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.

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.

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.

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.

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.

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.

Hepatic outflow obstruction (Budd-Chiari syndrome). Experience with 177 patients and a review of the literature.

Budd-Chiari syndrome (BCS) may not be as uncommon as was once believed. Our study has substantiated the existence of 2 major clinical forms. The acute syndrome is invariably associated with extensive blockage of the major hepatic veins, resulting in congestive liver cell necrosis. In a small, but significant, number of patients the inferior vena cava (IVC) is also occluded. The important etiologic factors are related to hypercoagulability of blood. Immediate placement of a shunt improves survival. The chronic syndrome is characterized by portal hypertension and is associated with a variable abnormal vascular anatomy. The causes of the chronic syndrome are not clear, but a substantial number of cases are related to the presence of an IVC membrane. Shunt surgery is effective but procedures aimed at the primary pathology are likely to be even more so. The natural history of BCS should be viewed over a long period of time. The very long survival of several patients urges a more cautious approach to surgical remedies. Budd-Chiari syndrome probably represents a spectrum of disease caused primarily by a hypercoagulable state and having a varied presentation depending on the balance between rate of formation and the extent of the thrombosis and the body's own rate of thrombolysis and recanalization. The extent and efficacy of the individual's collateral circulation and the rate of development of liver fibrosis are other determinants. It is thus possible to view BCS as a continuum of a single pathogenetic spectrum. Pregnancy-related BCS in India probably has strong social determinants, and is usually acute and fulminant. We have, however, documented a chronic form not described earlier. Children usually do not have acute BCS, but chronic BCS in children and adolescents is similar to that in adults. Membranous obstruction of the inferior vena cava (MOVC) is common and was found even at a young age. The association of MOVC with hepatocellular carcinoma, however, did not appear to be as clear as was previously believed. There has been a wide geographical variability in the causes and manifestations of BCS. Our study has clearly shown that--Kipling's categorical statement to the contrary--East and West do meet in India, in the Budd-Chiari syndrome.

The pathology of noncirrhotic portal fibrosis: a review of 32 autopsy cases.

A wide spectrum of clinical and morphologic changes in 32 autopsy cases of noncirrhotic portal fibrosis have been described. The disease frequently occurs in younger patients with a long history of splenomegaly, usually with a history of hematemesis. Females are affected almost equally as often as males in contrast to cirrhosis. The patients tolerate the bleeding episodes well. Death is usually due to massive hemorrhage. The diagnosis is achieved through a process of exclusion. A critical analysis of hemodynamic data, a splenoportogram, liver function tests (particularly Bromsulphalein retention) and angiographic data is mandatory. Needle biopsy of the liver appears to have limited value in making the diagnosis. The gross anatomic findings vary from a nearly normal liver to gross nodularity, seen particularly on the posteroinferior surface. In some cases these nodules are seen to physically impede the portal blood flow and contribute to portal hypertension. Phlebosclerosis of the smaller radicles of the portal vein and irregular scarring are the outstanding morphologic features of the disease. These changes are usually associated with irregular dilatation of some of the larger intrahepatic branches of the portal vein as well as fibroelastosis with or without occluding or organizing thrombi in both intra- and extrahepatic branches of the portal vein. The changes in hepatic venous radicles are characterized by irregular sclerosis, which seems to contribute significantly toward postsinusoidal block in advanced cases. The probable mode of evolution is discussed.

Perineal anal transplant in anorectal malformation in female patients.

Results of perineal anal transplant in 25 patients with anorectal malformations in female children have been analyzed. The present study shows that the operation is ill advised in patients with intermediate anomalies but can be undertaken safely in patients with low anomalies. In our series best results were obtained when the transplant was performed in patients who were past 5 years of age. Colostomy, though helpful in reducing the severity of the immediate complications, does not influence the late results.

Perineal anal transplant in low anorectal anomalies.

Data on 67 female infants who received perineal and transplants for for treatment of low anorectal anomalies over a period of 12 years are presented. The procedure is considered to be safe in neonates and infants. When performed in the first year of life, it does not require a preliminary colostomy and gives good functional and cosmetic results. In older children it should be preceded by a diversion colostomy. The immediate complications are few and are not life threatening; the late results are satisfactory.

Echinococcus multilocoularis infection in India: First case report proved at autopsy.

