Small dense LDL cholesterol in human subjects with different chronic inflammatory diseases.

BACKGROUND AND AIMS: Chronic inflammatory diseases (CID) are associated with a profound increase in cardiovascular (CV) risk resulting in reduced life expectancy. However, LDL-cholesterol is reported to be low in CID patients which is referred to as the "LDL paradoxon". The aim of the present study was to investigate whether LDL-particles in CID exhibit an increased content of the highly atherogenic small-dense LDL subfraction (sdLDL). METHODS AND RESULTS: In this prospective, single center, observational study we enrolled 141 patients with CID (RA n = 59, inflammatory bowel disease (IBD) n = 35, ankylosing spondylitis (SpA) n = 25, Psoriasis n = 22) in 2011 through 2013 to evaluate sdLDL levels before as well as 6 and 26 weeks after initiation of different anti-cytokine therapies (anti-TNFα, anti-IL-6R antibodies). sdLDL levels were compared to 141 healthy individuals in a case control design. Compared to healthy controls, all CID patients displayed a significantly higher sdLDL content within the LDL cholesterol fraction: RA 35.0 ± 9.2% (p < 0.001), SpA 42.5 ± 10.5% (p < 0.001), IBD 37.5 ± 7.1% (p < 0.001), Psoriasis 33.6 ± 4.6% (p < 0.01). Furthermore, the sdLDL/LDL ratio was significantly higher in male compared to female RA subjects (p < 0.05). Neither anti-TNFα nor anti-IL6R medication altered sdLDL levels despite a significant improvement of disease activity. CONCLUSION: In several different chronic inflammatory disease entities, LDL-cholesterol is shifted toward a pro-atherogenic phenotype due to an increased sdLDL content which might in part explain the LDL paradoxon. Since premature CV disease is a major burden of affected patients, specifically targeting lipid metabolism should be considered routinely in clinical patient care. CLINICAL TRIALS: Registration at German Clinical Trial Register (DRKS): DRKS00005285.

The wingless-related integration site-5a/secreted frizzled-related protein-5 system is dysregulated in human sepsis.

Sepsis and type 2 diabetes exhibit insulin resistance as a common phenotype. In type 2 diabetes we and others have recently provided evidence that alterations of the proinflammatory wingless-related integration site (wnt)-5a/anti-inflammatory secreted frizzled-related protein (sFRP)-5 system are involved in the pathogenesis of insulin resistance. The aim of the present study was to investigate whether this novel cytokine system is dysregulated in human sepsis, which may indicate a potential mechanism linking inflammation to metabolism. In this single-centre prospective observational study, critically ill adult septic patients were examined and proinflammatory wnt5a and wnt5a inhibitor sFRP5 were measured in serum samples by enzyme-linked immunosorbent assay (ELISA) at admission to the intensive care unit (ICU) and 5 days later. Sixty sepsis patients were included, and 30 healthy individuals served as controls. Wnt5a levels were found to be increased significantly in septic patients compared to healthy controls (2·21 ± 0·33 versus 0·32 ± 0·03 ng/ml, P < 0·0001). In contrast, sFRP5 was not altered significantly in septic patients (19·72 ± 3·06 versus 17·48 ± 6·38 ng/ml, P = 0·07). On admission to the ICU, wnt5a levels exhibited a significant positive correlation with the leucocyte count (rs = 0·3797, P = 0·004). Interestingly, in patients recovering from sepsis, wnt5a levels declined significantly within 5 days (2·17 ± 0·38-1·03 ± 0·28 ng/ml, P < 0·01). In contrast, if sepsis was worsening, wnt5a levels increased in the same time-period by trend (2·34 ± 0·59-3·25 ± 1·02 ng/ml, P > 0·05). sFRP5 levels did not change significantly throughout the study period. The wnt5a/sFRP5 system is altered in human sepsis and might therefore be of interest for future studies on molecular pathophysiology of this common human disease.

An Internet-based program to enhance motivation to change in females with symptoms of an eating disorder: a randomized controlled trial.

