Modulation of cadmium induced alterations in murine thymocytes by piperine: oxidative stress, apoptosis, phenotyping and blastogenesis

Piperine, a main component of Piper longum Linn. and Piper nigrum Linn., is a plant alkaloid with a long history of medicinal use in Indian medicine. It is known to exhibit a variety of biological activities which include anti-pyretic, anti-inflammatory, anti-depressant, hepatoprotective and antitumor. Its immunomodulatory role has so far been limited to humoral response. The influence of piperine on murine thymocytes, immunocompromised by cadmium has been reported by us in this investigation. The various biochemical parameters such as oxidative stress markers (ROS and GSH), Bcl-2 protein expression, mitochondrial membrane potential, caspase-3 activity, DNA damage, blastogenesis and T lymphocyte phenotypes were determined. Cadmium (25 microM) induced apoptosis earliest at 6 h. Alterations in ROS and GSH preceded mitochondrial membrane depolarization and caspase-3 activation followed by apoptosis. The phenotypic changes occurred at 18 h and blastogenesis at 72 h. Various conc. of piperine (1, 10 and 50 microg/ml) when added along with Cd (25 microM) from 1.5 to 72 h, caused a dose and time dependent amelioration in all the cellular events mentioned above. Modulation of oxidative stress has earlier been reported to reduce Cd induced apoptosis in murine lymphocytes. Inhibition of the ROS production and replenishment of GSH by piperine, may in part be responsible for the suppression of downstream cascade of events, i.e. apoptosis, blastogenesis and T lymphocyte phenotyping. The study clearly demonstrated the anti-oxidative, anti-apoptotic, and restorative ability against cell proliferative mitogenic response and phenotypic alterations by piperine, suggesting its therapeutic usefulness in immunocompromised conditions.     

Oxidative stress and apoptotic changes in murine splenocytes exposed to cadmium

Cadmium being a potent immunotoxicant, affects both humoral and cell mediated immunity. However, its effect on spleen is not clearly understood. Hence, to delineate the action of Cd, mouse splenic lymphocytes were exposed to Cd (10, 25 and 50 microM) for 60 min, 1.5, 3, 6 and 18 h. At 6 h, apoptosis was reflected by DNA fragmentation, increased sub-G1 population (apoptotic DNA) and apoptotic cells (Annexin V binding assay). The early stage markers of apoptosis, i.e. decreased mitochondrial membrane potential and caspase-3 activation were observed as early as 1.5 h by the highest dose of Cd (50 microM). Significant ROS production by 25 and 50 microM Cd at 60 min occurred prior to the lowering of mitochondrial membrane potential, suggesting involvement of ROS in causing mitochondrial membrane damage. N-acetylcysteine and pyrrolidine dithiocarbamate (thiol antioxidants) lowered the sub-G(1) population, inhibited the ROS generation and raised the GSH levels induced by Cd. Buthionine sulfoximine (GSH depletor) on the other hand, enhanced the ROS production as well as the sub-G1 fraction. These results imply that ROS is a critical mediator of Cd-induced apoptosis and that cadmium may compromise splenic immune function by accelerating apoptosis.     

Statistical model for predicting non-heme iron bioavailability from vegetarian meals

Availability of non-heme iron has been extensively discussed when meals comprise heme as well as non-heme iron, but seldom so for exclusively vegetarian meals. The present study aimed to develop a statistical model for predicting non-heme iron availability from a composite vegetarian meal. Radioisotopic measurements of in vitro iron dialyzability of 208 out of 274 meals representing vegetarian diets from Asia, Africa, Europe and Latin America and the meal contents of iron, zinc, copper, ascorbic acid, beta-carotene, riboflavin, thiamin, folic acid, tannic acid, fiber and degraded phytate forms (IP6-IP1) were used for development of the model. A multiple regression model weighted for calorie contents was developed for the percentage iron dialyzability with the possible predictors as meal contents along with plausible interaction terms. The model was validated with in vitro iron dialyzability of 66 meals and in vivo iron absorption in five ileostomized adults. Application of the model was demonstrated using data on the daily dietary intake of 215 young adults whose hemoglobin levels were estimated twice in 3 weeks. Weighted multiple regression model was: ln(% Fe dialyzability)=1.340-0.259xln(IP2 [mg])+0.188xln(IP3 [mg])-0.278xln(IP5 [mg])+0.0912xln(ascorbic acid [mg])+0.06693xln(tannins [mg])+0.09552xln(beta-carotene [microg])+0.137xln(hemicellulose [g]) (P<0.01, R2=0.51). Good agreement was seen between observed and predicted dialyzability (r=0.90) and human absorption (r=0.89). The model would be useful to estimate bioavailable iron intakes of vegetarian populations and to identify at-risk individuals.     

