qRT-PCR Analysis of GLUT-4 and Assessment of Trolox as an Effective Antioxidant in Diabetic Cardiomyoblasts.

A high global prevalence of diabetes and its implications on the heart in vivo and in vitro tools have been pursued to alleviate the complications of high glucose. This chapter oulines the methods used for maintaining H9C2 cardiomyoblasts in vitro and for stimulating hyperglycemic situation. In addition, we present a method to assess cellular GLUT-4 expression using qRT-PCR. This cellular model also allows us to examine the therapeutic approach of an antioxidant, Trolox, for upregulating GLUT-4 and uptake of glucose under hyperglycemic condition.

Flow Cytometric Analysis of Hyperglycemia-Induced Cell Death Pathways in Cardiomyoblasts.

Type-2 diabetes, characterized by hyperglycemia causing various symptoms of metabolic disorders in the heart, kidneys, and brain, has many underlying molecular mechanisms leading to functional insufficiency of these organs. We describe protocols wherein we have optimized conditions for maintenance of hyperglycemic H9c2 cell lines and design to assess the effect of a water-soluble vitamin, Trolox, on the apoptotic pathway. Primarily, the design provides researchers to analyze apoptosis by flow cytometry.

Early Detection of Skin Cancer Using Melanoma Segmentation technique.

The significance of pattern recognition techniques is widely enhanced in image processing and medical applications. Thus, lesion segmentation method is an essential technique of pattern recognition algorithms to detect the melanoma skin cancer in patients at earliest stage, otherwise, in further stages it becomes one of the deadliest disease and its mortality rate is very high. Therefore, a precise melanoma segmentation technique is introduced based on the Gradient and Feature Adaptive Contour (GFAC) model to detect melanoma skin cancer in earliest stage and diagnosis of dermoscopic images. In the proposed image segmentation technique pre-processing and noise elimination techniques are introduced to decrease noise and make execution faster. This technique helps in separating the required entity from the background and gather the information from the adjacent pixels of similar classes. Multiple Gaussian distributed patterns are adopted to extract efficient features and to get precise segmentation. The proposed GFACmodel is noise free and consist of smoother border. The segmentation model efficiency is tested on PH2 dataset. The superiority of the proposed modified gradient and feature adaptive contour model can be verified against various state-of-art-techniques in terms of segmented image, error reduction and efficient feature extraction.

Longitudinal analyses of anti-JCV antibody index for risk assessment of progressive multifocal leukoencephalopathy.

Risk assessment for natalizumab-associated progressive multifocal leukoencephalopathy (Nat-PML) comprises the anti-JC virus (JCV) antibody index (AI). The anti-JCV AI was longitudinally determined in a natalizumab-treated MS cohort (Nat-MS, n = 468) and samples of Nat-PML patients (n = 15). In Nat-MS, the median AI was 0.8 (25th to 75th percentile, 0.2-2.8) with an intra-individual coefficient of variation (CV) of 9.8% (4.8-17.6). Patients with an AI ≤ 0.9 exhibited higher CV. The AI was higher (3.4 (3.1-3.6)) in samples before Nat-PML diagnosis than in seropositive Nat-MS (2.4 (1.0-3.4), n = 298, p = 0.010). AIs ≥ 3.0 were associated with a 14.5-fold (95% CI 2.3-90.4) increased PML risk (p = 0.002). Groups with an AI below 1.5 exhibit higher variability or even serostatus fluctuation. AI dynamics require further investigation.

Anti-JC virus (JCV) antibody prevalence in the JCV Epidemiology in MS (JEMS) trial.

BACKGROUND AND PURPOSE: Progressive multifocal leukoencephalopathy (PML) is caused by reactivation of JC virus (JCV) infection due to combined host and viral factors. Anti-JCV antibodies provide a means to assess JCV exposure and stratify PML risk. The reported seroprevalence of anti-JCV antibodies varies from 39% to 91% depending on assay methodology and population studied. A two-step anti-JCV antibody assay (STRATIFY JCV™; Focus Diagnostics, Cypress, CA, USA) detected anti-JCV antibodies in approximately 55% of multiple sclerosis (MS) patients. This study describes the prevalence of anti-JCV antibodies in a large, multinational MS population. METHODS: This cross-sectional epidemiology study was designed to enroll a minimum of 2000 patients with an MS diagnosis of any type, irrespective of treatment, from Europe, Canada and Australia. Anti-JCV antibody prevalence was determined by STRATIFY JCV; the effects of demographic and disease characteristics were evaluated. RESULTS: A total of 7724 patients from 10 countries participated in the study. Overall anti-JCV antibody prevalence was 57.1% (95% confidence interval 56.0%-58.2%). Seroprevalence was significantly associated with increasing age, gender and country of current residence (P < 0.0001). No significant differences in anti-JCV antibody prevalence were associated with MS disease characteristics, including duration and type of MS and number and duration of MS therapies. CONCLUSIONS: Overall seroprevalence of anti-JCV antibodies in MS patients from Europe, Canada and Australia was consistent with previous studies using the STRATIFY JCV assay. Anti-JCV prevalence differed significantly by age, gender and country, but no geographical pattern was evident. Disease and treatment type were not associated with differences in anti-JCV antibody status.

