Non-inertial lift induced migration for label-free sorting of cells in a co-flowing aqueous two-phase system.

Label free sorting of circulating tumor cells (CTCs) often remains a challenge due to their rarity in peripheral blood and identical morphology to white blood cells. We present a novel label-free passive microfluidic technique for isolation of cancer cells (EpCAM+ and CD45-) from peripheral blood mononuclear cells (PBMCs) (CD45+ and EpCAM-) in aqueous two-phase system (ATPS). Our technique involves non-inertial lift induced lateral cell migration across liquid-liquid interface that is employed for sorting cells of different size and stiffness. The interplay between lift force and interfacial tension (IFT) force governs cell migration phenomena. We estimate the order of magnitude of the lift force and find it to be higher than the IFT for cancer cells above a critical strain rate parameter ([small gamma, Greek, dot above]/h). The effect of spreading parameter and viscoelastic force was found to have negligible effect on lateral migration of cells. We demonstrated isolation of two different types of cancer cells, namely, MCF-7 and MDA-MB-231 from PBMCs and quantify our sorting results by tagging the cells with EpCAM and CD45 and using fluorescence imaging. With 102-104 cancer cells in 105-107 PBMCs, we achieved a processing rate of >25 000 cells per min at a sorting efficiency of ∼99%. Moreover, we demonstrated that cancer cells isolated from PBMCs using the proposed technique remain viable and can be cultured for downstream analysis.

Theories and management of aging: modern and ayurveda perspectives.

Aging is a complex phenomenon, a sum total of changes that occur in a living organism with the passage of time and lead to decreasing ability to survive stress, increasing functional impairment and growing probability of death. There are many theories of aging and skin remains the largest organ of the study. Skin aging is described as a consequence of intrinsic and extrinsic factors. The most common amongst visible signs of skin aging are wrinkles and there are various therapies including antiaging cosmeceuticals, sunscreens, chemical peeling, injectable agents, such as botox, fibrel, autologous fat grafting as also few surgical procedures have been used. Ayurveda, the Indian traditional medicine, describes aging with great details. This review provides modern and Ayurvedic perspectives on theories and management of aging.

Gastric, pancreatic, and ureteric duplication.

We report a case of an 8-month-old, asymptomatic child who was incidentally detected to have two cystic structures in the abdomen. Surgical exploration revealed a gastric and pancreatic duplication cyst along with a blind-ending duplication of the right ureter. Excision of the duplications was relatively straightforward, and the child made an uneventful recovery. This constellation of duplications has not been reported before.

Pharmacodynamics & toxicological profile of PartySmart, a herbal preparation for alcohol hangover in Wistar rats.

BACKGROUND & OBJECTIVE: PartySmart is a herbal preparation intended for the management of alcohol hangover and other related toxic effects in clinical situation. The present study was designed to investigate the pharmacodynamics and oral toxicity of PartySmart, a herbal formulation in rats. METHODS: Effect of PartySmart on blood acetaldehyde and alcohol levels was evaluated at doses of 125, 250 and 500 mg/kg b.wt. in rats. Acute toxicity study was conducted with PartySmart at a limit test dose of 2000 mg/kg b.wt., p.o. In repeated dose 90 day study, PartySmart was administered at doses of 500 and 1000 mg/kg b.wt. once-a-day, orally throughout the study period. RESULTS: PartySmart dose-dependently decreased blood ethanol and acetaldehyde levels as compared to control. PartySmart at a dose of 500 mg/kg b.wt. significantly reduced the area under curve (AUC) of ethanol and acetaldehyde levels. It increased the hepatic alcohol dehydrogenase (ADH) at 500 mg/kg b.wt. and aldehyde dehydrogenase (ALDH) activities at doses of 250 and 500 mg/kg b.w. significantly. Acute toxicity study showed no clinical signs and pre-terminal deaths. The LD(50) of PartySmart was found to be greater than 2000 mg/kg b.wt. No significant differences in PartySmart-treated groups were observed on body weight, food intake, haematological and clinical chemistry, and organ weight ratios as compared to control group in the repeated dose study. Histopathological examination of all target organs showed no evidence of lesions attributing to drug toxicity. INTERPRETATION & CONCLUSION: PartySmart enhanced acetaldehyde metabolism by increasing ADH and ALDH activity without any side effects. These findings indicate that PartySmart may exert beneficial role in the management of alcohol hangover without any toxicity.