BACKGROUND: Previous research has demonstrated an association between low motivation to change and an unfavorable treatment outcome in patients with an eating disorder. Consequently, various studies have examined the effects of motivational enhancement therapy (MET) on motivation to change and treatment outcome in eating disorders. In each of these studies, MET was administered in a face-to-face setting. However, because of its anonymity and ease of access, the internet provides several advantages as the format for such an intervention. Therefore, the current study investigated the effects of an internet-based program ('ESS-KIMO') to enhance motivation to change in eating disorders. METHOD: In total, 212 females were accepted for participation and assigned randomly to the intervention condition (n = 103) or waiting-list control condition (n = 109). The intervention consisted of six online MET sessions. Before and after the intervention or waiting period respectively, participants completed the Eating Disorder Examination Questionnaire (EDE-Q), the Stages of Change Questionnaire for Eating Disorders (SOCQ-ED), the Pros and Cons of Eating Disorders Scale (P-CED), the Self-Efficacy Scale (SES), and the Rosenberg Self-Esteem Scale (RSES). A total of 125 participants completed the assessment post-treatment. Completer analyses and intent-to-treat analyses were performed. RESULTS: Significant time × group interactions were found, indicating a stronger increase in motivational aspects and self-esteem, in addition to a stronger symptom reduction on some measures from pre- to post-treatment in the intervention group compared to the control group. CONCLUSIONS: Internet-based approaches can be considered as useful for enhancing motivation to change in eating disorders and for yielding initial symptomatic improvement.

Caloric restriction increases serum testosterone concentrations in obese male subjects by two distinct mechanisms.

The concentration of serum testosterone is mainly regulated by the testicular function, which is under control of the central hypothalamic-pituitary-gonadal axis. A certain amount of testosterone is converted into ß-estradiol by adipose tissue. Obesity in men is often associated with decreased androgen levels. The aim of the present study was to examine the effect of caloric restriction on serum testosterone levels in obese men. Dietary intervention study was performed with a very low calorie diet (800 kcal/d) for 12 weeks. Thirteen obese human male subjects (median body mass index: 42.7 kg/m2) were included. Body composition was assessed by impedance analysis. Insulin sensitivity was estimated by leptin-to-adiponectin ratio (LAR). Testosterone (T), ß-estradiol, albumin, sex hormone-binding globulin (SHBG), LH, and FSH serum concentrations were measured by enzyme immunoassays. Statistical analysis was performed on baseline and values after 3 months. Caloric restriction significantly increased total testosterone (6.97 nmol/l to 13.21 nmol/l; p=0.001) and SHBG (22.11 nmol/l to 42.12 nmol/l; p=0.001) concentrations in serum. This is caused by a significant improvement of the testicular function (LH/T: 0.36-0.20; p=0.005) and a significant reduction of the T/ß-estradiol conversion rate (73.59-104.29; p=0.003). There was a significant negative correlation of improvement of testicular function and LAR (rs=-0.683 (p=0.042)). In obese men caloric restriction significantly increases the serum testosterone concentration. This is achieved by 2 distinct mechanisms, that is, improvement of testicular function and reduced conversion of testosterone to ß-estradiol by aromatase activity of the adipose tissue.

Weight loss due to a very low calorie diet differentially affects insulin sensitivity and interleukin-6 serum levels in nondiabetic obese human subjects.

Inflammatory mechanisms are involved in the pathogenesis of type 2 diabetes with interleukin (IL)-6 being particularly important. While long term exercise has been shown to be associated with reduction in IL-6 serum levels in several reports, the discussion on the effect of dietary intervention on IL-6 serum levels is controversial. In the present study, we aimed to investigate the effect of weight loss due to a very low calorie diet (VLCD) on insulin sensitivity and IL-6 serum levels in nondiabetic obese human individuals. 10 patients with obesity were examined during 12 weeks of a VLCD (800 kcal/d). Body composition was measured by impedance analysis. Blood samples were taken before, during, and after the dietary intervention. Leptin, adiponectin, and IL-6 serum levels were measured by ELISA. The body weight decreased significantly from 123.9±6.2-103.5±5.6 kg with a significant reduction in body fat content (43.2±2.3-36.1±3.1%). Leptin levels exhibited a significant decrease from 56.8±5.6-27.9±5.6 ng/ml while adiponectin levels increased significantly from 7.5±0.9-10.6±1.1 µg/ml. Thereby the leptin-to-adiponectin ratio, a novel marker for insulin sensitivity, significantly improved. Mean IL-6 serum concentrations were within the normal range (3.2±0.8 pg/ml) before the study and were not significantly altered by the nutritional therapy. Despite improvement of insulin sensitivity, IL-6 serum levels did not change throughout the study period, suggesting that in nondiabetic obese human subjects IL-6 might have only a minor role in the impairment of insulin sensitivity.

