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Biomedical subjects

R C Tripathi

Publications and source records attributed to R C Tripathi.

At least 37 records · Page 2Linked to original sources
PubMed1996

Pharmacokinetics of TCDD in veterans of Operation Ranch Hand: 10-year follow-up.

Using multiple measurements from serum collected over 10 yr (1982, 1987, and 1992), we estimated the half-life of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 213 veterans of Operation Ranch Hand, the Air Force unit responsible for the aerial spraying of Agent Orange in Vietnam. The potential influences of age, percent body fat, and changes in percent body fat on the half-life estimate were also examined. The mean decay rate of TCDD for these veterans is 0.0797 per year with 95% confidence interval 0.0727 to 0.0868 per year; the corresponding half-life estimate is 8.7 yr with 95% confidence interval 8.0-9.5 yr. Half-life increased significantly with increasing body fat, but not with age or relative changes in percent body fat.

Adipose Tissue↗
PubMed1996

Expression of growth factor mRNAs by human Tenon's capsule fibroblasts.

By using the techniques of reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern hybridization, we demonstrated that human Tenon's capsule fibroblasts in primary cultures, express the messenger RNA (mRNA) transcripts encoding transforming growth factor-beta 1 (TGF-beta 1), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF), but not those coding for aFGF and TGF-beta 2. Two PCR products were obtained for TGF-beta 1: a major fragment of 161 base pairs that corresponded to the expected size, and a minor sequence of 400 base pairs. An antisense oligonucleotide probe specific for TGF-beta 1 detected only the band of 161 base pair of PCR-amplified sequence. For bFGF, PDGF-A and PDGF-B, we obtained only a single PCR product with the anticipated length of 222, 225, and 217 base pairs, respectively. Southern blotting experiments revealed that these PCR-amplified fragments were specific for the respective growth factors. The negative control experiments without template did not reveal any amplification. No product for aFGF or TGF-beta 2 was detected. By radioimmunoassay, TGF-beta 1 protein was detected at the level of 24-30 pg ml-1 per 2 million Tenon's fibroblasts during a 24-hour period in acid-activated conditioned medium. These results indicate that human Tenon's capsule fibroblasts in primary cultures express TGF-beta 1 at the translational as well as at the transcriptional levels, and they also have the capacity to synthesize bFGF and PDGF. Considering the significant effects of bFGF, TGF-beta 1, and PDGF in wound healing response, these growth factors are implicated in tissue repair process after glaucoma filtration surgery that contributes to the failure of the procedure.

Aged↗
PubMed1996

Transforming growth factor-beta 1 and -beta 2 positively regulate TGF-beta 1 mRNA expression in trabecular cells.

PURPOSE: To determine whether transforming growth factor (TGF)-beta 1 and -beta 2 and basic fibroblast growth factor (bFGF) induce the gene expression of TGF-beta 1 in the first-passage trabecular meshwork cells of the eye. METHODS: Trabecular meshwork cells were cultured from fresh porcine eyes and treated with 1 ng/ml of TGF-beta 1, TGF-beta 2, or bFGF for 1 hour. Cells maintained in serum-free medium were used as controls. Total cellular RNA was extracted, and the first-strand cDNA was synthesized. Multiplex polymerase chain reaction (PCR) and competitive PCR were performed on aliquots of the cDNAs by using either endogenous (glyceraldehyde-3-phosphate dehydrogenase [G3PDH]) or exogenous sequence (PCR mimic for TGF-beta 1) as internal standards, respectively. The obtained products were quantitated by laser densitometry, and statistical analysis was performed. RESULTS: The findings show that trabecular cells in vitro express the TGF-beta 1 messenger RNA constitutively. Both the techniques of multiplex PCR and competitive PCR demonstrated that the addition of either TGF-beta 1 or TGF-beta 2 at a concentration present in normal aqueous humor increased the mRNA levels of TGF-beta 1 by 2.82-to 3.07-fold over the controls, and these results were statistically significant (P < 0.01). Basic fibroblast growth factor did not have an effect on TGF-beta 1 expression (P < 0.05). CONCLUSIONS: Transforming growth factor-beta 1 activates its own gene expression in trabecular cells. Considering the multifunctional property of this cytokine, which includes increased deposition of extracellular matrix material and growth inhibition of trabecular cells, a change in its concentration within the eye would have a profound effect because of this autoinductive activity. Transforming growth factor-beta 2 treatment of trabecular cells also increased their expression of the TGF-beta 1 gene. The authors previously showed that the level of TGF-beta 2 in the aqueous humor of glaucomatous eyes is significantly higher than that of age-matched nonglaucomatous controls. The current finding suggests that this growth modulator may exert its effects directly on the trabecular cells or that it may act indirectly through upregulating the production of TGF-beta 1.