Assessment and reporting of the clinical immunogenicity of therapeutic proteins and peptides-harmonized terminology and tactical recommendations.

Immunogenicity is a significant concern for biologic drugs as it can affect both safety and efficacy. To date, the descriptions of product immunogenicity have varied not only due to different degrees of understanding of product immunogenicity at the time of licensing but also due to an evolving lexicon that has generated some confusion in the field. In recent years, there has been growing consensus regarding the data needed to assess product immunogenicity. Harmonization of the strategy for the elucidation of product immunogenicity by drug developers, as well as the use of defined common terminology, can benefit medical practitioners, health regulatory agencies, and ultimately the patients. Clearly, understanding the incidence, kinetics and magnitude of anti-drug antibody (ADA), its neutralizing ability, cross-reactivity with endogenous molecules or other marketed biologic drugs, and related clinical impact may enhance clinical management of patients treated with biologic drugs. To that end, the authors present terms and definitions for describing and analyzing clinical immunogenicity data and suggest approaches to data presentation, emphasizing associations of ADA development with pharmacokinetics, efficacy, and safety that are necessary to assess the clinical relevance of immunogenicity.

Protection against oxidative stress caused by intermittent cold exposure by combined supplementation with vitamin E and C in the aging rat hypothalamus.

This study investigated the effects of combined supplementation with vitamin E and C against oxidative stress (OS) caused by intermittent cold exposure (ICE) in the hypothalamus (HY) of aging male Wistar rats [adult (3-months), middle-aged (18-months) and old (24-months)]. Each age was divided into sub-groups: control (CON), cold-exposed at 10 °C (C10), cold-exposed at 5 °C (C5), supplemented control (CON+S) and supplemented cold-exposed at either 5 °C (C5+S) or 10 °C (C10+S). The supplement was a daily dose of 400 mg vitamin C and 50 IU of vitamin E/kg body weight. Cold exposure lasted 2 h/day for 4 weeks. All age groups exposed to cold showed increase in body mass and feeding efficiency. Feeding efficiency in the supplemented old group showed a statistically significant increase in the cold (p < 0.001). Age-related increases in levels of hydrogen peroxide (H2O2), protein carbonyl (PrC), advanced oxidation protein products and thiobarbituric acid reactive substances (TBARS) were further increased by cold in the HY. Cold reduced thiol(P-SH) levels and increased superoxide dismutase (SOD) and, catalase (CAT) activities as well as Hsp72 levels. However, supplementation lowered H2O2, PrC and TBARS with decreases in Hsp72 levels and in SOD and CAT activities. These changes were concomitant with elevations in P-SH, vitamin E and C levels. The results show that the OS caused by ICE in the HY and its subsequent protection following supplementation is related to the intensity of ICE as well as age of the animal. Immunohistochemical studies are underway to examine the findings on ICE-induced oxidative injury in the HY, and the prospects for vitamin E and C supplementation in the senescent.

Protective role of vitamins E and C against oxidative stress caused by intermittent cold exposure in aging rat's frontoparietal cortex.

This study examined the role of vitamins E and C in combating oxidative stress (OS) caused by intermittent cold exposure (ICE) in the frontoparietal cortex (FPC) of adult (3 months), late-adult (12 months), middle-aged (18 months) and old (24 months) male Wistar rats. Each age group was divided into sub-groups, control (CON), cold-exposed at 5°C (C5), control supplementees (CON+S) and cold-exposed supplementees (C5+S). The supplement was a daily dose of 400mg vitamin C and 50I.U.of vitamin E/kg body weight. Cold exposure lasted 2h/day for 4 weeks. All age groups except the old showed an increase in the final body mass in the cold-exposed. The feeding efficiency was higher in the cold-exposed irrespective of age. OS as reflected in age-related increased levels of hydrogen peroxide, protein carbonyl, advanced oxidation protein products and malondialdehyde showed further increase with ICE in the FPC. However, vitamins E and C supplementation attenuated the ICE-induced OS. ICE depleted the levels of tissue vitamins E and C while supplementation resulted in increased levels. Further age emerged as a significant factor in ICE-induced stress and also the response to vitamins E and C supplementation. Behavioral studies are underway to examine the findings on ICE-induced oxidative injury in the FPC, and the prospects for using vitamins E and C in cold exposures in the aged.