Adipose tissue macrophages inhibit adipogenesis of mesenchymal precursor cells via wnt-5a in humans.

In patients with obesity and type 2 diabetes, adipose tissue is infiltrated by macrophages known to alter adipogenesis of mesenchymal precursor cells via secretion of proinflammatory cytokines. Recently, it has been shown that under certain conditions, immune cells can also express wnt-5a, a factor known to inhibit adipogenesis in humans. Therefore, in this study we aimed to investigate whether macrophages affect adipogenesis of mesenchymal precursor cells via wnt-5a. Wnt-5a was found to be expressed in adipose tissue macrophages in obese and type 2 diabetic human subjects in vivo by immunohistochemistry of adipose tissue biopsies. Furthermore, wnt-5a was detectable in circulating CD14(+) blood monocytes of human subjects with obesity and type 2 diabetes on RNA level by real-time PCR. Besides expression analysis in vivo, we also performed functional studies to explore the role of wnt-5a in low-grade inflammation of adipose tissue. In a cell culture experiment, macrophage-conditioned differentiation medium inhibited adipogenesis of 3T3-L1 cells. This inhibitory effect was restored by adding neutralising anti-wnt-5a antibodies. In conclusion, our data indicate that macrophages alter adipogenesis of 3T3-L1 cells not only via classical proinflammatory cytokines, but also via wnt signalling molecules.

Changes in neuronal correlates of body image processing by means of cognitive-behavioural body image therapy for eating disorders: a randomized controlled fMRI study.

BACKGROUND: Previous neuroimaging studies have demonstrated abnormalities in visual body image processing in anorexia and bulimia nervosa, possibly underlying body image disturbance in these disorders. Although cognitive behavioural interventions have been shown to be successful in improving body image disturbance in eating disorders, no randomized controlled study has yet analysed treatment-induced changes in neuronal correlates of visual body image processing. METHOD: Altogether, 32 females with eating disorders were randomly assigned either to a manualized cognitive behavioural body image therapy consisting of 10 group sessions, or to a waiting list control condition. Using functional magnetic resonance imaging, brain responses to viewing photographs of one's own and another female's body taken from 16 standardized perspectives while participants were wearing a uniform bikini were acquired before and after the intervention and the waiting time, respectively. RESULTS: Data indicate a general blood oxygen level dependent signal enhancement in response to looking at photographs of one's own body from pre- to post-treatment, whereas exclusively in the control group activation decreases from pre- to post-waiting time were observed. Focused activation increases from pre- to post-treatment were found in the left middle temporal gyrus covering the coordinates of the extrastriate body area and in bilateral frontal structures including the middle frontal gyrus. CONCLUSIONS: Results point to a more intense neuronal processing of one's own body after the cognitive behavioural body image therapy in cortical regions that are responsible for the visual processing of the human body and for self-awareness.

The whey fermentation product malleable protein matrix decreases triglyceride concentrations in subjects with hypercholesterolemia: a randomized placebo-controlled trial.

Malleable protein matrix (MPM) is a unique whey-derived ingredient obtained through a fermentation process using proprietary lactic acid bacteria strains from the Lactobacillus kefiranofaciens species. Because evidence from animal models suggests that MPM decreases serum lipid concentrations, the purpose of the present trial was to assess the hypothesis that MPM exerts lipid-lowering effects in humans. A total of 161 subjects (50% male; age 54.5 ± 9.8 yr, body mass index 26.3 ± 3.6 kg/m(2)) with hypercholesterolemia with baseline low-density lipoprotein cholesterol (LDL-C) levels of 181 ± 30 mg/dL and normal triglyceride (TG) levels (131 ± 55 mg/dL) were randomized to receive MPM (2 × 15 g/d) or matching placebo. A 6-wk run-in phase was followed by a double-blind 12-wk treatment phase after randomization. The data were analyzed on an intention-to-treat basis. The primary outcome measure was the percentage change of LDL-C. The secondary outcome measures were changes in TG and high-density lipoprotein cholesterol concentrations as well as changes in other cardiovascular risk factors. After 12 wk of treatment, the relative TG decrease from baseline reached 9.8%, whereas LDL-C was slightly decreased (by 1.5%) following MPM treatment compared with placebo in the intention-to-treat cohort. The treatment effect on TG reduction was much higher in the subset of subjects having TG levels at baseline of 150 mg/dL or above (n=42), reaching 20.0% compared with placebo. High-density lipoprotein cholesterol concentrations, blood pressure, and fasting blood glucose remained unchanged, whereas a positive treatment effect was seen on hemoglobin A(1c). The MPM product was tolerated well without severe adverse events. In conclusion, MPM has significant TG-lowering properties in subjects with combined hypercholesterolemia and higher TG levels. Its effects on LDL-C concentrations and glucose metabolism deserve further investigation.