Animals↗
PubMed1994

Identification of type VI collagen in the trabecular meshwork and expression of its mRNA by trabecular cells.

To investigate the nature of the 140 kDa glycoprotein in the trabecular meshwork, polypeptides were extracted with either urea/sodium dodecyl sulfate (SDS)/beta-2-mercaptoethanol (BME) or guanidine hydrochloride followed by pepsin digestion. After electrophoresis and immunoblotting with anti-type-VI-collagen antibodies, a single fraction of molecular weight 140 kDa was identified in the urea/SDS/BME extracts. Pepsin solubilization revealed two immunoreactive fractions (molecular weights 75 and 85 kDa) that comigrated with purified, pepsin-solubilized type VI collagen. By using the polymerase chain reaction (PCR) and primers specific for the alpha 2(VI) chain of type VI collagen, a single PCR product was obtained, which corresponded to the expected size of 137 base pairs, from the total RNA extracted from the trabecular meshwork ex vivo. Southern hybridization with the antisense oligonucleotide probe of the alpha 2(VI) chain confirmed that the amplified sequence was specific. The results show that the trabecular meshwork contains a significant amount of type VI collagen and that trabecular cells express the mRNA coding for the alpha 2(VI) chain of this glycoprotein. The presence of type VI collagen in the trabecular meshwork is implicated in cell-extracellular matrix interactions at this site, and its abnormal accumulation in glaucomatous and aging eyes probably signifies a defect in the function of the trabecular cells in these eyes.

Amino Acid Sequence↗
PubMed1994

Trabecular cells express the TGF-beta 2 gene and secrete the cytokine.

We investigated whether trabecular cells contain TGF-beta 2 mRNA and whether they synthesize this cytokine. By using a primer pair for TGF-beta 2 and the highly specific and sensitive techniques of reverse transcriptase-polymerase chain reaction, a single amplified sequence was obtained from cultured trabecular cells and tissue samples of trabecular meshwork, corneal epithelium, iris and ciliary body ex vivo from fresh porcine eyes. Regression analysis with standard DNA size markers indicated that the products were of the anticipated size of 310 base pairs. Southern hybridization further confirmed that all of the products were specific for TGF-beta 2. Enzyme-linked immunosorbent assay (ELISA) with a specific antibody against TGF-beta 2 revealed that this cytokine was secreted by cultured trabecular cells in the conditioned medium. The TGF-beta 2 produced by trabecular cells was in a latent form, and no intrinsically active protein was detected. The results suggest that TGF-beta 2 has an autocrine and/or paracrine action in the trabecular meshwork system, and that trabecular cells could contribute to the presence of TGF-beta 2 in their microenvironment as well as in aqueous humor. It is hypothesized that abnormal changes in the production of TGF-beta s by trabecular cells and/or in the activation of these growth modulators contribute to the excess accumulation of extracellular matrix components in the aqueous outflow system as observed in aging and glaucomatous eyes.

Animals↗
PubMed1994

Aqueous humor in glaucomatous eyes contains an increased level of TGF-beta 2.

