A peptide inhibitor of C-jun N-terminal kinase modulates hepatic damage and the inflammatory response after hemorrhagic shock and resuscitation

Hemorrhage and resuscitation (H/R) leads to phosphorylation of mitogen-activated stress kinases, an event that is associated with organ damage. Recently, a specific, cell-penetrating, protease-resistant inhibitory peptide of the mitogen-activated protein kinase c-JUN N-terminal kinase (JNK) was developed (D-JNKI-1). Here, using this peptide, we tested if inhibition of JNK protects against organ damage after H/R. Male Sprague-Dawley rats were treated with D-JNKI-1 (11 mg/kg, i.p.) or vehicle. Thirty minutes later, rats were hemorrhaged for 1 h to a MAP of 30 to 35 mmHg and then resuscitated with 60% of the shed blood and twice the shed blood volume as Ringer lactate. Tissues were harvested 2 h later. ANOVA with Tukey post hoc analysis or Kruskal-Wallis ANOVA on ranks, P < 0.05, was considered significant. c-JUN N-terminal kinase inhibition decreased serum alanine aminotransferase activity as a marker of liver injury by 70%, serum creatine kinase activity by 67%, and serum lactate dehydrogenase activity by 60% as compared with vehicle treatment. The histological tissue damage observed was blunted after D-JNKI-1 pretreatment both for necrotic and apoptotic cell death. Hepatic leukocyte infiltration and serum IL-6 levels were largely diminished after D-JNKI-1 pretreatment. The extent of oxidative stress as evaluated by immunohistochemical detection of 4-hydroxynonenal was largely abrogated after JNK inhibition. After JNK inhibition, activation of cJUN after H/R was also reduced. Hemorrhage and resuscitation induces a systemic inflammatory response and leads to end-organ damage. These changes are mediated, at least in part, by JNK. Therefore, JNK inhibition deserves further evaluation as a potential treatment option in patients after resuscitated blood loss.

Direct analysis of peptide binding to cell-associated MHC class I molecules.

Exogenously added synthetic peptides can mimic endogenously produced antigenic peptides recognized on target cells by MHC class I-restricted cytolytic T lymphocytes. While it is assumed that exogenous peptides associate with class I molecules on the target cell surface, direct binding of peptides to cell-associated class I molecules has been difficult to demonstrate. Using a newly developed binding assay based on photoaffinity labeling, we have investigated the interaction of two antigenic peptides, known to be recognized in the context of H-2Kd or H-2Db, respectively, with 20 distinct class I alleles on living cells. None of the class I alleles tested, with the exception of H-2Kd or H-2Db, bound either of the peptides, thus demonstrating the exquisite specificity of peptide binding to class I molecules. Moreover, peptide binding to cell-associated H-2Kd was drastically reduced when metabolic energy, de novo protein synthesis or protein egress from the endoplasmic reticulum was inhibited. It is thus likely that exogenously added peptides do not associate with the bulk of class I molecules expressed at the cell surface, but rather bind to short-lived molecules devoid of endogenous peptides.

Protective effects of hypercapnic acidosis on ventilator-induced lung injury.