Radioimmunoassay for plasma cortisol,testosterone, estradiol-17β, and estrone in the catfish Heteropneustes fossilis (Bloch): Development and validation

Radioimmunoassay (RIA) techniques for cortisol, testosterone, estradiol-17β, and estrone in the catfish plasma have been developed. The assays have been validated by assessing their accuracy, sensitivity, precision, and specificity. The sensitivity of the assays was 5 pg for testosterone, 10 pg for estrone, 25 pg for estradiol-17β, and 50 pg for cortisol. Comparison of plasma levels of various steroids after purification on Celite columns with those not subjected to Celite chromatography reveals very close correspondence between the two during the various phases of the reproductive cycle. Thus the RIA on unfractionated plasma extract is as reliable as that following Celite chromatography when specific antisera are used. The fact that the chromatography step can be eliminated without any appreciable loss of precision or accuracy of the assay, considerably simplifies the RIA for cortisol, testosterone, estradiol-17β, and estrone during experimental studies on this catfish.     

Orbital tuberculoma extending into the cranium

Orbital tuberculoma is not uncommon in the developing countries, but intracranial extension of orbital tuberculoma is extremely rare. Our case, a 14-year-old girl, presented with proptosis and progressive painless diminution of vision eventually leading to loss of vision. MRI showed a mass with peripheral enhancement of contrast, separate from the optic nerve and extending into the cranium through the optic foramen. Early decompression and chemotherapy resulted in marked visual recovery. Histopathology of the excised lesion confirmed tuberculosis. The case is reported to highlight both the rare presentation as well as remarkable visual recovery in a patient with orbital tuberculosis.     

Clinical outcome of autologous cultivated limbal epithelium transplantation

PURPOSE: To report the clinical outcome of autologous cultivated limbal epithelial transplantation. METHODS: Eighty-six patients' records and their clinical photographs were reviewed for demographics, primary etiology, type of limbal transplantation, ocular surface stability, visual acuity, final outcome and possible factors affecting outcome and complications. RESULTS: Eighty-eight eyes of 86 patients with limbal stem cell deficiency (LSCD) underwent autologous cultivated limbal epithelium transplantation between March 2001 and May 2003, with a mean follow-up of 18.3 months. The etiology of LSCD was alkali burns in 64% patients. Sixty-one eyes had total LSCD. Thirty-two of the 88 eyes had undergone amniotic membrane transplantation and 10 eyes had previously undergone limbal transplantation with unfavorable outcome. Nineteen eyes underwent penetrating keratoplasty, of which 11 grafts survived at the final follow-up. Finally, 57 eyes (73.1%, 95% CI: 63.3-82.9) had a successful outcome with a stable ocular surface without conjunctivalization, 21 eyes (26.9%, 95%CI: 17.1-36.7) were considered failures and 10 patients were lost to follow-up. CONCLUSION: LSCD can be successfully treated by autologous cultivated limbal epithelium transplantation in majority of the cases.     

A technique for accurate and safe injection of steroid in trigger digits using ultrasound guidance

Steroid injections have long been the main stay of conservative treatment of trigger digits. This procedure gives variable results, which is dependent on a number of factors. The injection of the steroid in the right place improves the success rate and also prevents complications associated with the procedure. We describe a technique using ultrasound for accurate injection of steroid to maximise its beneficial effects in treatment of trigger digits.     

Tissue Inhibitor of Metalloproteinases-1 Stimulates Gene Expression in MDA-MB-435 Human Breast Cancer Cells by Means of its Ability to Inhibit Metalloproteinases

Summary Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a widely expressed, secreted protein that functions primarily to inhibit members of a large family of metalloproteinases (MPs). Because of the ability of TIMP-1 to inhibit MPs, it functions in many of the same pathophysiological processes as these enzymes, e.g. wound healing, ovulation, angiogenesis, and cancer cell metastasis. TIMP-1 can also stimulate proliferation ([³H]thymidine incorporation) and cellular anabolic processes (Alamar Blue reduction). This stimulation has been shown to be dependent on the MP-inhibitory ability of TIMP-1 in the human breast cancer cell line MDA-MB-435 (Porter et al., Br J Cancer 90: 463, 2004). To shed light on the mechanism by which TIMP-1 stimulates cellular anabolic processes, an oligonucleotide microarray analysis was performed over a time course of TIMP-1 treatment of MDA-MB-435 cells. Fifteen genes whose mRNAs were differentially regulated were identified. Six (Importin-7, MGC10471, FOXC1, subunit p20 of Arp2/3 complex, mitochondrial ribosomal protein L32, and the serine/threonine kinase-4 (MST1)) of these genes were confirmed by quantitative real time PCR. These same mRNAs were shown to be regulated by the synthetic hydroxamate MP-inhibitor GM6001 but not by its inactive derivative GM6001*, suggesting that the differential regulation occurs through the MP-inhibitory ability of TIMP-1. These results suggest a complex action of TIMP-1 on cancer cells mediated by constitutively active cell surface metalloproteinases that release factors regulating cell signaling pathways; they may account for the paradoxical observation that elevated levels of TIMP-1 in tumors can correlate with an adverse prognosis.