The occurrence of Echinococcus multilocularis is reported in India for the first time. The patient was a young man, various clinical diagnoses were made and he finally died after an attempted membranotomy for suspected membranous obstruction in the inferior vena cava. Autopsy revealed classical E. multilocularis infection of the liver with direct spread of the inferior vena cava, the right atrium and through the diaphragm into the base of the left lung. It also had caused an outflow tract obstruction to the hepatic venous flow by direct physical pressure distorting the proximal intrahepatic portion of the inferior vena cava. In addition the patient had multi-valvular lesions of rheumatic origin and a terminal infective endocarditis due to staphyloccal infection.

An improved determination of total glycosaminoglycans in body fluids by formation of complexes with quinacrine: changes in amniotic fluid total glycosaminoglycans during normal pregnancies and in pregnancies at risk for mucopolysaccharidoses.

Acidic glycosaminoglycans form insoluble complexes with quinacrine and this has been exploited for their analysis in blood, urine and amniotic fluid. The method is specific for glycosaminoglycans including keratan sulphate and the samples do not have to be deproteinized. Values for normal urine, serum and amniotic fluid are presented. Urinary total glycosaminoglycans excreted by patients with mucopolysaccharidoses were also determined. The normal changes in amniotic fluid total glycosaminoglycans have been measured between 14 weeks' gestation and term, and values are given for amniotic fluid total glycosaminoglycans in several pregnancies at risk for mucopolysaccharidoses. It is suggested that this method is a potentially valuable analytical tool in the pre-natal diagnosis of mucopolysaccharidoses.

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.

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.

Anxiogenic activity of isatin, a putative biological factor, in rodents.

Isatin (2,3-dioxoindole) has been proposed as a new biological factor, responsible for at least part of the activity of tribulin, an endogenous monoamine oxidase and benzodiazepine receptor binding inhibitory factor, which may serve as an endocoid marker of stress and anxiety. The putative anxiogenic activity of isatin was investigated in rats and mice. The doses chosen for the study, namely 15 mg/kg i.p. in mice and 20 mg/kg i.p. in rats, were based on preliminary behavioural studies. Yohimbine, a well established anxiogenic agent, was used for comparison and used at doses of 2 and 2.5 mg/kg i.p. in mice and rats, respectively. The experimental paradigms chosen have been shown to stand the tests of validity and reliability. Isatin induced significant anxiogenic activity in the open-field and elevated plus-maze tests in mice, and the social interaction test in rats, which were comparable to those induced by yohimbine. In addition, both isatin and yohimbine attenuated the effects of the anxiolytic agent diazepam in the open-field test. The investigations indicate that isatin has significant anxiogenic effect and support the contention that it and/or its biotransformation products may be responsible for at least part of the activity of tribulin demonstrated previously in animal models and in clinical situations of stress and anxiety.

Protective effect of Prostane in experimental prostatic hyperplasia in rats.

AIM: Prostane, a polyherbal formulation, was evaluated for its efficacy on 5alpha-reductase inhibition, alpha-adrenergic antagonistic activity and testosterone-induced prostatic hyperplasia. METHODS: 5alpha-reductase inhibition was evaluated using rat prostate homogenate as an enzyme source. Adrenergic antagonistic activity was evaluated using isolated rat vas deferens. Experimental prostatic hyperplasia was induced in rats by giving testosterone 3 mg/kg sc for 21 days. RESULTS: Prostane dose-dependently inhibited 5alpha-reductase activity and exhibited alpha-adrenergic antagonistic activity. Treatment with Prostane at 250, 500 and 750 mg/kg body wt, po for 21 days significantly reduced the prostatic weight, the epithelial height and the stromal proliferation in experimental prostatic hypertrophy. CONCLUSION: Prostane is effective in the treatment of experimental prostatic hypertrophy in rats and may be passed on to clinical trials on benign prostatic hypertrophy after necessary toxicological evaluations.

Multiple cytomegalovirus-related intestinal perforations in patients with acquired immunodeficiency syndrome. Report of two cases and review of the literature.

We present two cases of patients with acquired immunodeficiency syndrome who, in the course of their disease, suffered multiple intestinal perforations that were directly related to cytomegalovirus infection. Biopsy and surgical specimens and autopsy findings in both cases revealed extensive lesions of gastroenteritis; the gastroenteritis was characterized by randomly distributed deep ulcers, resulting in multiple perforations. The main characteristic histopathologic finding was the association of intestinal lesions with a severe form of cytomegalovirus-related occlusive vasculitis. This report provides evidence that supports the contention that cytomegalovirus is the primary causal agent of gastrointestinal lesions affecting immunocompromised patients.