By using the highly sensitive and specific technique of enzyme-linked immunosorbent assay, we investigated the presence and amount of transforming growth factor-beta 2 (TGF-beta 2) in samples of aqueous humor obtained from 15 patients who had a clinically established diagnosis of advanced primary open-angle glaucoma (POAG), as well as from ten age-matched normal human subjects undergoing cataract surgery. The total amount of TGF-beta 2 in the samples of normal aqueous humor ranged from 0.41 to 2.24 ng ml-1 (mean +/- S.D.: 1.48 +/- 0.68 ng ml-1) of which 4.88 to 37.05% (11.99 +/- 9.95%) was intrinsically active. Compared with normal subjects, the aqueous humor from POAG patients had a statistically significantly greater amount of total TGF-beta 2 (2.70 +/- 0.76 ng ml-1, P < 0.01), as well as a higher level of intrinsically active TGF-beta 2 (0.45 +/- 0.28 ng ml-1, P < 0.05) which corresponded to 1.09 to 60.84% (18.33 +/- 15.50%) of the total amount. No linear correlation was found between the age of the subjects and the protein concentration of the aqueous humor from either normal or glaucomatous eyes, nor between the age of the patient and the total amount of TGF-beta 2. The negligible amount of TGF-beta 2 present in serum argues against its influx into the aqueous humor after breakdown of the blood-aqueous barrier that is known to occur in glaucomatous eyes; rather, our present findings support the concept of the intraocular derivation of this cytokine.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1993

Trabecular cells of the eye express messenger RNA for transforming growth factor-beta 1 and secrete this cytokine.

PURPOSE: To determine whether trabecular tissue in vivo and cultured trabecular cells have the messenger RNA transcript for transforming growth factor-beta 1 (TGF-beta 1), and to examine whether these cells synthesize and secrete TGF-beta 1 in vitro. METHODS: Total RNA was isolated from the trabecular meshwork, iris, and ciliary body freshly excised from porcine eyes as well as from cultured trabecular cells, and the reverse transcriptase-polymerase chain reaction and Southern hybridization were used for detection of TGF-beta 1 messenger RNA. The amount of TGF-beta 1 secreted by trabecular cells in culture was determined by radioimmunoassay. RESULTS: Excised whole trabecular tissue, iris, and ciliary body, as well as cultured trabecular cells expressed messenger RNA transcripts for TGF-beta 1. On the ethidium bromide-stained agarose gel, two PCR-amplified products (161 and 400 base pairs) were found in the total RNA isolated from cultured trabecular cells. The oligonucleotide probe specific for TGF-beta 1 detected only one band with the expected length of 161 base pairs. The secretion of TGF-beta 1 into conditioned medium was at the level of 16.7-20 pg/ml per 2 million trabecular cells during a 24-hr period. CONCLUSIONS: These investigations show that the trabecular meshwork, iris, and ciliary body in vivo express the messenger RNA transcript for TGF-beta 1, and that trabecular cells in vitro synthesize and secrete this cytokine. The TGF-beta 1 present in normal aqueous humor may be derived locally, at least in part, from the cells of the trabecular meshwork, iris, and ciliary body. Abnormal synthesis, secretion, activation, and clearance of TGF-beta 1 may contribute to the pathogenesis of many ocular disorders, including primary open-angle glaucoma.

Animals↗
PubMed1993

Trabecular cells express receptors that bind TGF-beta 1 and TGF-beta 2: a qualitative and quantitative characterization.