Ameliorative effect of Partysmart in rat model of alcoholic liver disease.

Present study was designed to investigate the effect of polyherbal formulation PartySmart in experimental model of alcoholic liver disease in male Wistar strain rats. Alcohol plus fish oil were administered to animals for 8 weeks to induce liver injury. PartySmart was administered at doses of 250 and 500 mg/kg body weight. After 8 weeks, parameters such as liver weight, liver function serum markers alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) and lipid peroxidation were studied. Livers from all the groups were subjected for histological evaluation. Treatment with PartySmart at the dose of 500 mg/kg body weight showed significant reduction in the levels of serum ALT, AST and ALP with a decrease in liver weight as compared to ethanol-fed rats. A significant decrease was also observed in malondialdehyde levels following treatment with PartySmart at 500 mg/kg body weight. Histological profile of liver tissue in PartySmart-treated animals showed lesser vacuolar degeneration and intactness of hepatic architecture along with improved glycogen deposition as demonstrated by PAS staining. PartySmart ameliorated alcohol-induced liver injury by preventing cell membrane disturbances, reduction of oxidative stress by free radical scavenging and antioxidant activity and normalization of altered intracellular redox status. Thus, PartySmart can be beneficial in the treatment of alcohol-induced liver damage.

Distinct FAK-Src activation events promote alpha5beta1 and alpha4beta1 integrin-stimulated neuroblastoma cell motility.

Signals from fibronectin-binding integrins promote neural crest cell motility during development in part through protein-tyrosine kinase (PTK) activation. Neuroblastoma (NB) is a neural crest malignancy with high metastatic potential. We find that alpha4 and alpha5 integrins are present in late-stage NB tumors and cell lines derived thereof. To determine the signaling connections promoting either alpha4beta1- or alpha5beta1-initiated NB cell motility, pharmacological, dominant negative and short-hairpin RNA (shRNA) inhibitory approaches were undertaken. shRNA knockdown revealed that alpha5beta1-stimulated NB motility is dependent upon focal adhesion kinase (FAK) PTK, Src PTK and p130Cas adapter protein expression. Cell reconstitution showed that FAK catalytic activity is required for alpha5beta1-stimulated Src activation in part through direct FAK phosphorylation of Src at Tyr-418. Alternatively, alpha4beta1-stimulated NB cell motility is dependent upon Src and p130Cas but FAK is not essential. Catalytically inactive receptor protein-tyrosine phosphatase-alpha overexpression inhibited alpha4beta1-stimulated NB motility and Src activation consistent with alpha4-regulated Src activity occurring through Src Tyr-529 dephosphorylation. In alpha4 shRNA-expressing NB cells, alpha4beta1-stimulated Src activation and NB cell motility were rescued by wild type but not cytoplasmic domain-truncated alpha4 re-expression. These studies, supported by results using reconstituted fibroblasts, reveal that alpha4beta1-mediated Src activation is mechanistically distinct from FAK-mediated Src activation during alpha5beta1-mediated NB migration and support the evaluation of inhibitors to alpha4, Src and FAK in the control of NB tumor progression.

Safety and efficacy of oral HD-03/ES given for six months in patients with chronic hepatitis B virus infection.

AIM: To investigate the safety and efficacy of the formulation HD-03/ES capsules in the management of patients with chronic hepatitis B infection. METHODS: A total of 25 patients were recruited to the study and were given HD-03/ES, two capsules twice daily for six months. Clinical assessment of symptoms and signs were done using the " clinical observation table" once a month before and after the treatment. Biochemical investigations of total bilirubin, ALT, AST, serum protein for liver function tests were done every month after initiating treatment. Serum was analyzed for HBV markers for HBsAg, HBeAg and HBV DNA at baseline, 4 and 6 mo after therapy using ELISA kits from Roche. RESULTS: After 6 mo of therapy with HD-03/ES, a significant reduction of ALT values from 66.5 +/- 11.1 to 39.1 +/- 5.2 (P < 0.01) and a significant HBsAg loss (52%, P < 0.001), HBeAg loss (60%, P < 0.05) and HBV DNA loss (60%, P < 0.05) was observed. Adverse effects were mild and never warranted withdrawal of the drug. CONCLUSION: The results of this pilot study indicate that HD-03/ES might be a safe and effective treatment for chronic hepatitis B infection and a long-term multicentric comparator trial is warranted and under way.