Visfatin/PBEF/Nampt and resistin expressions in circulating blood monocytes are differentially related to obesity and type 2 diabetes in humans.

Low-grade inflammation is important in the development of obesity related pathologies such as insulin resistance and type 2 diabetes, and also cardiovascular disease. Visfatin/PBEF/Nampt and resistin are proinflammatory adipokines secreted from adipocytes, monocytes, and macrophages, and have been linked to atherosclerotic plaque formation, recently. The aim of the present study was to investigate if the expression of these molecules in circulating blood monocytes is altered in obese and/or type 2 diabetic human subjects. Monocytes were isolated by CD14-antibody based magnetic cell sorting from blood samples of 17 lean controls, 20 obese nondiabetic subjects, and 19 obese patients with type 2 diabetes. FACS analysis was performed to test purity of the cell preparations. Expression of the different adipokines was measured by multiplex real-time PCR on RNA-level. Visfatin/PBEF/Nampt was found to be very strongly expressed in monocytes, whereas resistin levels were significantly lower. Furthermore, visfatin/PBEF/Nampt expression was significantly upregulated in obese type 2 diabetic patients, whereas obese nondiabetics exhibited similar levels compared to lean controls, indicating that visfatin/PBEF/Nampt levels are related to type 2 diabetes rather than to obesity. In contrast, resistin expression displayed a different pattern being significantly increased in obese subjects compared to controls but not related to type 2 diabetes. These data suggest a differential role for these two proinflammatory adipokines in linking metabolic diseases to atherosclerosis with visfatin/PBEF/Nampt being more important in patients with type 2 diabetes and resistin in obese but nondiabetic human subjects.

Magnetic nanoparticles encapsulated within a thermoresponsive polymer.

This study describes a facile two-step approach to modify the surface of nanoparticles, thereby imparting a core-shell structure to the system. The core consists of magnetic nanoparticles and the shell is composed of thermoresponsive hydroxypropyl cellulose, using a coupling agent to covalently bind the core to the shell. Hydroxypropyl cellulose is known for its biocompatibility and biodegradability, and its thermoresponsive properties make it an excellent candidate for fabricating biocompatible stimuli-responsive magnetic nanoparticles. We report the synthesis of magnetic nanoparticles and the successful binding of the polymer to them. X-ray diffraction studies show that the surface modification of the magnetic nanoparticles does not result in any phase change and the size of the magnetic core thus calculated (7 nm) reveals that such hybrid core-shell system is superparamagnetic in nature, as further confirmed by magnetization measurements. The size obtained by X-ray diffraction is in good agreement with that obtained by transmission electron microscope. Evidence of binding is given by Fourier transform infrared spectroscopy and a quantitative analysis of the polymeric content obtained by thermogravimetry analysis. Dynamic light scattering as a function of temperature reveals the thermoresponsive behavior of the particles with a lower critical solution temperature around 41 degrees C, which is also the temperature at which cellulose undergoes a coil-to-globule transition.

[Surveillance of nosocomial infections in a long-term care facility. Incidence and risk factors]. / Surveillance nosokomialer Infektionen in einem Altenpflegeheim. Inzidenz und Risikofaktoren.