PURPOSE: To quantitate the receptors for transforming growth factor (TGF)-beta 1 on trabecular cells in culture and to determine the relative affinities of TGF-beta 1 and TGF-beta 2 for these receptors. METHODS: We quantitated the receptors for TGF-beta 1 by Scatchard analysis of radioligand binding of 125I-TGF-beta 1 to cultured porcine trabecular cells. We established the relative affinities of TGF-beta 1 and TGF-beta 2 for the receptors by competitive binding of 125I-TGF-beta 1 with increasing concentrations of the unlabeled TGF-beta 1 or TGF-beta 2. We also investigated the binding of 125I-TGF-beta 1 after pre-treatment of trabecular cells with heparinase. RESULTS: Trabecular cells expressed approximately 4,000 high-affinity receptors per cell for TGF-beta 1, with a dissociation constant (Kd) of 15.8 +/- 7.6 pmol/l. By varying the concentrations of the unlabeled growth factors, we determined that the relative affinities of TGF-beta 1 and TGF-beta 2 for the receptors were 16 pmol/l and 50 pmol/l, respectively. Heparinase treatment of the trabecular cells did not change the binding affinity of the receptor for 125I-TGF-beta 1. CONCLUSIONS: Our findings show that trabecular cells express heparinase-insensitive TGF-beta receptors that have an approximately threefold greater affinity for TGF-beta 1 than for TGF-beta 2. Based on the present investigation, together with our previous data on the molecular weights of the binding sites, we conclude that trabecular cells do possess types II and III receptors but not type I receptors.

Animals↗
PubMed1992

Familial total ophthalmoplegia with iris transillumination (a neurocristopathy).

A family with total (internal and external) ophthalmoplegia had associated iris transillumination. No abnormal visual-evoked response brain lateralization indicative of albinism was found. On the basis of avian chimera experiments showing iris muscles to be derived from neural crest cells, we proposed a neurocristopathic theory to explain all clinical findings in this family.

Female↗
PubMed1992

Clinicopathologic correlation and pathogenesis of ocular and central nervous system manifestations in Hallervorden-Spatz syndrome.

We have correlated the clinical and histopathologic features of the eyes and central nervous system in a patient with Hallervorden-Spatz syndrome who died at age 11 years. The main ocular findings included degeneration of photoreceptors, marked thinning of the outer nuclear and outer plexiform layers, retinal gliosis, narrowing and obliteration of blood vessels with a perivascular cuffing of pigment cells, and degenerative changes in the retinal pigment epithelial cells with accumulation of melanolipofuscin. The positive findings in the brain included a symmetrical, partially destructive lesion of the globus pallidus, especially in its internal fibers and neurons; in addition, we noted gliosis, widely disseminated axonal spheroidal bodies, which were most numerous in the globus pallidus and pars reticulata, as well as deposits of iron. Our histopathologic findings implicate three possible mechanisms, namely, lipid peroxidation, a deficiency of fatty acid membrane components, and increased cGMP which, either singly or in combination, are responsible for a pathogenesis that is common to the eye and brain in Hallervorden-Spatz syndrome.

Brain↗
PubMed1992

Detection, quantification, and significance of basic fibroblast growth factor in the aqueous humor of man, cat, dog and pig.

By using a highly specific and sensitive heterologous radioimmunoassay, we determined the content of basic fibroblast growth factor (bFGF) in fresh samples of aqueous humor obtained from human (n = 18), feline (n = 8), canine (n = 12), and porcine (n = 10) eyes by paracentesis. The content of bFGF in human aqueous humor ranged from 0.480 to 1.44 ng ml-1 (mean +/- S.D. = 1.074 +/- 0.297 ng ml-1); in feline samples, from 0.672 to 1.152 ng ml-1 (1.016 +/- 0.158 ng ml-1); in canine samples, from 0.640 to 1.232 ng ml-1 (1.026 +/- 0.171 ng ml-1); and in porcine samples, from 0.627 to 0.957 ng ml-1 (0.728 +/- 0.115 ng ml-1). These values were confirmed by means of a dot/slot-blot technique. For all species, the aqueous samples had normal protein levels that ranged from 5 to 19 mg dl-1. There was no correlation of the content of bFGF with the level of protein or with age of the human subjects. The similarity in the concentrations of bFGF in the aqueous humor as well as the stability of the blood-aqueous barriers of all four species indicate that cats, dogs, and pigs can serve as suitable animal models for the study of the role of bFGF in health and disease. We suggest the possible involvement of bFGF in the pathogenesis of anterior-segment disorders, such as neovascular glaucoma, and in the wound-healing response of limbal tissues after glaucoma filtration surgery.

