Amit K. Dinda

Concentration-dependent toxicity of iron oxide nanoparticles mediated by increased oxidative stress

Iron oxide nanoparticles with unique magnetic properties have a high potential for use in several biomedical, bioengineering and in vivo applications, including tissue repair, magnetic resonance imaging, immunoassay, drug delivery, detoxification of biologic fluids, cell sorting, and hyperthermia. Although various surface modifications are being done for making these nonbiodegradable nanoparticles more biocompatible, their toxic potential is still a major concern. The current in vitro study of the interaction of superparamagnetic iron oxide nanoparticles of mean diameter 30 nm coated with Tween 80 and murine macrophage (J774) cells was undertaken to evaluate the dose- and time-dependent toxic potential, as well as investigate the role of oxidative stress in the toxicity. A 15–30 nm size range of spherical nanoparticles were characterized by transmission electron microscopy and zeta sizer. MTT assay showed >95% viability of cells in lower concentrations (25–200 μg/mL) and up to three hours of exposure, whereas at higher concentrations (300–500 μg/mL) and prolonged (six hours) exposure viability reduced to 55%–65%. Necrosis-apoptosis assay by propidium iodide and Hoechst-33342 staining revealed loss of the majority of the cells by apoptosis. H2DCFDDA assay to quantify generation of intracellular reactive oxygen species (ROS) indicated that exposure to a higher concentration of nanoparticles resulted in enhanced ROS generation, leading to cell injury and death. The cell membrane injury induced by nanoparticles studied using the lactate dehydrogenase assay, showed both concentration- and time-dependent damage. Thus, this study concluded that use of a low optimum concentration of superparamagnetic iron oxide nanoparticles is important for avoidance of oxidative stress-induced cell injury and death.

Fabrication and characterization of PCL/gelatin composite nanofibrous scaffold for tissue engineering applications by electrospinning method

In the present study, composite nanofibrous tissue engineering-scaffold consisting of polycaprolactone and gelatin, was fabricated by electrospinning method, using a new cost-effective solvent mixture: chloroform/methanol for polycaprolactone (PCL) and acetic acid for gelatin. The morphology of the nanofibrous scaffold was investigated by using field emission scanning electron microscopy (FE-SEM) which clearly indicates that the morphology of nanofibers was influenced by the weight ratio of PCL to gelatin in the solution. Uniform fibers were produced only when the weight ratio of PCL/gelatin is sufficiently high (10:1). The scaffold was further characterized by Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric (TG) analysis, and X-ray diffraction (XRD). FT-IR and TG analysis indicated some interactions between PCL and gelatin molecules within the scaffold, while XRD results demonstrated crystalline nature of PCL/gelatin composite scaffold. Cytotoxicity effect of scaffold on L929 mouse fibroblast cells was evaluated by MTT assay and cell proliferation on the scaffold was confirmed by DNA quantification. Positive results of MTT assay and DNA quantification L929 mouse fibroblast cells indicated that the scaffold made from the combination of natural polymer (gelatin) and synthetic polymer (PCL) may serve as a good candidate for tissue engineering applications.

Efficacy and Safety of Treatment with Rituximab for Difficult Steroid-Resistant and -Dependent Nephrotic Syndrome: Multicentric Report

BACKGROUND AND OBJECTIVES The treatment of idiopathic nephrotic syndrome is often complicated by a refractory and relapsing course, with risk of drug toxicity and progressive renal failure. We report the efficacy and safety of rituximab in patients with steroid-resistant (SRNS) and steroid-dependent nephrotic syndrome (SDNS) refractory to standard therapy. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This was a cohort study in academic, tertiary care centers in India and the United States. Patients with SRNS or SDNS, not responding to medications or showing calcineurin inhibitor toxicity, treated with two to four doses of intravenous rituximab, and followed ≥12 months were included. Remission was termed as complete, partial, or no response. RESULTS Thirty-three patients with SRNS (24 initial, 9 late resistance) and 24 with SDNS, with mean ages of 12.7 ± 9.1 and 11.7 ± 2.9 years, respectively, were included. Six months after rituximab therapy, 9 (27.2%) patients with SRNS showed complete remission, 7 (21.2%) had partial remission, and 17 (51.5%) had no response. At 21.5 ± 11.5 months, remission was sustained in 15 (complete: 7, partial: 8) patients. Of 24 patients with SDNS, remission was sustained in 20 (83.3%) at 12 months and in 17 (71%) at follow-up of 16.8 ± 5.9 months. The mean difference in relapses before and 12 months after treatment with rituximab was 3.9 episodes/patient per year. CONCLUSIONS Therapy with rituximab was safe and effective in inducing and maintaining remission in a significant proportion of patients with difficult SRNS and SDNS.