Peroxyl-induced oxidative stress in aging erythrocytes of rat.

This study aims at determining the possible changes in intracellular calcium (Ca (i) (2+) ), plasma membrane calcium ATPase (PMCA) activity and phosphatidylserine (PS) along with glutathione (GSH) level in response to an oxidant challenge in vitro. Erythrocytes were isolated on Percoll and incubated with 2, 2'azobis (2-aminopropane) hydrochloride (AAPH) as well as with vitamin C preceding AAPH incubation. Membrane integrity in terms of hemolysis was negatively related to acetylcholine esterase (AChE) activity with the extent of reduction under OS being higher in the old erythrocyte than in the young. A divergent pattern was seen towards lower PMCA and higher (Ca (i) (2+) ) in the young and old cells. However, the PMCA activity in the stressed young cell was high when pre-treated with vitamin C. PS externalization in the young under OS is perhaps analogous to normal aging, with vitamin C preventing premature death. These findings suggest that young erythrocytes may benefit from vitamin C in therapies addressed towards the mechanisms underlying the reduced effects of OS.

Taking immunogenicity assessment of therapeutic proteins to the next level.

Therapeutic proteins provide innovative and effective therapies for numerous diseases. However, some of these products are associated with unwanted immunogenicity that may lead to clinical consequences such as reduced or loss of efficacy, altered pharmacokinetics (PK), general immune and hypersensitivity reactions, and neutralisation of the natural counterpart (e.g. the physiological hormone). Regulatory guidance on immunogenicity assessment needs to take into consideration a great diversity of products, indications and patient populations as well as constantly advancing manufacturing technologies. Such guidance needs to be sufficiently specific while, at the same time, allowing interactive discussion and adjusted benefit-risk weighing of each product on a case-by-case basis, e.g. for a unique treatment of a life threatening disease acceptable treatment risks may differ considerably from the ones in case of less serious disease. This theme was the focus of the international conference "Taking immunogenicity assessment of therapeutic proteins to the next level", held at the Paul-Ehrlich-Institut in Langen, Germany, on the 10-11. June 2010. The objectives of the conference were to highlight how the field could move from that of a mere description of risk factors to a system of risk assessment and mitigation, as well as an understanding of the impact of unwanted immunogenicity on the overall benefit/risk consideration for a medicinal product. More than 150 experts from industry, academia and regulatory authorities worldwide discussed the phenomenon of undesired immunogenicity from different perspectives. The conference focussed on issues relevant to three areas: (1) new European guidelines that are currently the subject of discussion; (2) testing strategies for immunogenicity assessment; and (3) scientific progress on the product-related factors that may contribute to the development of pathogenesis of immunogenicity, in particular in the field of protein aggregation and post-translational modifications. This report provides an overview of issues, insights, and conclusions that were discussed and achieved during the meeting.

Oxidative stress and intracellular pH in the young and old erythrocytes of rat.

The effects of oxidative stress (OS) on the rat erythrocytes (RBCs) that were fractionated on the percoll/BSA gradient into young and old cells were studied to find out if the altered Na+/H+ and Cl⁻/ HCO3⁻ antiporters and in turn the intracellular pH (pHi) could act as one of the promoters of cell death. Old cells were more spherical with lesser surface area, more fragile osmotically and had lesser protein sulphydryl content than the young cells. OS was induced in RBCs by 2,20-azobis (2-amidinopropane) dihydrochloride (AAPH). AAPH increased the superoxide dismutase (SOD) activity and MDA level and, the changes between the young and old. Interestingly, vitamin C was effective in reducing MDA in the old. Further, in the old a rapid Na+-dependent acidification in the presence of AAPH and a marginal acidosis in the presence of vitamin C were evident. Old RBCs exhibited higher acidosis and vitamin C was less effective in lowering the stress-induced acidosis compared to the young. Our studies suggest that increased acidosis followed by low intracellular pH could be one of the determinant factors for the disappearance of old RBCs from circulation, and perhaps of the young too under OS.

Age-related responses of the rat cerebral cortex: influence of vitamin E and exercise on the cholinergic system.

In this study, we have assessed the impact of vitamin E and exercise on acquisition and retention of spatial memory for a given task in aging rats, using a T-maze. Acetylcholine esterase (AChE) and cholineacetyl transferase (ChAT) activities and acetylcholine (ACh) were measured in the cerebral cortex (CC) of male Wistar rats of 4- (adult), 12- (middle-aged) and 18-months (old) of age. Animals were categorized into sedentary [(SEC (N)], sedentary supplemented [SEC (+E)], swim trained [SWT (N)] and swim trained supplemented [SWT (+E)]. In the old, ChAT activity increased in the SEC (+E). AChE activity was highest in the adults, irrespective of training or supplementation. By contrast, ACh concentration remained unaltered with age, exercise and supplementation. Middle-aged and old rats were benefited in terms of a better acquisition and retention in the case of those that were trained and supplemented with Vitamin E. Adults showed better retention in all the groups after 7 and 15 days, while in the middle-aged, training was beneficial after 15 days. We observed decreased AChE activity when old rats were trained with the supplement. Our results also suggest that this regimen may be analogous to the AChE inhibitors that are widely advocated to derive positive benefits in up-regulating the possible reduction in ACh and in turn age-associated memory deficits.

