Visualization of live endothelial cells ex vivo and in vitro

The present study describes quick and effective methods that allow visualization of the vascular endothelium in living networks within dissected pieces of human tissue or in primary cultures containing heterogeneous cell populations. Fresh human uterine and subcutaneous gluteal fat tissues were directly labelled using fluorescently conjugated Ulex Europaeus Agglutinin I (UEA-1) to visualize the three-dimensional nature of the vascular network. Using conventional epi-illuminescence microscopy, the convoluted architecture demonstrating branch points within capillaries, between capillaries and larger vessels, were clearly observed in uterine and subcutaneous gluteal fat samples. In adult endometrial tissue where angiogenesis occurs on a monthly basis, complex anastamosis of vessels and tenuous structures were clearly seen. Three-dimensional rendered surface models formed by examination of confocal z-stacks demonstrated the existence of the lumen within microvessels. Tissue prelabelled with UEA-1 was used to assist and verify the presence of endothelial cells in culture, during and after the isolation procedure. Additionally, UEA-1 was added to a uterine fibroblast-microvascular-endothelial cell coculture model to allow daily vital observations of changes in the phenotype of the endothelium. The simple techniques described here demonstrate the ease with which fluorescently labelled UEA-1 can be used as a vital marker of endothelial cells either in tissue or in a tube-forming human uterine microvascular culture model.     

Evidence for metabolic and endocrine abnormalities in subjects recovered from anorexia nervosa

Subjects with anorexia nervosa (AN) at low weight display metabolic, endocrine, and behavioral abnormalities. Whether these various differences are a consequence of the condition and persist after recovery is unclear. We tested the hypothesis that abnormalities in the insulin and leptin axes and in the desire to eat persisted in subjects who had recovered from AN in terms of body mass index (BMI) and menstrual function. Endocrine, metabolic, and psychological parameters were assessed by sampling under fasting conditions and serially in response to a standard meal. Subjects included 18 females recovered from AN and 18 female controls and measures included plasma insulin, leptin, glucose and beta-hydroxybutyrate (beta-HBA) concentrations together with desire to eat. Fasting glucose concentrations were normal in both groups, but fasting insulin concentrations were significantly lower and the fasting glucose/insulin ratio significantly higher in the recovered subjects. The glucose concentration was significantly higher at the end of the meal period in the recovered group. The peak increase of insulin during the meal was significantly less in the recovered group and in response to the meal, glucose/insulin ratios were significantly higher for the first 45 minutes indicating a delayed insulin response. Fasting beta-HBA concentrations were not significantly different between groups, but postmeal decreases were significant and larger in the recovered AN group. Fasting and meal-related leptin concentrations were not significantly different between the groups and in both groups were correlated with BMI. In controls, but not in recovered subjects, the reported desire to eat was correlated with plasma glucose and leptin concentrations. The insulin, glucose and beta-HBA data indicated the presence of insulin hypersensitivity in the recovered subjects. As the insulin response to the meal was blunted and apparently delayed, there may be a persistent alteration in pancreatic function as a long-term pathological consequence of the anorexia. Alternatively, these data indicate a possible trait marker for AN.     

Human cerebrospinal fluid central memory CD4+ T cells: evidence for trafficking through choroid plexus and meninges via P-selectin

Cerebrospinal fluid (CSF) from healthy individuals contains between 1,000 and 3,000 leukocytes per ml. Little is known about trafficking patterns of leukocytes between the systemic circulation and the noninflamed CNS. In the current study, we characterized the surface phenotype of CSF cells and defined the expression of selected adhesion molecules on vasculature in the choroid plexus, the subarachnoid space surrounding the cerebral cortex, and the cerebral parenchyma. Using multicolor flow cytometry, we found that CSF cells predominantly consisted of CD4+/CD45RA-/CD27+/CD69+-activated central memory T cells expressing high levels of CCR7 and L-selectin. CD3+ T cells were present in the choroid plexus stroma in autopsy CNS tissue sections from individuals who died without known neurological disorders. P- and E-selectin immunoreactivity was detected in large venules in the choroid plexus and subarachnoid space, but not in parenchymal microvessels. CD4+ T cells in the CSF expressed high levels of P-selectin glycoprotein ligand 1, and a subpopulation of circulating CD4+ T cells displayed P-selectin binding activity. Intercellular adhesion molecule 1, but not vascular cell adhesion molecule 1 or mucosal addressin cell adhesion molecule 1, was expressed in choroid plexus and subarachnoid space vessels. Based on these findings, we propose that T cells are recruited to the CSF through interactions between P-selectin/P-selectin ligands and intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 in choroid plexus and subarachnoid space venules. These results support the overall hypothesis that activated memory T cells enter CSF directly from the systemic circulation and monitor the subarachnoid space, retaining the capacity to either initiate local immune reactions or return to secondary lymphoid organs.     

