Microfluidics enables multiplex evaluation of the same cells for further studies.

OBJECTIVE: The continuous discovery of biomarkers and their evolving use for the diagnosis and guidance of therapy for patients with cancer has increased awareness of the need to triage biospecimens properly. On occasion, cytology samples are the only type of biospecimen available for analysis. Often, the current approach for these latter specimens is cytopathology-centric, with cells limited to examination by bright field microscopy. When specimens are paucicellular, there is often insufficient material for ancillary testing. Therefore, a need exists to develop an alternative approach that allows for the multiplexed analysis of cells when they are limited in number. In recent previous publications, we demonstrated that clinically derived cells from tissue are suitable for evaluation in a microfluidic device. In our current endeavour, we seek to expand upon those findings and determine if those same cells can be recovered for further analysis. METHODS: A microfluidic channel was designed, fabricated and tested using cytology specimens generated from tissue specimens. The cytological features of the cells tested were examined prior to entering the channel; they were then compared to similar cells while in the channel, and upon recovery from the channel. Recovery of DNA and proteins were also tested. RESULTS: The morphology of the tested cells was not compromised in either the channel or upon recovery. More importantly, the integrity of the cells remained intact, with the recovery of proteins and high molecular weight DNA possible. CONCLUSIONS: We developed and tested an alternative approach to the processing of cytopathology specimens that enables multiplexed evaluation. Using microfluidics, cytological examination of biopecimens can be performed, but in contrast to existing approaches, the same cells examined can be recovered for downstream analysis.

The time-dependent alteration of anti-diuretic hormone system in hindlimb unloaded rats.

It is important to understand the mechanism on the fluid shift and volume regulation occurring in astronauts after spaceflight for future life in space. In the present study, we examined the time-dependent alteration of anti-diuretic hormone (ADH) concentrating on the water reabsorption system in hindlimb unloaded rats. Male Sprague-Dawley rats were hindlimb unloaded for 1 (HU1), 7(HU7), 14 days (HU14) or rested in the ground for 3 days after HU14 (HU14+3). The plasma ADH and angiotensin II level showed peak value at HU7, and the alterations were restored at HU14. However, several serum electrolytes (Na, K, Cl) were not changed regardless of HU period. In the immunohistochemical study, we examined that ADH and c-Fos immunoreactivities (IR) were maximized at HU7 in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). Aquaporin 2 (AQP2) IR also was increased in the renal collecting duct for water re-absorption at HU7 showing a similar pattern with ADH. These results present a series of physiological ADH system alteration following to period of hindlimb unloading stimulus, indicating that ADH system is activated significantly at HU7. In addition, our results suggest that ADH system activation may be involved in anti-diuretic phenomenon in early spaceflight period. Furthermore, it is speculated that ADH system may require 14 days for adaptation to microgravity.

Serum 1,5-anhydroglucitol is associated with diabetic retinopathy in Type 2 diabetes.

AIMS: To determine whether there is a relationship between 1,5-anhydroglucitol (1,5-AG), a marker of postprandial hyperglycaemia and glycaemic variability, and the presence of diabetic retinopathy and albuminuria in patients with Type 2 diabetes. METHODS: Five hundred and sixty-seven patients with Type 2 diabetes (serum creatinine < 133 µmol/l), who were enrolled in the Seoul Metro-City Diabetes Prevention Program (SMC-DPP), were cross-sectionally assessed by multivariate logistic regression analysis. RESULTS: After controlling for age, sex, binary HbA(1c) levels, duration of diabetes, triglyceride, systolic blood pressure, smoking status, history of hypertension and dyslipidaemia, and the use of angiotensin-converting enzyme inhibitor/angiotensin receptor blocker medication, the odds ratios (95% CI) of diabetic retinopathy were 2.86 (1.12-7.25) for the first (lowest) quartile of 1,5-anhydroglucitol, 2.87 (1.25-6.61) for the second quartile and 0.88 (0.35-2.22) for the third quartile compared with the fourth quartile (P for trend = 0.010). Conversely, the associations between 1,5-anhydroglucitol and clinical albuminuria were non-significant after adjustment. Subjects with low 1,5-anhydroglucitol (< 10.0 µg/ml) were more likely to experience diabetic retinopathy than those with high 1,5-anhydroglucitol (≥ 10.0 µg/ml) under moderate glucose control (HbA(1c) < 8%, 64 mmol/mol) and there were no significant differences in the prevalence of diabetic retinopathy between the subgroup with HbA(1c) < 8% (64 mmol/mol) and low 1,5-anhydroglucitol and the subgroup with HbA(1c) ≥ 8% (64 mmol/mol). CONCLUSIONS: 1,5-Anhydroglucitol levels show close associations with diabetic retinopathy, especially among patients under moderate glucose control, but not with albuminuria. These results suggest that 1,5-anhydroglucitol might be a complementary marker for targeting higher risk group.