NCB-02 (standardized Curcumin preparation) protects dinitrochlorobenzene- induced colitis through down-regulation of NFkappa-B and iNOS.

AIM: To evaluate the efficacy and mechanism of action of NCB 02, a standardized Curcumin preparation, against 2, 4 dinitrochlorobenzene (DNCB) induced ulcerative colitis in rats. METHODS: Ulcerative colitis was induced in male rats by sensitizing with topical application of DNCB in acetone for 14 d and intra-colonol challenge with DNCB on day 15. A separate group of animals with vehicle treatment in similar fashion served as control group. Colitis rats were divided into different groups and treated with NCB-02 at doses of 25, 50 and 100 mg/kg b.wt p.o. for 10 d. Sulfasalazine at a dose of 100 mg/kg b.wt for 10 d served as a reference group. On day 10 after respective assigned treatment, all the animals were euthanized and the length of the colon, weight of entire colon and distal 8 cm of the colon were recorded. The distal part of the colon was immediately observed under a stereomicroscope and the degree of damage was scored. Further distal 8 cm of the colon was subject to the determination of colonic myeloperoxidase (MPO), lipid peroxidation (LPO) and alkaline phosphatase (ALP) activities. A small piece of the sample from distal colon of each animal was fixed in 10% neutral buffered formalin and embedded in paraffin wax and sectioned for immunohistochemical examination of NFkappa-B and iNOS expression. RESULTS: NCB-02 showed a dose dependent protection against DNCB-induced alteration in colon length and weight. NCB-02 treatment also showed a dose dependent protection against the elevated levels of MPO, LPO and ALP, induced by DNCB. NCB-02 demonstrated a significant effect at a dose of 100 mg/kg b.wt., which was almost equipotent to 100 mg/kg b.wt. of sulfasalazine. Treatment with sulfasalazine and curcumin at a dose of 100 mg/kg b.wt. inhibited the DNCB-induced overexpression of NFkappa-B and iNOS in the colon. CONCLUSION: Curcumin treatment ameliorates colonic damage in DNCB induced colitic rats, an effect associated with an improvement in intestinal oxidative stress and downregulation of colonic NFkappa-B and iNOS expression.

Pharmacognostical and physicochemical characteristics of roots of lesser known medicinal plant caesalpinia digyna rottl.

Caesalpinia digyna Rottl. (Caesalpiniaceae) is shrubby perennial climber found in Eastern Ghats. Roots are astringent and used in Ayurveda and Unani systems of medicines. Bergenin, Caesalpinine A and Caesalpinine C were isolated from the roots. However, this medicinal plant has not been studied pharmacognostically. Hence, the present investigation reports pharmacognostical and physicochemical properties of roots of Caesalpinia digyna.

Intrinsic FAK activity and Y925 phosphorylation facilitate an angiogenic switch in tumors.

Elevated focal adhesion kinase (FAK) expression occurs in advanced cancers, yet a signaling role for FAK in tumor progression remains undefined. Here, we suppressed FAK activity in 4T1 breast carcinoma cells resulting in reduced FAK Y925 phosphorylation, Grb2 adaptor protein binding to FAK, and signaling to mitogen-activated protein (MAP) kinase (MAPK). Loss of a FAK-Grb2-MAPK linkage did not affect 4T1 cell proliferation or survival in culture, yet FAK inhibition reduced vascular endothelial growth factor (VEGF) expression and resulted in small avascular tumors in mice. This FAK-Grb2-MAPK linkage was essential in promoting angiogenesis as reconstitution experiments using Src-transformed FAK-null fibroblasts revealed that point mutations affecting FAK catalytic activity (R454) or Y925 phosphorylation (F925) disrupted the ability of FAK to promote MAPK- and VEGF-associated tumor growth. Notably, in both FAK-inhibited 4T1 and Src-transformed FAK-null cells, constitutively activated (CA) mitogen-activated protein kinase kinase 1 (MEK1) restored VEGF production and CA-MEK1 or added VEGF rescued tumor growth and angiogenesis. These studies provide the first biological support for Y925 FAK phosphorylation and define a novel role for FAK activity in promoting a MAPK-associated angiogenic switch during tumor progression.

