the agglutination reaction is monitored at 340 nm. The sample volume is 100 microL and the calibration curve covers the range 2-250 mg/L. Evaluation of commercial scale reagents against the Beckman Array nephelometric immunoassay system gave a Deming regression correlation of aca = 0.87 x Beckman + 8.5, r = 0.995, n = 145. Mean analytical recovery was 104+/-4.5%, n = 20, and there was no evidence of a lack of parallelism. Interassay precision was 8.8% at 10.0 mg/L and <2.5% at >65 mg/L. Calibrator stability was in excess of 60 days. A small reference range study (24-h urine collections, n = 27) gave a mean of 5.6 mg/L with a range of 0.5-16.2 mg/L. Analytical sensitivity (2.5 SD from zero) was 0.40 mg/L.

Albuminuria↗
PubMed1996

Electronic self-help and support groups.

OBJECTIVES: The authors examined the impact of a voice bulletin board on the following: (1) participation in self-help efforts, (2) expression of emotional support, (3) development of solidarity within the group, (4) utilization of health-care services, and (5) health status of subjects. METHODS: Subjects were 53 pregnant women who abused drugs. A quasi-experimental design with matched control group and observations before and after intervention were carried out. Clients in the control group were asked to participate in biweekly face-to-face meetings. Clients in the experimental group participated in the voice bulletin board. Experimental subjects had previous experience with computer services. Subjects reported their level of drug use, health status, and utilization of health services. They also reported on their symptoms, attitudes toward use of physician services, loneliness, willingness to disclose information in groups, and sense of solidarity with their group. The content of the communication among the experimental group was recorded and the utterances were classified as to the type of communication. Exit interviews were done 4 months after baseline interviews were conducted. Clients were paid to complete the baseline and the exit questionnaires, and 94% completed the exit questionnaires. The dependent variables were utilization or health status at exit; the co-variate was utilization or health status at baseline; and the independent variable was the group in which the subject participated. RESULTS: Clients were eight times more likely to participate in the voice bulletin board than in the face-to-face meeting (alpha < 0.01). The majority of the comments left on the bulletin board (54.6%) were for emotional support of each other; no "flaming" or overt disagreements occurred. The more clients participated in the voice bulletin board, the more they felt a sense of solidarity with each other (alpha < 0.001). Members of the experimental group reported significantly lower rates of visiting outpatient clinics than members of the control group (alpha < 0.05). Lower utilization did not lead to poor health status or more drug use: There were no statistically significant differences in the health status and drug use between the experimental and the control groups. CONCLUSIONS: Voice bulletin boards may be an effective method of providing support to mothers who have a history of drug use. Use of these services may lead to lower cost without worsening patients' health.

Adult↗
PubMed1995

Serum cystatin C measured by automated immunoassay: a more sensitive marker of changes in GFR than serum creatinine.

Serum cystatin C has been suggested as a new marker of GFR. For the introduction of this marker into clinical use a rapid and automated method is required. We have developed and validated an assay for serum cystatin C using latex particle-enhanced immunoturbidimetry. Intra- and inter-assay precision were < 3% and < 5% across the assay range. Analytical recovery was 93 +/- 3.8% and no lack of parallelism was demonstrated. Regression analysis of a method comparison with an enzyme-enhanced radial-immunodiffusion method, gave PETIA = 0.074 + 0.93 x SRID, r = 0.98, N = 100. Inter-assay precision profiles showed cystatin C was measured with two-fold better precision than creatinine on the same analyzer. Cystatin C measurement was neither interfered with by icterus nor by hemolysis. 1/cystatin C versus 1/creatinine concentrations gave r = 0.67, N = 469. Comparison of Cr EDTA GFR with 1/cystatin C and 1/creatinine gave r = 0.81 and 0.50, respectively, N = 206. Calculating diagnostic sensitivity for abnormal GFR showed cystatin C to be significantly (P < 0.05) more sensitive than creatinine (71.4 vs. 52.4%). Cystatin C measurement using PETIA technology can be automated on the same instruments used routinely for the measurement of creatinine and offers better analytical performance and probably improved clinical sensitivity as a screening test for early renal damage.

Biomarkers↗
PubMed1995

Use of urine albumin measurement as a replacement for total protein.

We have investigated the replacement of urine total protein estimations for the assessment of glomerular permeability, by the measurement of urine albumin excretion using a latex particle enhanced immunoturbidimetric assay. An initial screen was performed using Albustix to assess the sample pre-dilution necessary for immunoanalysis. A total of 167 24-hour urine samples were analysed and urine albumin concentration correlated well with that of urine total protein (r = 0.93) over the range 0-16,800 mg/l. This protocol provides a more cost effective and analytically valid assessment of glomerular permeability.

Albuminuria↗
PubMed1994

Hyperandrogenism in polycystic ovary syndrome. Evidence of dysregulation of 11 beta-hydroxysteroid dehydrogenase.