The role of serum adipocyte fatty acid-binding protein on the development of metabolic syndrome is independent of pro-inflammatory cytokines.

BACKGROUND AND AIM: Adipocyte fatty acid-binding protein (FABP4) is abundantly expressed in adipocytes and plays a role in glucose homeostasis. We analysed the relationship between serum FABP4 levels and the progression of metabolic syndrome in healthy adults. METHODS AND RESULTS: A total of 465 subjects were selected from participants in a medical check-up programme at a Health Promotion Center. Baseline serum FABP4 levels were measured, and the subjects were evaluated for the presence of metabolic syndrome (MetS) according to the recommendations of the American Heart Association/National Heart, Lung, and Blood Institute. The subjects were re-evaluated 4 years later. Baseline FABP4 concentrations were significantly higher in subjects with MetS than in those without MetS (P<0.001). At the 4-year follow-up, subjects in the highest FABP4 tertile at baseline exhibited higher values for body mass index, fat mass and percent body fat, as well as blood pressure, fasting glucose, total cholesterol, triglycerides, low-density lipoprotein (LDL)-cholesterol, insulin, homeostasis model assessment of insulin resistance, monocyte chemoattractant protein-1 and tumor necrosis factor-α levels (all P<0.05). The subjects with higher FABP4 levels had lower HDL-cholesterol concentrations (P<0.05). After adjustment for age, sex, change in percent body fat and baseline values for other metabolic and inflammatory parameters, FABP4 levels at baseline were shown to be strongly associated with the development of MetS by year 4 (odds ratio (OR), 5.75; 95% confidence interval (CI), 2.71-12.23 for highest tertile vs. lowest tertile, P<0.001) CONCLUSION: Baseline serum FABP4 levels appear to be a significant predictor for the future development of MetS, independent of pro-inflammatory cytokines.

Serine palmitoyltransferase inhibitor myriocin induces growth inhibition of B16F10 melanoma cells through G(2) /M phase arrest.

OBJECTIVES: Melanoma is the most aggressive form of skin cancer, and it resists chemotherapy. Candidate drugs for effective anti-cancer treatment have been sought from natural resources. Here, we have investigated anti-proliferative activity of myriocin, serine palmitoyltransferase inhibitor, in the de novo sphingolipid pathway, and its mechanism in B16F10 melanoma cells. MATERIAL AND METHODS: We assessed cell population growth by measuring cell numbers, DNA synthesis, cell cycle progression, and expression of cell cycle regulatory proteins. Ceramide, sphingomyelin, sphingosine and sphingosine-1-phosphate levels were analysed by HPLC. RESULTS: Myriocin inhibited proliferation of melanoma cells and induced cell cycle arrest in the G(2) /M phase. Expressions of cdc25C, cyclin B1 and cdc2 were decreased in the cells after exposure to myriocin, while expression of p53 and p21(waf1/cip1) was increased. Levels of ceramide, sphingomyelin, sphingosine and sphingosine-1-phosphate in myriocin-treated cells after 24 h were reduced by approximately 86%, 57%, 75% and 38%, respectively, compared to levels in control cells. CONCLUSIONS: Our results suggest that inhibition of sphingolipid synthesis by myriocin in melanoma cells may inhibit expression of cdc25C or activate expression of p53 and p21(waf1/cip1) , followed by inhibition of cyclin B1 and cdc2, resulting in G(2) /M arrest of the cell cycle and cell population growth inhibition. Thus, modulation of sphingolipid metabolism by myriocin may be a potential target of mechanism-based therapy for this type of skin cancer.