Intrinsic focal adhesion kinase activity controls orthotopic breast carcinoma metastasis via the regulation of urokinase plasminogen activator expression in a syngeneic tumor model.

Expression of focal adhesion kinase (FAK) is elevated in malignant breast cancer, yet the role of intrinsic FAK activity in promoting tumor progression remains undefined. Here, we have inhibited FAK activity or expression in murine 4T1 breast carcinoma cells via dominant-negative focal adhesion kinase-related non-kinase (FRNK) or anti-FAK short hairpin RNA (shRNA) expression, respectively. Neither FRNK nor FAK shRNA ( approximately 80% reduced FAK levels) affected 4T1 proliferation in culture, whereas reduced FAK activity or expression blocked 4T1 cell invasion through Matrigel and resulted in 2-3-fold lower urokinase plasminogen activator (uPA) expression. Control 4T1 cells implanted into mammary fat pads of BALB/c mice exhibited spontaneous metastasis to the lungs, to the peritoneal cavity, and resulted in 90% lethality within 21 days. Whereas FAK shRNA-expressing 4T1 cells formed tumors in mice with low levels of apoptosis, when mammary-injected, these cells did not exhibit lung metastasis after 21 days and caused only 40% lethality up to 60 days. Transient re-expression of wild-type but not kinase-dead FAK in 4T1 FAK shRNA cells promoted uPA production and mammary to lung metastasis within 7 days. In fact, stable human uPA overexpression in 4T1 FAK shRNA cells promoted Matrigel invasion and lung metastasis equal to 4T1 controls. Conversely, treatment with plasminogen activator inhibitor-1 or neutralizing antibody to uPA blocked Matrigel invasion of 4T1 control cells. These studies provide the first direct proof that FAK catalytic activity can facilitate metastatic breast cancer progression by regulating uPA expression.

Induction and evaluation of atherosclerosis in New Zealand white rabbits.

Atherosclerosis was experimentally induced in New Zealand white rabbits by feeding a high cholesterol diet for 12 weeks for screening of drugs against atherosclerosis. After 12 weeks, blood was collected from ear vein for evaluation of total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL) and very low density lipoprotein (VLDL) levels, then the animals were sacrificed to collect the livers for estimation of cholesterol, and aorta for gross and histopathological evaluations. The elevated levels of serum and liver parameters accompanied by gross and histopathological changes like accumulation of foam cells, atheromatous plaque formation and replacement fibrosis supported the successful induction of atherosclerosis in New Zealand white rabbits.

The role of research in a technical assistance agency: the case of the 'German Agency for Technical Co-operation'.

Technical assistance agencies have a sustainable impact on the health systems of the countries they are operating in. As well as policy-makers at the national level, technical assistance agencies see themselves confronted that their interventions should be based on evidence, usually meaning the results of research. This study has the aim to analyse role of research in the implementation of technical assistance. We sent a questionnaire to all health project managers of the 'German Agency for Technical Co-operation' and performed a qualitative case study in one of the health projects. Forty-seven of 80 (58.8%) of the questionnaires were completed and sent back. The managers considered publications of International Organisations (IOs), scientific articles and local research as most important for their work. The case study showed application problems in the daily work. Research use not only depends on the relevance of the data but also on analytical skills, linguistic barriers and technical access to research by the potential users. The role of knowledge and information management has to be clearly defined in an organisation of technical assistance. The specific needs at the different levels have to be analysed so that skills and resources can be allocated adequately.