BACKGROUND: Hyperandrogenemia is the hallmark of the polycystic ovary syndrome, yet the relative contributions of the adrenal cortex and ovary to the overproduction of androgen remain unclear. To identify possible causes of adrenocortical overactivity, we studied the metabolism of adrenal and ovarian steroid hormones in women with this disorder. METHODS: We measured 24-hour urinary excretion of steroid hormone metabolites by high-resolution capillary gas chromatography in 65 women with the polycystic ovary syndrome and 45 normal women matched for body-mass index. RESULTS: After adjustment for body-mass index, the urinary excretion of testosterone and androstenedione metabolites was 1.9 times higher in the women with the syndrome than in the normal women, and the excretion of dehydroepiandosterone metabolites (C19 steroid sulfates) and cortisol metabolites was 1.5 and 1.3 times higher, respectively (P < 0.01 for all comparisons). The affected women also had significantly higher ratios of 11-oxo (oxygenated) metabolites to 11-hydroxy metabolites of cortisol (1.4 times higher, P < 0.001) and of 11-oxo to 11-hydroxy metabolites of corticosterone (1.8 times higher, P < 0.001). In the group with the polycystic ovary syndrome, 55 percent of the nonobese women and 24 percent of the obese women had ratios above the upper limit of normal; the ratios in the obese women did not differ significantly from those in the nonobese women. CONCLUSIONS: Adrenal secretion of cortisol and androgens is increased in women with the polycystic ovary syndrome. The increases may be explained by dysregulation of 11 beta-hydroxysteroid dehydrogenase causing increased oxidation of cortisol to cortisone, which cannot be accounted for by obesity.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1991

Development of a rapid latex enhanced turbidimetric assay for retinol binding protein in urine.

We describe a simple, rapid and sensitive homogeneous immunoassay for urinary retinol-binding protein (RBP) using latex particle-enhanced turbidimetric immunoassay. Rabbit anti-human RBP is covalently coupled to 40 nm latex particles and the assay performed on the IL Monarch 2000 centrifugal analyser, with a 20 microL sample volume and the reaction monitored at 340 nm over an 8 min period. The assay range is 0-6 mg/L with a detection limit of 25 micrograms/L. The within and between assay coefficients of variation are less than 1.5% and less than 2.5%, respectively. Comparison with radioimmunoassay for RBP showed good agreement.

Immunoassay↗
PubMed1991

Stabilization of turbidimetric immunoassay by covalent coupling of antibody to latex particles.

Turbidimetric immunoassay is commonly used to quantify serum proteins. Latex-particle enhancement of this type of assay has been primarily associated with increasing assay sensitivity. However, covalent coupling of an antibody to a latex particle can offer other advantages that are also pertinent in measurement of high concentrations of analytes. By using a common antibody with IgG as a model analyte, we describe the development of a nonenhanced and a latex-particle-enhanced turbidimetric assay for measuring serum IgG. Both assays show adequate analytical recovery and parallelism, and results compare well with those by rate nephelometry. The latex-enhanced assay has equivalent sensitivity, working range, and interassay precision, but much greater signal change and calibration stability than the nonenhanced assay. In addition, with latex particles, less antiserum is needed. Coupling antibodies to latex particles offers considerable advantages, even when an improved assay detection limit is not required.

Humans↗
PubMed1988

Diagnostic limitations of region-specific parathyroid hormone assays in the investigation of hypercalcaemia.

Assays for N-terminus, C-terminus and mid-molecule parathyroid hormone have been assessed with respect to their sensitivity and specificity in the diagnosis of primary hyperparathyroidism (1 degree HPT). The mid-molecule assay was the most sensitive method studied and only failed to identify one out of 30 patients with histologically proven 1 degree HPT. In all three assays there was some degree of overlap between results observed in 1 degree HPT and in patients with non-parathyroid hypercalcaemia, with results in the latter group sometimes falling well within the hyperparathyroid range. This study highlights the limitations of currently available methods and emphasises the need for caution in the interpretation of parathyroid hormone (PTH) measurements.

Adult↗
PubMedDate not supplied

The use of optical sensors to understand cellular interactions with renal cells.

Optical biosensor technology has revolutionized the assessment of receptor binding, enabling the characterization of low affinity interactions in real time. We report the application of the LAsys Optical Biosensor to the investigation of the affinity and specificity of the putative proximal tubular scavenging receptor for protein reabsorption and the specificity of AGE-modified protein interactions with primary human mesangial cells. Using the LLCPK cell line, the carboxy-methyl dextran cuvette surface and five different proteins (ranging in size and charge), we have shown that there is evidence to support the existence of a single scavenging receptor for all the proteins tested. The proteins competed with each other differing only in their relative binding affinity for the common receptor. We have also shown that human mesangial cells can bind to AGE-modified human serum albumin (AGE-HSA) immobilized onto the carboxylate surfaced planar cuvette and that binding can be inhibited using increasing concentrations of soluble AGE-HSA. However, increasing concentrations of soluble Non-AGE modified HSA can also inhibit binding to a similar extent which implies that there is relatively little AGE-receptor (RAGE) expression on cultured primary human mesangial cells. These results demonstrate the exciting potential of this technology as a tool to explore cellular interactions with renal cells.

Animals↗