4-(4-Bromo-phenyl-hydrazono)-1-(5-bromo-pyrimidin-2-yl)-3-methyl-2-pyrazolin-5-one.

The asymmetric unit of the title compound, C(14)H(10)Br(2)N(6)O, contains two crystallographically independent mol-ecules. The pyrazole ring of one mol-ecule makes dihedral angles of 22.0 (3) and 3.5 (3)° with the pyrimidine and benzene rings, respectively; the corresponding values in the other mol-ecule are 9.2 (3) and 2.1 (3)°, respectively. The mol-ecules are linked by N-H⋯O, C-H⋯N and C-H⋯Br hydrogen bonds.

1-Methyl-5-(4-methyl-phen-yl)-3-oxo-cyclo-hexane-1-carbonitrile.

In the title mol-ecule, C(15)H(17)NO, the cyclo-hexane ring adopts a chair conformation. The cyano and methyl groups at position 1 have axial and equatorial orientations, respectively. The benzene ring has an equatorial orientation. A C-H⋯π inter-action involving the benzene ring is found in the crystal structure.

5-(4-Chloro-phen-yl)-1-methyl-3-oxocyclo-hexa-necarbonitrile.

In the title mol-ecule, C(14)H(14)ClNO, the cyclo-hexane ring adopts a chair conformation. The cyano group and the methyl group have axial and equatorial orientations, respectively. The benzene ring has an equatorial orientation. A C-H⋯π inter-action involving the benzene ring is found in the crystal structure.

3-Methyl-5-(3-phenoxy-phen-yl)cyclo-hex-2-enone.

In the title mol-ecule, C(19)H(18)O(2), the cyclo-hexene ring adopts an envelope conformation, with all substituents equatorial. The dihedral angle between the benzene and phenyl rings is 83.75 (16)°. No classical hydrogen bonds are found in the crystal structure.

3-Methyl-5-(4-methyl-phen-yl)cyclo-hex-2-enone.

In the title mol-ecule, C(14)H(16)O, the cyclo-hexene ring adopts an envelope conformation, with all substituents equatorial. Mol-ecules are linked by C-H⋯O hydrogen bonds. A C-H⋯π inter-action involving the benzene ring is also found in the crystal structure. The H atoms of both methyl groups are disordered equally over two positions.

(E)-1-[4-(Methyl-sulfan-yl)phen-yl]-3-phenyl-prop-2-en-1-one.

In the title mol-ecule, C(16)H(14)OS, the dihedral angle between the phenyl and benzene rings is 3.81 (15)°. The H atoms of the central enone group are trans. The propenone unit makes dihedral angles of 11.73 (18) and 11.62 (17)° with the benzene and phenyl rings, respectively. The crystal structure is stabilized by weak C-H⋯O and C-H⋯π inter-actions.

In vitro models of oxidative stress in rat erythrocytes: effect of antioxidant supplements.

The present study was designed to induce oxidative stress in lipid and aqueous phases through azo bis(2-amidinopropane)dihydrochloride (AAPH), 2,2'-azobis 2,4-dimethylvaleronitrile (ADVN) and hydrogen peroxide (H(2)O(2)) either alone or in combination with vitamin C or vitamin El and to assess the vulnerability of rat erythrocytes to oxidative stress. While AAPH acted equally on cell membrane and cytosol, ADVN increased OS in the membrane. The extent of hemolysis and increased membrane fragility caused was more in the case of azo compounds than of H(2)O(2). While vitamin E (2mM) reduced oxidative stress in the membrane, vitamin C (60mM) was more effective in the lysates. The concentration of malondialdehyde and advanced oxidation protein products was lowered by antioxidants. The level of lipofuscin, a product of lipid peroxidation was also increased by ADVN and H(2)O(2). Antioxidants, did, however, reduce the accumulation of protein carbonyl content in cells exposed to azo compounds although they were ineffective in inhibiting oxidation of membrane band 3 protein and sulphydryl content. Taken together, our study demonstrated the antioxidative property of vitamin E and vitamin C in reducing oxidative stress in aqueous as well as lipid phases of erythrocytes and further suggested the feasibility of in vitro models in evaluating the mechanisms of oxidative injury.