In Germany, surveillance of nosocomial infections is obligatory by law in hospitals, but not in long-term care facilities. In long-term care facilities, neither X-ray nor laboratory facilities are available; therefore, special criteria for infection surveillance in these facilities were developed by Mc Geer et al. (1991), based only on observations of the medical staff and foregoing laboratory methods. The practicability of these criteria was assessed in a long-term care facility with an electronic medical documentation system. In a retirement home in Frankfurt/Main, Germany, the residents' symptoms according to McGeer et al. were recorded from January, 1 to June 31, 2006. The study included 278 residents, 45,710 resident days, including 4413 "urinary catheter days" und 6009 "gastric tube days". Based on the symptoms documented in the electronic medical documentation system, the respective diagnoses were obtained. Data on gastrointestinal, bronchial, urinary, and eye infections are reported here, including number of cases as well as incidences per 1000 resident days. The overall incidence rate was 5.07/1000 resident days, including 1.9 gastrointestinal infections, 0.95 bronchitis/pneumonia, and 0.44 urinary infections. Incidence of urinary infections in residents with an indwelling urinary catheter was 2.26/1000 catheter days compared to 0.242/1000 days for those without a catheter. Urinary catheter, however, was not only a risk factor for urinary infections (OR 9.4, 95CI 3.4-25.8) but also for bronchial infections (OR 3.0, 95CI 1.3-6.8), and eye infections (OR 1.6, 95CI 0.4-5.8). Gastric tubes were associated with bronchial infections (OR 3.7; CI 1.7-7.9), eye infections (OR 5.4, CI 1.9-15.2), and urinary infections (OR 2.7, 95CI 0.9-8.2). Urinary or fecal incontinence were not associated with any infections, and age (>80 years) was negatively associated with urinary infections (OR 0.35, significant) and bronchial infections (OR 0.51, significant). Compared with published studies, the incidence rates were comparable. The practicability of the criteria of McGeer et al. could be confirmed - with only slight modification - in a long-term care facility with electronic medical documentation.

[Marginal protection of retinal cells by bisperoxovanadium : Appropriate therapy in the model of retinal ischemia?] / Marginale Protektion retinaler Zellen durch Bisperoxovanadium : Geeignete Therapie im Modell der retinalen Ischämie?

BACKGROUND: Ischemic processes usually lead to the destruction of retinal cells and therefore play a key role in a multitude of eye diseases. OBJECTIVE: The aim of this study was to investigate whether bisperoxovanadium has a potential neuroprotective effect in an ischemia/reperfusion animal model. MATERIAL AND METHODS: Initially, ischemia was induced in one eye of an ischemia/reperfusion model and 3 days later, a 14-day medication-based treatment was initiated. Bisperoxovanadium was administered intraperitoneally every 3 days. Subsequently, the number of ganglion cells, the rate of apoptosis, amacrine cells, macroglia, microglia, and their activation state, as well as photoreceptors were determined by histological and immunohistochemical analyses. RESULTS: In comparison to the control group, a significant retinal ganglion cell loss, a significant reduction of the inner layers as well as a decrease in photoreceptor and amacrine cell numbers could be determined in the ischemic eyes. In addition, there was an increase in the number of microglia in these animals. The rats treated with bisperoxovanadium did not exhibit a significant neuroprotective effect regarding the number of ganglion cells, the rate of apoptosis, macroglia, amacrine cells, or photoreceptors; however, a low structural degeneration of photoreceptors could be observed as an effect of the treatment. Additionally, fewer microglia and activated microglia were observed after bisperoxovanadium treatment. CONCLUSION: Bisperoxovanadium seems to have only a marginal neuroprotective effect on ischemic retinae. It needs to be examined whether earlier therapy onset, higher dose or different route of administration would significantly improve the results or whether this therapeutic approach is unsuitable.

Misexpression of the Emx-related homeobox genes cVax and mVax2 ventralizes the retina and perturbs the retinotectal map.

The mechanisms that establish the dorsal-ventral (D-V) axis of the eye are poorly understood. We isolated two homeobox genes from mouse and chicken, mVax2 and cVax, whose expression during early eye development is restricted to the ventral retina. In chick, ectopic expression of either Vax leads to ventralization of the early retina, as assayed by expression of the transcription factors Pax2 and Tbx5, and the Eph family members EphB2, EphB3, ephrinB1, and ephrinB2, all of which are normally dorsally or ventrally restricted. Moreover, the projections of dorsal but not ventral ganglion cell axons onto the optic tectum showed profound targeting errors following cVax misexpression. mVax2/cVax thus specify positional identity along the D-V axis of the retina and influence retinotectal mapping.

[First results with catheter and magnetically guided and detached polymerized ferromagnetic particle filaments and heat-induced particle release using the Stereotaxis Niobe system]. / Erste In-vitro-Anwendung katheter- und magnetgesteuerter ferromagnetischer Polymerfilamente aus Nanopartikeln mit hitzeinduzierter Partikelabgabe unter Einsatz des Stereotaxis-Niobe-Systems.