Prevention of radiocontrast-induced nephropathy with N-acetylcysteine in patients undergoing coronary angiography

OBJECTIVES: Acetylcysteine in patients undergoing computerized tomography with intravenous contrast reduces the incidence of acute renal dysfunction. We examined the effect of N-acetylcysteine in patients undergoing coronary angiography. METHODS: Fifty-five consecutive patients receiving 3 doses of N-acetylcysteine prior to cardiac catheterization were compared to 55 historical controls. All patients in both groups had baseline serum creatinine > 1.2 mg/dl and received intravenous hydration before and after the procedure. Serum creatinine levels at baseline and 48 hours after the procedure were compared. RESULTS: Univariate analysis of clinical variables revealed no significant differences between the groups except for a higher baseline creatinine in the treatment group (2.0 0.7 vs. 1.8 0.4 mg/dl; p = 0.04). There was no difference in the amount or type of contrast used. The mean change in creatinine after 48 hours was -0.4 0.3 versus +0.1 0.3 mg/dl for treatment and control groups (p < 0.001). In patients with baseline creatinine > 2 mg/dl, the benefit was larger (-0.4 0.4 vs. +0.5 0.3 mg/dl; p < 0.001). Multivariate analysis confirmed pre-treatment with N-acetylcysteine as an independent predictor of renal protection (p < 0.001). CONCLUSIONS: Prophylactic use of acetylcysteine prevented reduction of renal function after coronary angiography. The benefit was greater in patients with baseline serum creatinine > 2 mg/dl.     

Comparison of desipramine or carbamazepine to placebo for crack cocaine-dependent patients

The authors compared the effects of desipramine or carbamazepine to placebo in an intensive outpatient program for cocaine abuse. Subjects recruited from an urban drug treatment program were randomly assigned to a double-blind, placebo-controlled, eight-week trial of desipramine, carbamazepine, or placebo. Patient ratings, urine drug screens, and blood samples were obtained weekly. Using survival analysis, the three groups did not differ in time to drop out of treatment. While subjects improved over time on all self-ratings related to cocaine use, mood, and craving, only two items related to mood were significantly different over time as a function of treatment group. Subjects in the two treated groups reported significantly more improvement on self-ratings of depression and irritability. No treatment differences were noted for sustained abstinence or for proportion of positive urine drug screens. Desipramine subjects who attained a minimum blood level were retained in treatment significantly longer than placebo or other non-compliant treatment groups. This finding supports previous reports of a possible role for desipramine in cocaine abuse treatment.     

Fibroblast growth factor receptor-1 and neonatal compensatory lung growth after exposure to 95% oxygen

Neonatal rats exposed to 95% oxygen (O2) for 7 days from birth had inhibited lung growth, DNA synthesis, and secondary septation. These parameters were rapidly restored by a period of recovery in air. Northern and Western blot analysis and immunohistochemistry were used to screen for the fibroblast growth factor receptor-1 (FGF-R1) and its high affinity ligand, basic fibroblast growth factor (bFGF), which could have a role in this recovery process. Expression of bFGF in the lung was significantly reduced at the end of the 7-day exposure to 95% O2 and was increased after 3 days of recovery in air. Expression of FGF-R1 was not affected by exposure to 95% O2 or recovery in air. We hypothesized that the increase in bFGF after removal from 95% O2, acting through the FGF-R1, would be critical for compensatory growth. Intraperitoneal injection of soluble truncated FGF-R1 at the onset of the recovery phase arrested compensatory lung DNA synthesis and secondary septation seen in control animals after 3 days of recovery, confirming a role for FGF-R1 in this model of compensatory neonatal lung growth.     

The obesity epidemic: can we turn the tide?

Obesity has reached epidemic proportions in the UK. It is important because of the associated co-morbidities, which include cardiovascular disease, type 2 diabetes, and osteoarthritis. The prevalence of obesity has increased because of a combination of excessive calorific intake (for example, from increased intake of energy dense foods) and insufficient energy expenditure (associated with a sedentary lifestyle). Weight loss of 5-10%, which can be achieved in primary care, is associated with significant health benefits. Obesity treatment in primary care includes lifestyle modification and drug treatment. The prevention and treatment of obesity cannot, however, be left solely to health professionals. Action is needed by government, the food industry, and society as a whole.     