Impact of non-alcoholic fatty liver disease on microalbuminuria in patients with prediabetes and diabetes.

BACKGROUND: It is unknown whether microalbuminuria is associated with non-alcoholic fatty liver disease (NAFLD) among patients with prediabetes and type 2 diabetes mellitus (DM). This study investigated the association of NAFLD with microalbuminuria among patients with prediabetes and diabetes. METHODS: We evaluated 1361 subjects who had an abnormal oral glucose tolerance test (OGTT) on routine screening. All participants were divided into two groups, prediabetes and newly diagnosed type 2 DM, and the association of NAFLD with metabolic parameters on microalbuminuria was analysed. RESULTS: The patients with NAFLD had higher prevalence rates of microalbuminuria (6.3% vs 19%; P = 0.001 in prediabetes, 4.5% vs 32.6%; P < 0.001 in diabetes) and also had a greater albumin-to-creatinine ratio (14.6 +/- 52.0 microg/mg Cr vs 27.7 +/- 63.9 microg/mg Cr; P = 0.051 in prediabetes, 11.4 +/- 21.4 microg/mg Cr vs 44.7 +/- 76.4 microg/mg Cr; P < 0.001 in diabetes) than those without NAFLD. The logistic regression analysis showed that NAFLD was associated with increased rates of microalbuminuria (odds ratio 3.66; 95%confidence interval (CI) 1.31-10.20, P = 0.013 in prediabetes, odds ratio 5.47;95% CI 1.01-29.61, P = 0.048 in diabetes), independently of age, sex, body mass index, waist circumference, liver enzymes, lipid profiles, HbA1c, insulin resistance as estimated by homeostasis model assessment, hypertension,smoking status and the metabolic syndrome. CONCLUSIONS: The results of our study revealed a strong relationship between microalbuminuria and NAFLD in the patients with prediabetes and newly diagnosed diabetes. Further studies are required to confirm whether NAFLD is a predictor of the development of microalbuminuria in patients with prediabetes and diabetes.

Impact of circulating bone-resorbing cytokines on the subsequent bone loss following bone marrow transplantation.

Cytokines including IL-6 and TNF-alpha play an important role in the pathogenesis of postmenopausal osteoporosis. However, the relationship between changes in the cytokine levels and subsequent bone loss in patients undergoing a bone marrow transplantation (BMT) is unclear. A total of 46 patients undergoing an allogeneic BMT were prospectively investigated. The bone turnover markers and the serum cytokines were measured before BMT and serially after BMT. Bone mineral density (BMD) was measured before and 1 year after BMT. At 1 year after BMT, the lumbar spine BMD had decreased by 4.8%, and the total proximal femoral BMD had decreased by 12.3%. The serum IL-6 and TNF-alpha levels increased until 2 and 3 weeks after BMT, respectively. The lumbar BMD was significantly decreased as the serum IL-6 and TNF-alpha levels increased by post-BMT 3 weeks. The lumbar BMD decreased significantly as the cumulative prednisolone and cyclosporine dose increased. Patients with GVHD > or =grade II had higher lumbar bone loss than patients with GVHD

Relationship between circulating cytokine levels and thyroid function following bone marrow transplantation.

The relation between thyroid hormone changes and cytokines in bone marrow transplantation (BMT) patients has not been studied. This prospective study was designed to determine the relation between thyroid hormones and cytokine levels after BMT and their effects on the mortality. We studied 80 patients undergoing allogeneic BMT. Serum thyroid hormone parameters and cytokine levels were measured before and serially during 6 months after BMT. Serum T(3) decreased to a nadir 3 weeks post-BMT and serum T(4) was lowest at 3 months post-BMT. Serum thyroid stimulating hormone (TSH) sharply decreased to a nadir at 1 week and recovered. Serum interleukin-6 increased for 2 weeks after BMT and declined thereafter. Serum tumor necrosis factor-alpha increased for 3 weeks after BMT and declined thereafter. After 3 weeks post-BMT, both cytokine levels were negatively correlated with serum T(3) and T(4) levels. A total of 29 patients died before 1 year post-BMT and 51 patients survived longer than 1 year. Those patients who died before 1 year post-BMT had significantly lower levels of T(4) at 3 weeks, 3 and 6 months than surviving patients. In conclusion, increased levels of serum IL-6 and TNF-alpha were negatively correlated with thyroid hormone concentrations in BMT recipients suggesting the role of these cytokines in euthyroid sick syndrome.