Protection against acute adriamycin-induced cardiotoxicity by garlic: Role of endogenous antioxidants and inhibition of TNF-α expression

BackgroundOxidative stress is the major etiopathological factor in adriamycin-induced cardiotoxicity. Relatively low amounts of endogenous antioxidant makes the heart vulnerable to oxidative stress-induced damage. Chronic oral administration of garlic has been reported to enhance the endogenous antioxidants of heart. We hypothesized that garlic-induced enhanced cardiac antioxidants may offer protection against acute adriamycin-induced cardiotoxicity.ResultsRats were either administered freshly prepared garlic homogenate (250 and 500 mg/kg daily, orally, for 30 days) or probucol (cumulative dose, 120 mg/kg body weight divided in 12, i.p. over a period of 30 days) or double distilled water (vehicle), followed by a single dose of adriamycin (30 mg/kg i.p.). In the adriamycin group, increased oxidative stress was evidenced by a significant increase in myocardial TBARS (thiobarbituric acid reactive substances) and decrease in myocardial SOD (superoxide dismutase), catalase and GPx (glutathione peroxidase) activity. Histopathological studies showed focal as well as subendocardial myocytolysis with infiltration of macrophages, lymphocytes and edema. Immunocytochemistry showed marked expression of TNF-α (tumor necrosis factor-alpha) in the myocardium. Increase in myocardial TBARS and decrease in endogenous antioxidants by adriamycin was prevented significantly in the garlic treated rat hearts, which was comparable to the probucol-treated group. Histopathological evidence of protection was also evident in both garlic-treated and probucol-treated groups. Probucol, 250 mg/kg and 500 mg/kg of garlic reduced adriamycin induced TNF-α expression in the myocardium and was associated with reduced myocyte injury.ConclusionsIt is concluded that chronic garlic administration prevents acute adriamycin-induced cardiotoxicity and decreases myocardial TNF-α expression.

Dose-dependent induction of endogenous antioxidants in rat heart by chronic administration of garlic

The inhibitory property of garlic on free radical generation and lipid peroxidation has been reported in a number of in vitro studies. However, the in vivo effects of chronic garlic intake on the antioxidant milieu of heart has not been reported. Therefore, the present study was designed to investigate the effect of chronic garlic homogenate administration on myocardial endogenous antioxidants and lipid peroxidation at five different dosage levels (125, 250, 500, 1000 and 2000 mg/kg; B, C, D, E, F groups respectively). Garlic homogenate was administered orally to Wistar albino rats (150-200 gms) of either sex 6 days/week for 30 days. Myocardial TBARS (Thiobarbituric acid reactive substances) and antioxidants such as SOD (Superoxide Dismutase), catalase, GPx (glutathione peroxidase) and GSH (Reduced Glutathione) were estimated and histopathological changes were observed. Group F was excluded after 55% mortality occurred in 15 days. TBARS levels were significantly lower in groups B, C and D than that of control group (A). Catalase was increased significantly in groups C, D and E, whereas SOD increased significantly in groups B, C and D but decreased in group E. Significant increase in GSH in group E and significant reduction in GPx activity in group B were observed. Histopathological studies showed marked focal myocytolysis in group E. These results showed that chronic garlic intake dose dependently augmented endogenous antioxidants, which might have important direct cytoprotective effects on the heart, especially in the event of oxidant stress induced injury.