Neonatal 3,4-methylenedioxymethamphetamine (ecstasy) alters dopamine and serotonin neurochemistry and increases brain-derived neurotrophic factor in the forebrain and brainstem of the rat

Growing concerns surround the risk of fetal exposure to 3,4-methylenedioxymethamphetamine (MDMA; ecstasy). Prior animal studies using neonatal rats administered MDMA from postnatal days (P) 11-20 (a period approximating third trimester brain development in humans) have demonstrated long-lasting decrements in serotonin (5-HT) and learning; however, no studies have examined the acute post-MDMA response of the brain at this early age. Specifically, it is of interest whether MDMA administration to neonatal rats produces the expected depletion of monoamines and whether the brain exhibits any ameliorative response to the pharmacologic insult. In the current study, this model was employed to determine whether forebrain and brainstem dopamine (DA) and 5-HT neurochemistry were altered 24 h after the last injection (P21), and whether brain-derived neurotrophic factor (BDNF) was upregulated in response to MDMA exposure. All forebrain structures examined (frontal cortex, hippocampus, and striatum) showed significant MDMA-induced reductions in 5-HT and its metabolite, 5-HIAA, and significant increases in the DA metabolite, HVA, as well as DA turnover (HVA/DA). In the brainstem, there were significant increases in 5-HIAA, HVA and DA turnover. BDNF was significantly increased (19-38%) in all forebrain structures and in the brainstem in MDMA-exposed neonates versus saline controls. These data suggest that MDMA exposure to the developing rat brain from P11-20 produces similar alterations in serotonin and dopamine neurochemistry to those observed from adult administrations. In addition, a compensatory increase in BDNF was observed and may be the brains ameliorative response to minimize MDMA effects. This is the first report demonstrating that MDMA exposure results in increased levels of BDNF and that such increases are correlated with changes in monoamine levels. Future research is needed to elucidate any deleterious effects MDMA-induced increases in trophic activity might have on the developing brain and to examine earlier gestational exposure periods in order to assess the risk throughout pregnancy.     

Is mycophenolate mofetil less safe than azathioprine in elderly renal transplant recipients?

BACKGROUND: Mycophenolate mofetil (MMF) is a potent immunosuppressive agent that has been shown to be superior to azathioprine in preventing early acute rejection in the general renal transplant population. However, it is uncertain whether these benefits also apply to older renal transplant recipients, who are known to be more susceptible to infectious complications and have considerably lower rates of rejection and immunological graft loss. METHODS: A retrospective analysis was undertaken of all elderly (> or =55 years old) renal transplant recipients who underwent renal transplantation at the Princess Alexandra Hospital (1994-2000) and received either MMF (n=60) or azathioprine (n=55) in combination with prednisolone and cyclosporin. Data were analyzed on an intention-to-treat basis using a multivariate Cox proportional hazards model. RESULTS: The azathioprine- and MMF-treated groups were well matched at baseline with respect to demographic characteristics, end-stage renal failure causes and transplant characteristics. Compared with the MMF cohort, azathioprine-treated patients experienced a shorter time to first rejection [hazard ratio (HR) 4.47, 95% CI 1.53-13.1, P<0.01]. However, azathioprine-treated patients were also less likely to develop opportunistic infections (HR 0.11, 95% CI 0.03-0.41, P=0.001). No differences were observed between the two groups with respect to hospitalization rates, intensive care admissions, hematological complications, or posttransplant malignancies. Actuarial 2-year survival rates for the azathioprine- and MMF-treated patients were 100 and 87%, respectively (P<0.001). The principal cause of death in the MMF cohort was infection. Using a multivariate Cox regression analysis of patient survival, an adjusted hazard ratio of 0.01 (95% CI 0.001-0.08, P=0.001) was calculated in favor of azathioprine. Overall graft survival also tended to be better in patients receiving azathioprine (HR 0.27, 95% CI 0.06-1.33, P=0.11), CONCLUSIONS: In elderly renal transplant recipients, the combination of MMF, cyclosporin, and prednisolone appears to result in a worse outcome compared with the less potent combination of azathioprine, cyclosporin, and prednisolone. Future prospective studies need to specifically evaluate the risk/benefit ratios of newer, more potent immunosuppressive protocols, such as MMF-based regimens, in this important and sizeable patient subgroup.