The role of cytokines in the changes in bone turnover following bone marrow transplantation.

Osteoporosis is a common disease among patients undergoing transplantation and a loss of bone mass is usually detected after bone marrow transplantation (BMT), particularly during the immediate post-BMT period. Post-BMT bone loss is primarily related to gonadal dysfunction and immunosuppression. Cytokines, especially interleukin 6, play an important role in the pathogenesis of postmenopausal osteoporosis. However, the pathogenetic role of cytokines in post-BMT bone loss is unknown and data on the changes of cytokines in accordance with bone turnover markers are scarce. The aim of this study was to assess the relationship between bone turnover markers and cytokines, which are regularly sampled at peripheral blood and bone marrow before and after allogeneic BMT. This prospective study included two analyses. The first was a study of 46 BMT recipients (M/F 28/18), examining the relationship between bone turnover markers and serum cytokines that were measured before and at 1 week, 2 weeks, 3 weeks, 4 weeks and 3 months after BMT. Serum intact parathyroid hormone was measured before BMT and at 3 weeks after BMT and its relation to other cytokines and bone turnover markers was evaluated. The second analysis was a study of 14 (M/F 9/5) of 46 patients in whom bone marrow plasma cytokines [interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha)] were measured at 3 weeks after BMT. The relationship between bone marrow plasma cytokines and bone turnover markers was studied because bone marrow is the microenvironment where the real changes in bone turnover occur. Serum type I collagen carboxyterminal telopeptide (ICTP), a bone resorption marker, increased progressively until 4 weeks (peak) after BMT and then decreased thereafter. Serum osteocalcin, a bone formation marker, decreased progressively until 3 weeks after BMT and then increased thereafter. Serum IL-6 increased until 2 weeks after BMT and declined thereafter. Serum TNF-alpha increased until 3 weeks after BMT and declined thereafter. There was a significant positive correlation between serum ICTP and bone marrow IL-6 levels at 3 weeks after BMT, when a marked change in bone metabolism occurs following BMT. However, a correlation between bone turnover markers and bone marrow TNF-alpha or peripheral blood cytokines was not found. At 3 months after BMT, there was a significant negative correlation between the mean daily steroid dose and the serum osteocalcin level (r = -0.43, p < 0.05). The correlation between the Mean daily steroid dose and serum ICTP was also significant (r = 0.41, p < 0.05). Our data suggest that the progressive increase in bone resorption during the immediate post-BMT period is related to both steroid dose and the increase in bone marrow IL-6, which is a potent stimulator of bone resorption in vivo.

Mode of antiviral activity of water soluble components isolated from Elfvingia applanata on vesicular stomatitis virus.

A preparation of water soluble components (EA) was made from carpophores of Elfvingia applanata (Pers.) Karst and its in vitro antiviral activity on vesicular stomatitis virus [(Indiana serotype, VSV(IND)] was investigated by plaque reduction assay. EA exhibited potent antiviral activity on VSV(IND) growth and negligible cytotoxicity on Vero cells, 50% effective concentration (EC50) of 104 microg/ml and 50% cytotoxic concentration (CC50) of 3,793 microg/ml, respectively. Selectivity index (SI, CC50/EC50) of EA on Vero cell and VSV(IND) was about 36.5. EA did not display either a direct virucidal effect on VSV(IND) or induction of antiviral substance by Vero cells upon its treatment. Thus, the mode of antiviral activity of EA was studied at steps of viral adsorption onto cell. When both EA and virus were added to cell monolayers, titer of cell-free virus in culture supernatant increased in ca. 30-40% compared with that of control group and titer of cell-associated virus was 60-100% higher than that of control group. These results suggested that antiviral activity of EA on VSV(IND) might be due to the hindrance of viral entry to cells at either endocytosis or loss of envelope.

Moderate dietary fat consumption as a risk factor for ischemic heart disease in a population with a low fat intake: a case-control study in Korean men.