Aged↗
PubMed1992

Corticosteroid treatment for inflammatory bowel disease in pediatric patients increases intraocular pressure.

Intraocular pressure (IOP) was measured in 54 pediatric patients (aged 7-21 years) with inflammatory bowel disease (IBD) who were treated with oral prednisone for 1-104 months. The difference in mean IOP between the treated patients (mean +/- SD, 15.62 +/- 4.11 mm Hg) and 55 age-matched controls (13.83 +/- 2.42 mm Hg) was statistically significant (P = 0.007). The IBD patients were classified as group I, IOP less than or equal to 19 mm Hg in both eyes, and group II, IOP greater than or equal to 20 mm Hg in either eye. Twelve of the 54 patients (22.2%) and none of the controls had IOP greater than or equal to 20 mm Hg (P less than 0.001). Seventeen of the 54 patients (31.5%) were characterized as "steroid responders" (IOP of greater than or equal to 20 mm Hg, change in IOP of greater than or equal to 6 mm Hg between visits, or difference in IOP of greater than or equal to 6 mm Hg between the two eyes). When the dose of prednisone was reduced to 0-10 mg/day 30 days or more before measurement, 9 steroid responders showed a decrease in IOP to within 2 SD of the mean control IOP; 7 of the 9 showed a decrease in IOP of greater than or equal to 6 mm Hg. These observations indicate that while prednisone is a causative factor in increasing the IOP, susceptibility to average doses of prednisone is highly variable, and patients need to be monitored on an individual basis. Because IBD is a chronic disorder that requires prolonged corticosteroid treatment, these children are at risk of developing steroid-induced glaucoma. Careful ophthalmologic monitoring of pediatric IBD patients, as well as of other pediatric patients who receive corticosteroid therapy, is recommended.

Adolescent↗
PubMed1992

Investigation of TCDD half-life heterogeneity in veterans of Operation Ranch Hand.

The half-life of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the contaminant of Agent Orange, has been recently estimated in 36 members of Operation Ranch Hand, the Air Force unit responsible for the aerial spraying of Agent Orange in Vietnam, as 7.1 yr with a 95% [corrected] confidence interval of 5.8-9.6 yr. We investigated the variability of TCDD half-life with percent body fat in these 36 Ranch Hand veterans who have two TCDD assay results from serum drawn in 1982 and 1987. Using a repeated measures linear model, we found a marginally significant change in half-life with percentage of body fat (p = .09) and no statistically significant change in half-life with relative changes in percentage of body fat from 1982 to 1987 (p = .60).

Body Composition↗
PubMed1992

Cytotoxicity of ophthalmic preservatives on human corneal epithelium.

Because the corneal epithelium invariably encounters the full concentration of the preservative that is contained in multi-dose topical ophthalmic preparations, we investigated the cytotoxicity of several of these agents by using a sensitive model of human corneal epithelial cells in vitro. Primary cultures of epithelial cells were prepared from freshly enucleated globes. At confluence, all experimental cultures received a single dose of preservative at the concentration present in marketed formulations. The serum in the culture medium simulated the possible neutralizing effect of proteins present in the tear film in vivo. The cells were observed continuously by phase-contrast microscopy and time-lapse videomicrography for 24 hrs. Benzalkonium chloride at a concentration of 0.01% and chlorobutanol at 0.5% caused immediate cell retraction, as well as cessation of normal cytokinesis, cell movement, and mitotic activity; the epithelial cells degenerated within 2 hrs and 8 hrs, respectively. Cultures treated with chlorobutanol developed conspicuous blebs on the cell surface after 3 to 5 hrs of exposure. Thimerosal (0.001%) caused cell retraction, cessation of mitotic activity, and total cell destruction within 9 hrs. Sorbic acid (0.1% and 0.2%) greatly reduced cell movement and suppressed mitotic activity, but no cell death occurred. At concentrations of 50 ppm and 30 ppm, H2O2 instantaneously caused a marked retraction of the cells, followed by cessation of cytokinesis, cell movement, and mitosis. Retraction and death of the epithelial cells occurred within 12-24 hrs after exposure to 1 ppm H2O2 in serum-free medium. Polyquaternium ammonium chloride (0.001%) and polyaminopropyl biguanide (0.00005%) had no discernible effects on cytokinetic movement or on the mitotic activity of the epithelial cells. We relate our findings in vitro to those reported in vivo and discuss the mechanism of cytotoxicity of the various preservatives.