PURPOSE: To develop a new technique for intravascular guidance and the release of magnetized ferromagnetic nanoparticles using a polymerized filament by means of an external magnetic field. MATERIALS AND METHODS: Ferromagnetic nanoscaled beads were embedded in temperature-sensitive gels to form filaments after polymerization. Deflection of the filaments was assessed in a Stereotaxis Niobe magnetic navigation system (MNS) in comparison with dedicated guide wires. The curvature was measured as a surrogate parameter for deflection. In combination with commercially available catheters, the filaments were navigated in a perfused aneurysmatic vessel model and a perfused branched vessel model under the influence of two permanent magnets of the Niobe MNS. The magnetic field vector was varied in all three dimensions. After positioning, the magnetic colloid-containing filaments were exposed to an electromagnetic field of 45 kA/m, 200 kHz for a period of 5 minutes for non-invasive heating. RESULTS: The filaments showed superior deflectability compared to the dedicated guide wires (p = 0.0091). The curvature was 0.54 +/- 0.12 mm(-1) for the filaments and 0.33 +/- 0.21 mm(-1) for the guide wires. In combination with angiography catheters, magnetic guidance and accumulation of specially designed filaments were possible in the perfused vessel model. Inductive heating allowed non-invasive disintegration and releasing of the nanoparticles in all filaments. CONCLUSION: This feasibility study shows that magnetic guidance and targeting of a specially designed magnetic colloid-containing filament and subsequent disintegration are feasible. This technique offers the potential for controlled local drug release.

Neutrophil cathepsin G is specifically decreased under vitamin A deficiency.

Vitamin A deficiency leads to an increased susceptibility to infections, increased severity of infections and increased mortality. Because the neutrophil is the first cell to respond to infection, this study explores the effect of vitamin A deficiency on neutrophil proteinases. We found that neutrophils from vitamin A-deficient rats had lower levels of two cathepsin G-like enzymes (28 and 24 kDa) when compared to neutrophils from weight-matched pair-fed rats, vitamin A-deficient rats which were repleted with retinyl palmitate and nonrestricted vitamin A complete diet rats. The 28 kDa cathepsin G-like enzyme, which migrated with the same mobility as elastase on SDS-polyacrylamide gels, was quantified using Western blots. The 24 kDa cathepsin G-like enzyme was quantified using zymogram gels. This activity was inhibited by chymostatin. Other neutrophil proteinases, elastase, plasminogen activators and gelatinase, were not altered significantly by vitamin A deficiency. The low levels of cathepsin G may contribute to differences in the inflammatory process observed under vitamin A deficiency.

Vitamin B6 intoxication after inappropriate supplementation with micronutrients following bariatric surgery.

A 50-year-old Caucasian woman was admitted to our hospital with intermittent diarrhoea, emesis and increasingly brown-coloured skin, mainly the in light-exposed areas, after biliopancreatic diversion for obesity treatment. Differential diagnoses such as adrenal insufficiency were ruled out, but biochemical analysis demonstrated unusual high pyridoxine serum levels (vitamin B6). History revealed the intake of 300 mg of vitamin B6 per day over 6 months as described by her general practitioner. All symptoms disappeared after the discontinuation of vitamin B6 supplementation. Importantly, in contrast to many other vitamins and supplements, there is no evidence in the literature of the occurrence of vitamin B6 deficiency after bariatric surgery. Therefore, supplementation of vitamins and supplements in bariatric patients has to be carefully considered according to the existing clinical guidelines, as uncritical oversupplementation of micronutrients might result in intoxication and serious illness as presented here.

Cycloheximide inhibits cellular, but not SV40, DNA replication.

We have prepared extracts from cycloheximide-treated cells for the study of simian-virus-40 (SV40)-DNA replication in vitro. When supplemented with the viral initiator protein (large T antigen), these extracts fully supported SV40-DNA replication. We also determined that SV40-DNA replication in vivo is much more resistant to cycloheximide than cellular DNA replication. SV40 encodes its own initiator protein, T antigen, which also functions as a DNA helicase, but depends on cellular functions for all additional replication reactions. Therefore, it appears to be quite likely that cycloheximide affects cellular DNA replication by blocking the synthesis of (a) cellular function(s) that is(are) performed by T antigen in SV40-DNA replication. Indeed, DNA fiber autoradiography and alkaline sucrose gradient centrifugation of pulse-labeled cellular DNA showed that cycloheximide treatment almost completely suppressed replicon initiation and reduced the rate of replication fork movement to about one third of the control.