BACKGROUND: Dietary fat intake is associated with the incidence of ischemic heart disease (IHD) in Western countries. In populations in which both the average dietary fat consumption and the incidence of IHD are lower than in Western countries, the association of dietary fat intake with IHD incidence remains unknown. OBJECTIVE: We conducted a case-control study to examine the association of dietary fat with IHD incidence in Korean men. DESIGN: The case group consisted of 108 patients with electrocardiogram-confirmed myocardial infarction or angiographically confirmed (> or =50% stenosis) IHD who were admitted to a university teaching hospital in Seoul, Republic of Korea. The controls were 142 age-matched patients admitted to the departments of ophthalmology and orthopedic surgery at the same hospital. Dietary fat intake was assessed by a nutritionist using a semiquantitative food-frequency questionnaire. Body mass index (BMI), cigarette use, alcohol intake, exercise, and history of disease were determined during an interview and examination. RESULTS: In a univariate analysis, the mean percentages of energy from total fat, saturated fatty acids, and monounsaturated fatty acids were significantly higher in the cases than in the controls. BMI, smoking, and a history of hypertension were associated with the occurrence of IHD. In multiple logistic analyses, total fat intake was a significant risk factor (odds ratio: 1.08 for 1% of energy intake; 95% CI: 1.02, 1.14) after adjustment for BMI and smoking. CONCLUSION: In a population with a relatively low fat intake (19% of energy intake), a moderate increase in total fat intake may be a risk factor for IHD.

Antiherpetic activities of acidic protein bound polysacchride isolated from Ganoderma lucidum alone and in combinations with interferons.

To investigate antiherpetic activity, an acidic protein bound polysaccharide (APBP) was isolated from carpophores of Ganoderma lucidum. This brownish APBP was isolated from water soluble substances of the carpophores by activity-guided isolation method. APBP was tested for its antiviral activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) by plaque reduction assay in tissue culture. APBP showed potent antiviral activity against HSV-1 and HSV-2 in Vero cells at its 50% effective concentration (EC(50)) of 300 and 440 microg/ml, respectively. APBP had no cytotoxicity on Vero cells at a concentration of 1x10(4) microg/ml. APBP exhibited a potent antiviral activity with selectivity index (SI) of more than 22.73. The combined antiherpetic effects of APBP with protein antiviral agents, interferon alpha (IFN alpha) and interferon gamma (IFN gamma), were examined on the multiplication of these two strains of herpesviruses in Vero cells by the combination assay. The results of combination assay were evaluated by the combination index (CI) that was calculated by the multiple drug effect analysis. The combinations of APBP with IFN alpha on HSV-1 and HSV-2 showed more potent synergistic effects with CI values of 0.30-0.62 for 50-90% effective levels than those of APBP with IFN gamma with CI values of 0.65-1.10. These results suggest the possibility of developing APBP as a new antiherpetic agent.

Dual effects of dextromethorphan on cocaine-induced conditioned place preference in mice.

Dextromethorphan (DM) at supra-antitussive doses might produce psychotomimetic effects in humans. In order to understand the underlying mechanisms responsible for the behavior induced by DM, we examined the effects of DM on cocaine-induced conditioned place preference (CPP) and locomotor pattern in mice, and Fos-related antigen immunoreactivity (FRA-IR) in the striatal complex (nucleus accumbens and striatum) of the mouse brain. The effects of DM (20 and 40 mg/kg, i.p.) on the CPP for 2.5, 5, 10, and 20 mg cocaine/kg, i.p. were assessed. Pretreatment with DM dose-dependently decreased the CPP for 20 mg cocaine/kg. Similarly, pretreatment with DM appeared to reduce the CPP for 10 mg cocaine/kg, but increase the CPP for 5 mg cocaine/kg. This finding was more pronounced for 2.5 mg cocaine/kg; DM significantly increased the CPP for 2.5 mg cocaine/kg in a dose-related manner. Furthermore, these results were correlated with alterations in the locomotor pattern (marginal activity) and FRA-IR in the striatal complex. Thus, our results suggest that DM exhibits a biphasic effect on the cocaine-induced CPP and locomotor pattern.

The short-term changes of bone mineral metabolism following bone marrow transplantation.