Mitochondrial Structural Changes and Dysfunction Are Associated with Experimental Allergic Asthma

An imbalance between Th1 and Th2 immune response is crucial for the development of pathophysiological features of asthma. A Th2-dominant response produces oxidative stress in the airways, and it is thought to be one of the crucial components of asthma pathogenesis. Although mitochondrion is a crucial organelle to produce endogenous reactive oxygen species, its involvement in this process remains unexplored as yet. We demonstrate in this study that OVA-induced experimental allergic asthma in BALB/c mice is associated with mitochondrial dysfunction, such as reduction of cytochrome c oxidase activity in lung mitochondria, reduction in the expression of subunit III of cytochrome c oxidase in bronchial epithelium, appearance of cytochrome c in the lung cytosol, decreased lung ATP levels, reduction in the expression of 17 kDa of complex I in bronchial epithelium, and mitochondrial ultrastructural changes such as loss of cristae and swelling. However, there was no change in the expression of subunits II and III of cytochrome c oxidase. Interestingly, administration of IL-4 mAb reversed these mitochondrial dysfunction and structural changes. In contrast, IFN-γ mAb administration neither reversed nor further deteriorated the mitochondrial dysfunction and structural changes compared with control asthmatic mice administered with isotypic control Ab, although airway hyperresponsiveness deteriorated further. These results suggest that mitochondrial structural changes and dysfunction are associated with allergic asthma. These findings may help in the development of novel drug molecules targeting mitochondria for the treatment of asthma.

Chronic garlic administration protects rat heart against oxidative stress induced by ischemic reperfusion injury

BackgroundOxidative stress plays a major role in the biochemical and pathological changes associated with myocardial ischemic-reperfusion injury (IRI). The need to identify agents with a potential for preventing such damage has assumed great importance. Chronic oral administration of raw garlic has been previously reported to augment myocardial endogenous antioxidants. In the present study, the effect of chronic oral administration of raw garlic homogenate on oxidative stress induced by ischemic-reperfusion injury in isolated rat heart was investigated.ResultsRaw garlic homogenate (125, 250 and 500 mg/kg once daily for 30 days) was administered orally in Wistar albino rats. Thereafter, hearts were isolated and subjected to IRI (9 min. of global ischemia, followed by 12 min of reperfusion; perfusion with K-H buffer solution; 37°C, 60 mm Hg.). Significant myocyte injury and rise in myocardial TBARS along with reduction in myocardial SOD, catalase, GSH and GPx were observed following IRI. Depletion of myocardial endogenous antioxidants and rise in TBARS were significantly less in the garlic-treated rat hearts. Oxidative stress induced cellular damage as indicated by ultrastructural changes, like disruption of myofilament, Z-band architecture along with mitochondrial changes were significantly less.ConclusionsThe study strongly suggests that chronic garlic administration prevents oxidative stress and associated ultrastructural changes, induced by myocardial ischemic-reperfusion injury.

Effect of chronic treatment with bark of Terminalia arjuna: a study on the isolated ischemic-reperfused rat heart

Dried pulverized bark of Terminalia arjuna Linn (TA) was administered orally to Wistar albino rats (120-150 g) in two doses [500 and 750 mg/kg in 2% carboxy methyl cellulose (CMC)], 6 days per week for 12 weeks. Thereafter, rats were sacrificed either for determination of baseline changes in cardiac endogenous antioxidant compounds [superoxide dismutase (SOD), reduced glutathione (GSH) and catalase (CAT)] or the hearts were subjected to oxidative stress associated with in vitro ischemic-reperfusion injury (IRI). There was significant increase in the baseline contents of thiobarbituric acid reactive substance (TBARS) (a measure of lipid peroxidation) with both doses of TA. However, only in the 500 mg/kg treated group, this was accompanied by a simultaneous increase in SOD, GSH and CAT levels, but not in the 750 mg/kg treated group, where only CAT was raised. Significant rise in myocardial TBARS and loss of SOD, CAT and GSH (suggestive of increased oxidative stress) occurred in the vehicle-treated hearts subjected to in vitro IRI. Only hearts, harvested from the 500 mg/kg rats treated rats, were significantly protected from oxidative stress, when subjected to in vitro IRI. The results suggest that crude bark of TA augments endogenous antioxidant compounds of rat heart and also prevents oxidative stress associated with IRI of the heart.