Posterior urethral valves: incidence and progress of vesicoureteric reflux after primary fulguration.

This is a prospective study of 20 cases of posterior urethral valves (PUV) presenting between the ages of 12 days and 5.5 years (median 15 months) in order to determine the incidence and progress of vesicoureteric reflux (VUR), hydronephrosis (HN), and renal functional status. The efficacy of fulguration as the sole modality of treatment for PUV was assessed in terms of improvement or disappearance of VUR over a 6-month follow-up period. VUR was present in 60% of the patients, being unilateral in 41.7%. Out of 19 renal units with VUR, reflux subsided in 31.5% by 3 months and 78.94% by 6 months. The blood urea and serum creatinine levels, which were raised in 50% of the patients at presentation, came down to normal by 6 months in all the cases. Improvement in glomerular filtration rates (GFR) was noted in all the children at each follow-up and was found to be statistically significant ( p<0.01). HN was present in all the patients at presentation and was bilateral in 90%. It decreased significantly during the follow-up period, though its complete disappearance was seen only in one case. Vesicoureteric reflux dysplasia syndrome (VURD) was present in two cases. Our study showed that VUR disappeared in a majority of the cases by 6 months once adequate urethral patency was restored, although hydronephrosis persisted.

Downregulation of hepatitis B surface antigen expression in human hepatocellular carcinoma cell lines by HD-03, a polyherbal formulation.

Hepatitis B virus is associated with chronic or acute liver diseases and with hepatocellular carcinoma. In the present study, we examined the activity of HD-03, a polyherbal formulation, on two hepatitis B surface antigen (HBsAg) expressing human hepatocellular carcinoma cell lines, PLC/PRF/5 and HepG2/A2. We observed that HD-03 downregulates HBsAg expression from these cell lines. Our studies have also shown that this effect is neither due to cytotoxicity on the cell lines nor due to blockade of the release of the antigen from the cells nor due to binding of the substance with the antigen. The possible mode of its antiviral activity is explained.

Histological changes in intestine in semichronic diarrhoea induced by lactose enriched diet in rats: effect of Diarex-Vet.

Efficacy of Diarex-Vet (The Himalaya Drug Company, Makali, Bangalore, India) was evaluated histologically in semichronic diarrhoea induced by lactose enriched diet in rats. The rats in different groups were given lactose enriched diet alone for 2 days before starting the treatment with Diarex-Vet at a dose of 250, 500 and 750 mg/kg body weight along with lactose enriched diet for 5 days. Animals were euthanised at the end of 5 days of treatment and histological changes were observed in the ileum, caecum and colon. Semiquantitative analysis of goblet cells in intestines showed dose dependent response among the treated groups. The morphological changes were comparable to normal in the group given 750 mg/kg body wt Diarex-Vet. The effects observed were attributed to the lactase like analogous activity of Diarex-Vet or the inhibition of the osmotic processes in the intestinal lumen thereby reducing the morphological changes in the intestines.

Tumor-specificity and radical scavenging activity of poly-herbal formula.

A total of 14 poly-herbal formula extracts were compared for their biological activities both in vivo and in vitro. Pretreatment of mice with the extracts protected them from E. coli infection to various extents. Among the extracts, the HD-12 and DLH-3073 extracts showed the highest cytotoxicity against both HIV-infected and mock-infected MT4 cells, without induction of any apparent anti-HIV activity. The extracts showed significantly higher cytotoxic activity against five human tumor cell lines (HSC-2, HSC-3, HSG, MT-4, HL-60) than against three normal human cell lines (HGF, HPC, HPLF). Agarose gel electrophoresis demonstrated that the HD-12 and DLH-3073 extracts induced intemucleosomal DNA fragmentation in HL-60 cells. ESR spectroscopy showed that all the extracts produced radicals and this was paralleled by their ability to scavenge the superoxide anion (produced by hypoxanthine-xanthine oxidase reaction), the hydroxyl radical (produced by Fenton reaction) and nitric oxide (produced by NOC- 7) in the presence of radical trapping agents. Higher and lower concentrations of extracts enhanced or reduced respectively, the radical intensity of sodium ascorbate, suggesting their bimodal actions. The tumor specificity and antioxidant properties of the herb extracts further suggest their medicinal efficacy.