The effects of high- and low-intensity percutaneous stimulation on nitric oxide levels and spike activity in the superficial laminae of the spinal cord.

Nitric oxide (NO) was measured using a new electrochemical method with a carbon fibre microelectrode at depths of up to 400 microm in the lumbar dorsal horn of the anaesthetised rat. The method allowed extracellular spike recording from single units together with the electrochemical recording at the same electrode. Thirty-six cells with low threshold cutaneous (brush/touch) or wide dynamic range receptive fields (brush/touch plus pinch) were studied. Adequate stimulation of the receptive fields did not alter the extracellular NO level for any cells. Percutaneous needle electrodes inserted into the receptive fields were used to stimulate the cells electrically. Twenty-one cells were stimulated using 10 mA current with 0.05 ms duration (low intensity) pulses to stimulate predominantly A-fibre afferents. Single shock stimuli gave short latency spike responses but no change in nitric oxide level. Tetanic bursts of stimuli (400 stimuli at 50 Hz) generated a burst of spikes (spike count 548+/-42) and a transient increase in NO (2.61+/-0.11 microM NO). Nitric oxide synthesis inhibition with N(G)-nitro-L-arginine methyl ester (L-NAME) nearly abolished the stimulus-evoked increase in nitric oxide and increased the response of the cells (spike count 694+/-34). However, the inhibition of nitric oxide synthesis had no effect on the receptive fields. Fifteen cells were stimulated with shocks using 5 ms pulses (high intensity), to recruit C-fibre afferents into the input volley. This more intense stimulation increased the evoked NO release to 3.63+/-0.15 microM and the spike response to 647+/-54 in control conditions. Following L-NAME, the evoked NO release was reduced and the evoked spike response was significantly decreased. These results show that tetanic activity in afferent fibres increases NO synthesis in the dorsal horn and that inhibition of nitric oxide synthesis may be associated with a selective attenuation of the spike responses to C-fibre inputs. NO may be necessary to maintain proper function of C-fibre afferent synapses when they are subjected to sustained or tetanic inputs.

Effect of vitamin A deficiency on the early response to experimental Pseudomonas keratitis.

PURPOSE: Vitamin A-deficient humans and animals are more susceptible to infections than are healthy humans and animals. This study compares the early corneal response (within 24 hours) to an experimental Pseudomonas aeruginosa infection between vitamin A deficient and control rats. METHODS: Male WAG/Rij/MCW rats were fed either a vitamin A- deficient diet (A-) or the same diet with retinyl palmitate added back in a nonrestricted manner (N) or under pair-fed conditions (A+) to yield weight-matched rats. Some A-rats were repleted wih retinyl palmitate 16 days before being killed and then given free access to the retinyl palmitate-supplemented diet (R). Twenty-four hours before being killed, the corneas of anesthetized rats were scratched and P. aeruginosa organisms were applied to the corneal surface. The rats were killed using an overdose of sodium pentobarbital. Corneas were either processed for light and electron microscopic examination or extracted for proteinase and myeloperoxidase determination. Corneal myeloperoxidase concentrations relative to neutrophil myeloperoxidase concentrations were used to determine the number of neutrophils in the cornea. Zymography was used to study caseinases, gelatinases, and plasminogen activators. Reverse zymography was used to detect proteinase inhibitors. Similar results were noted at early, mid, and late weight plateau stages of vitamin A deficiency. RESULTS: Ulceration occurred within 24 hours when low numbers of P. aeruginosa (10(4) cpu) were applied topically onto scratched A- corneas, whereas no ulceration was observed in the A+, R, and N corneas. When higher numbers of P. aeruginosa (10(7)-10(8)) were applied to the scratched corneas, all corneas became ulcerated within 24 hours. The extent of ulceration in the control corneas was greater than that in A- corneas by a factor of two. Only the A- corneas contained inflammatory cells with unusual striated deposits in phagolysosomes. The total number of neutrophils in the cornea and the concentrations of caseinases, plasminogen activators, and gelatinases in the infected corneal extracts were similar; however, the concentrations of cysteine proteinase inhibitors were elevated under A- conditions. CONCLUSIONS: Vitamin A deficiency alters the response of the cornea to a P. aeruginosa infection during the first 24 hours. The alterations observed are probably due to multiple factors: an insufficient tear film for bacterial clearance and migration of neutrophils, epithelial keratinization, alterations in corneal wound healing, and changes in polymorphonuclear function.