Cardioprotection from ischemia and reperfusion injury by Withania somnifera: A hemodynamic, biochemical and histopathological assessment

The efficacy of Withania somnifera (Ws) to limit myocardial injury after ischemia and reperfusion was explored and compared to that of Vit E, a reference standard known to reduce mortality and infarct size due to myocardial infarction. Wistar rats (150–200 g) were divided into six groups and received orally saline (sham, control group), Ws-50/kg (Ws control and treated group) and Vit E-100 mg/kg (Vit E control and treated group) respectively for 1 month. On the 31st day, rats of the control, Vit E and Ws treated groups were anesthetized and subjected to 45 min occlusion of the LAD coronary artery followed by 60 min reperfusion. Hemodynamic parameters: systolic, diastolic and mean arterial pressure (SAP, DAP, MAP), heart rate (HR), left ventricular end diastolic pressure (LVEDP), left ventricular peak (+) LVdP/dt and (−) LVdP/dt were monitored. Hearts were removed and processed for histopathological and biochemical studies: Myocardial enzyme viz, creatin phosphokinase (CPK), and antioxidant parameters: malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx) were estimated. Postischemic reperfusion produced significant cardiac necrosis, depression of left ventricular functions (MAP, LVEDP, (+) and (−) LVdP/dt) and a significant fall in GSH (p < 0.01), SOD, CAT(p < 0.05), LDH and CPK (p < 0.01) as well as an increase in MDA level (p < 0.05) in the control group rats as compared to sham group. The changes in levels of protein and GPx was however, not significant. Ws and Vit E favorably modulated most of the hemodynamic, biochemical and histopathological parameters though no significant restoration in GSH, MAP (with Vit E) were observed. Ws on chronic administration markedly augmented antioxidants (GSH, GSHPx, SOD, CAT) while Vit E did not stimulate the synthesis of endogenous antioxidants compared to sham. Results indicate that Ws significantly reduced myocardial injury and emphasize the beneficial action of Ws as a cardioprotective agent.

Prompt plasma exchanges and immunosuppressive treatment improves the outcomes of anti-factor H autoantibody-associated hemolytic uremic syndrome in children

Antibodies to complement factor H are an uncommon cause of hemolytic uremic syndrome (HUS). Information on clinical features and outcomes in children is limited. In order to explore this we studied a multicenter cohort of 138 Indian children with anti-complement factor H antibody associated HUS, constituting 56% of patients with HUS. Antibody titers were high (mean 7054 AU/ml) and correlated inversely with levels of complement C3, but not complement factor H. Homozygous deletion of the CFHR1 gene was found in 60 of 68 patients. Therapies included dialysis in 119 children, 105 receiving plasma exchanges and 26 intravenous immunoglobulin. Induction immunosuppression consisted of 87 children receiving prednisolone with or without intravenous cyclophosphamide or rituximab. Antibody titers fell significantly following plasma exchanges and increased during relapses. Adverse outcome (stage 4-5 CKD or death) was seen in 36 at 3 months and 41 by last follow up, with relapse in 14 of 122 available children. Significant independent risk factors for adverse outcome were an antibody titer over 8000 AU/ml, low C3 and delay in plasma exchange. Combined plasma exchanges and induction immunosuppression resulted in significantly improved renal survival: one adverse outcome prevented for every 2.6 patients treated. Maintenance immunosuppressive therapy, of prednisolone with either mycophenolate mofetil or azathioprine, significantly reduced the risk of relapses. Thus, prompt use of immunosuppressive agents and plasma exchanges are useful for improving outcomes in pediatric patients with anti-complement factor H-associated HUS.