Cell Death↗
PubMed1992

Ocular toxicity of prednisone in pediatric patients with inflammatory bowel disease.

We performed ocular examinations on 58 corticosteroid-treated pediatric patients with inflammatory bowel disease (IBD) and on 58 age-matched controls. Posterior subcapsular cataracts (PSC) were detected in 12 of the 58 treated patients (20.7%) and in none of the controls. The difference in mean intraocular pressure (IOP) between the treated patients (15.89 +/- 4.11 mm Hg) and control subjects (13.63 +/- 2.35 mm Hg) was significant statistically (P < 0.001). Twenty-one patients (36.2%) were characterized as "IOP responders" (IOP > or = 20 mm Hg, change in IOP > or = 6 mm Hg between visits, or a difference in IOP > or = 6 mm Hg between the two eyes). Formation of PSC was not correlated significantly (P > 0.05) with the total dose of prednisone, duration of treatment, average daily dose, or number of days on high doses (> or = 25 mg). Raised IOP was correlated (P = 0.005) only with average daily dose (12.4 +/- 10.9 mg/day; range, 0-47 mg/day) 30 days before examination. When the dose of corticosteroid was reduced to < 10 mg/day, 2 patients manifested regression of PSC, and 12 IOP responders showed a decrease in IOP to within 2 SD of the mean control IOP. Only 3 of the 58 treated patients (5.2%) manifested both PSC and raised IOP. A significant inverse correlation (P = 0.02) was established between IOP at first examination and formation of PSC. We propose that the mechanisms for steroid-induced lens opacities and raised IOP do not share the same genetic basis. Because 52% of these children developed either PSC or raised IOP with prednisone therapy, we advocate careful ophthalmologic monitoring of pediatric patients receiving corticosteroids for IBD or any other condition.

Adolescent↗
PubMed1992

Identification and partial characterization of TGF-beta 1 receptors on trabecular cells.

By using two specific receptor assays, we identified and partially characterized receptors for transforming growth factor-beta 1 (TGF-beta 1) on porcine trabecular cells in vitro. Cultured trabecular cells were incubated with labeled TGF-beta 1 and analyzed by flow cytometry. Pretreatment with trypsin or preincubation with cold TGF-beta 1 or a neutralizing antibody to TGF-beta 1 inhibited the binding of labeled TGF-beta 1. 125I-TGF-beta 1 was cross-linked covalently to cell surface receptors on the trabecular cells. By SDS-PAGE and autoradiography, we identified three labeled macromolecular species of receptors, two of which had apparent molecular weights greater than 212 kDa and one of which had an apparent molecular weight of approximately 80-90 kDa under reducing conditions. The low and high molecular weight species probably represent type II and type III TGF-beta 1 receptors, respectively. At concentrations of 0.5 and 1 ng/ml, activated TGF-beta 1 caused retraction and a marked decrease in the rate of proliferation and in the motility of trabecular cells in vitro. Our findings implicate TGF-beta 1 in the modulation of the functional homeostasis of trabecular cells and suggest that the aqueous humor contains a level of TGF-beta 1 which, once activated, is sufficient to exert a biologic effect on the trabecular meshwork.

Animals↗