Organ transplantation is now the treatment of choice for many patients with life-threatening chronic diseases. A new set of side effects unique to these groups of patients has become recognized, and bone disease is one of these complications. However, little is known about the effects of myeloablative treatment followed by bone marrow transplantation (BMT) on bone mineral metabolism. We have prospectively investigated 31 patients undergoing BMT for hematologic diseases. Serum concentrations of calcium, phosphorus, creatinine, gonadotropins, sex hormones, and the biochemical markers of bone turnover were measured. The samples were collected before BMT and 1, 2, 3, 4, and 12 weeks, 6 months, and 1 year after BMT. Bone mineral density (BMD) was measured with dual-energy X-ray absorptiometry before BMT and 1 year after BMT. The serum carboxy-terminal cross-linked telopeptide of type I collagen increased progressively until 4 weeks after BMT. Thereafter, it began to decrease and reached basal values after 1 year. Serum osteocalcin decreased progressively until 3 weeks after BMT. After that, it increased and reached basal values after 3 months. No distinct differences were observed in the serum biochemical turnover markers between males and females, or between patients who received total body irradiation and those who did not. One year after BMT, lumbar spine BMD had decreased by 2.2%, and total proximal femoral BMD had decreased by 6.2%. Eighty-six percent of the women (12/14) went into a menopausal state immediately after BMT. This was caused by high gonadotropin levels and low estradiol levels. In contrast, gonadotropin levels and testosterone levels did not change significantly in the male patients after BMT. In conclusion, the rapid impairment of bone formation and the increase in bone resorption, as shown by the biochemical markers in this study, might play a role in post-BMT bone loss.

Ginseng total saponin inhibits nicotine-induced hyperactivity and conditioned place preference in mice.

A single or repeated administration of nicotine in mice produced hyperactivity and conditioned place preference (CPP). Postsynapticdopamine (DA) receptor supersensitivity was also developed in nicotine-induced CPP mice. The hyperactivity induced by nicotine was evidenced by measuring the enhanced ambulatory activity using a tilting-type ambulometer. CPP effects were evaluated assessing the increased time spent by the mice to nicotine and the inhibition of CPP by the decreased time spent by the mice in the nonpreferred compartment. Postsynaptic DA receptor supersensitivity was evidenced by the enhanced response in ambulatory activity to the apomorphine, a DA receptor agonist. Administration of ginseng total saponin (GTS) prior to and during the nicotine treatment in mice inhibited not only nicotine-induced hyperactivity and CPP but also postsynaptic DA receptor supersensitivity in nicotine-induced CPP mice. These results suggest that inhibition by GTS of the nicotine-induced hyperactivity and CPP may be closely related with the inhibition of dopaminergic activation induced by nicotine and that the development of nicotine-induced CPP may be associated with the enhanced DA receptor supersensitivity. From these results, it is presumed that GTS may be useful for the prevention and therapy of these adverse actions of nicotine.

Cytotoxic activities of various fractions extracted from some pharmaceutical insect relatives.

This research was performed to screen the cytotoxic activities of some pharmaceutical insect relatives. Cytotoxic activities of total extract and fractions of hexane, ethyl acetate, methanol, water and boiling water were extracted from four pharmaceutical insect relatives: the Chinese gall, the cicada slough, the hornet nest and the batryticated silkworm. These extracts were investigated against the cancer cell lines of L1210, P388 and SNU-1in vitro tests. Results showed that, ED(50) against the cancer cell lines of L1210, P388 and SNU-1 were 0.55, 0.50, and 0.83 mug/ml in the ethyl acetate fraction from the Chinese gall; 1.07, 2.19, and 2.24 mug/ml in the ethyl acetate fraction, 1.51, 1.26, and 1.45 mug/ml in the water fraction and 1.48, 2.29, and 1.29 mug/ml in the boiling water fraction from the cicada slough; 3.31, 2.00, and 6.61 mug/ml in the water fraction from the hornet nest and 13.80, 19.95, and 31.62 mug/ml in the hexane fraction and 33.88, 21.88, and 25.12 mug/ml in the ethyl acetate fraction from the batryticated silkworm, respectively. All of the fractions metioned above showed high cytotoxic activities and could be suggested for further studiesin vivo tests.