Effect of poly-herbal formula on NO production by LPS-stimulated mouse macrophage-like cells.

Pretreatment of mice with lyophilized hot water extracts of five poly-herbal formula protected them from lethal infection by E. coli. ESR spectroscopy shows that these extracts produced radicals under alkaline condition, and scavenged radicals such as superoxide anion, hydroxyl radical and nitric oxide (NO) radical. There was a positive relationship between their radical intensity and radical scavenging activity. Among the extracts, HD-02 efficiently inhibited the production of NO and citrulline, and the expression of inducible NO synthase (iNOS) mRNA by LPS-stimulated mouse macrophage-like cells Raw 264.7. DLH-3073 not only inhibited the LPS-stimulated NO production at lower concentration, but also induced NO production at higher concentrations, suggesting the presence of two different antagonizing components in the DLH-3073 extract. These data suggest that poly-herbal extracts may alleviate radical-mediated diseases.

Inhibition of anchorage-independent cell growth, adhesion, and cyclin D1 gene expression by a dominant negative mutant of a tyrosine phosphatase.

PTP-S4/TC48 protein tyrosine phosphatase is localized in the nuclear and cytoplasmic membranes. To investigate the role of PTP-S4 in cell growth, adhesion, and transformation, normal and a catalytically inactive mutant form of this phosphatase were expressed in polyoma virus-transformed F111 fibroblast cell line, PyF. Expression of mutant PTP-S4 in PyF cells resulted in strong inhibition of anchorage-independent growth in soft agar but had no significant effect on growth in liquid culture. Tumor formation in nude mice was also reduced by mutant PTP-S4. Expression of normal PTP-S4 in PyF cells significantly increased anchorage-independent cell growth and tumor formation in nude mice. Overexpression of catalytically inactive mutant of PTP-S2/TC45 (a splice variant of PTP-S4 that is nuclear) did not inhibit anchorage-independent growth of PyF cells. Mutant PTP-S4-expressing cells were inhibited in adhesion and spreading on tissue culture plates compared to control cells. Expression of mutant PTP-S4 in PyF cells reduced the levels of cyclin D1 and cyclin A mRNA, whereas cyclin D2 mRNA level was not affected significantly. Expression of antisense cyclin D1 strongly inhibited anchorage-independent growth. Inhibition of anchorage-independent growth by mutant PTP-S4 was overcome to a large extent by coexpression of cyclin D1. These results suggest that mutant PTP-S4 inhibits anchorage-independent growth and adhesion of polyoma virus-transformed cells by interfering with the normal function of PTP-S4 upstream of cyclin D1 gene expression.

The protective effect of HD-03 in CCl4-induced hepatic encephalopathy in rats.

The liver is a major parenchymal organ involved in many functional activities in the body. Hepatic encephalopathy is a syndrome characterized by increased blood ammonia level and is one of the major complications of cirrhosis. In the present study the protective effect of HD-03, a poly-herbal formulation, was evaluated against CCl4-induced hepatic encephalopathy in rats. Hepatic encephalopathy was induced in Wistar rats by administration of CCl4 at a dose of 1 mL/kg orally in liquid paraffin (1:1) twice a week for 90 days. The liver enzymes (SGPT and SGOT) and blood ammonia levels were significantly (p < 0.001) higher in the CCl4-intoxicated group compared with the untreated control group. Administration of HD-03 at a dose of 750 mg/kg orally as an aqueous suspension significantly prevented the elevation of SGPT, SGOT and blood ammonia levels. Histomorphometric evaluation of liver and brain showed a protective effect of the HD-03 treatment, thus correlating with the changes in biochemical profiles. The protective effect of HD-03 against CCl4-induced encephalopathy may be due to the improved hepatocellular function, which in turn helps in regulating the metabolism of ammonia. However, further studies are required to measure the activity of enzymes involved in the urea cycle and brain aromatic amino acids in order to elucidate the exact mechanism of action of HD-03.