To investigate whether respiratory acidosis modulates ventilator-induced lung injury (VILI), we perfused (constant flow) 21 isolated sets of normal rabbit lungs, ventilated them for 20 min (pressure controlled ventilation [PCV] = 15 cm H(2)O) (Baseline) with an inspired CO(2) fraction adjusted for the partial pressure of CO(2) in the perfusate (PCO(2) approximately equal to 40 mm Hg), and then randomized them into three groups. Group A (control: n = 7) was ventilated with PCV = 15 cm H(2)O for three consecutive 20-min periods (T1, T2, T3). In Group B (high PCV/normocapnia; n = 7), PCV was given at 20 (T1), 25 (T2), and 30 (T3) cm H(2)O. The targeted PCO(2) was 40 mm Hg in Groups A and B. Group C (high PCV/hypercapnia; n = 7) was ventilated in the same way as Group B, but the targeted PCO(2) was approximately equal to 70 to 100 mm Hg. The changes (from Baseline to T3) in weight gain (Delta WG: g) and in the ultrafiltration coefficient (Delta K(f) = gr/min/ cm H(2)O/100g) and the protein and hemoglobin concentrations in bronchoalveolar lavage fluid (BALF) were used to assess injury. Group B experienced a significantly greater Delta WG (14.85 +/- 5.49 [mean +/- SEM] g) and Delta K(f) (1.40 +/- 0.49 g/min/cm H(2)O/100 g) than did either Group A (Delta WG = 0.70 +/- 0.43; Delta K(f) = 0.01 +/- 0.03) or Group C (Delta WG = 5.27 +/- 2.03 g; Delta K(f) = 0.25 +/- 0.12 g/min/cm H(2)O/ 100 g). BALF protein and hemoglobin concentrations (g/L) were higher in Group B (11.98 +/- 3.78 g/L and 1.82 +/- 0.40 g/L, respectively) than in Group A (2.92 +/- 0.75 g/L and 0.38 +/- 0.15 g/L) or Group C (5.71 +/- 1.88 g/L and 1.19 +/- 0.32 g/L). We conclude that respiratory acidosis decreases the severity of VILI in this model.

Anaplastic astrocytoma in adults.

Anaplastic astrocytoma is an uncommon disease in the adult population. Prognosis is influenced by age, symptom duration, mental status and Karnofsky performance status. A truly complete resection, which is a recognized independent prognostic factor, is not possible and recurrence in the surgical cavity is common. Based on randomized data available, chemotherapy has consistently failed to improve the outcome of patients with anaplastic astrocytoma, while a meta-analysis showed a small, but significant improvement in survival favouring the use of chemotherapy. Outside a clinical trial, postoperative radiotherapy (30 x 2 Gy) remains the standard adjuvant therapy for most patients. For elderly patients, the application of treatment is usually based on performance status and neurological function. In recurrent disease, chemotherapy with temozolomide has been proven to be active and well-tolerated in phase II trials, but no comparative phase III trials of other cytotoxic drugs have been conducted.

Anti-TNF Therapy in the Swiss Inflammatory Bowel Disease G8 Cohort

Background: A nti-TNF d rugs (Infliximab (IFX), Adalimumab (ADA), Certolizumab pegol (CZP)) are effective in inducing and maintaining response a nd remission in i nflammatory bowel disease (IBD). Insufficient response or side effects may lead to a switch o f the anti-TNF d rug. W e aimed to e valuate the frequency and reasons for anti-TNF switches. Methods: Analysis of data from the Swiss Inflammatory Bowel Disease Cohort (SIBDCS). Eighty percent of included patients were recruited in hospitals and 20% from private practice. Results: From 2,058 patients ( 1,172 with Crohn's disease (CD), 842 with ulcerative colitis (UC) and 44 with indeterminate colitis (IC)), 772 received at least one anti-TNF. Forty-eight % of patients w ith CD, 23% with U C, a nd 30% with IC w ere ever treated with an anti-TNF drug. There was no gender difference with respect to the frequency of a nti-TNF treatment. A total of 584 patients (76%) were treated with one, 142 (18%) with two, and 46 (6%) with three anti-TNF (of which 32 were female). A total of 89% patients were treated with IFX, 28% ADA and 13% with CZP. Overall response rate (defined as drop in CDAI >100 points) to anti-TNF was 50%, with best response rates for the first used anti-TNF. Reasons t o switch t he anti-TNF w ere in 11% a primary non-response, in 38% a loss of response and in 36% anti-TNF s ide effects o r intolerance ( reasons for 15% of treatment failures not documented). Conclusion: A nti-TNF d rugs were used in h alf of the CD patients a nd in o ne quarter of U C patients. Anti-TNF d rug switch d ue to insufficient response a nd/or side effects w as necessary in one quarter of IBD patients. IFX was mainly used as first-line therapy. Best response rates were observed for the first used anti-TNF. Following analyses will identify risk median treatment duration as well as risk factors for anti-TNF switch.

Molecular determinants for membrane association of the hepatitis C virus NS2 protease domain

Background: Hepatitis C virus (HCV) nonstructural protein 2 (NS2) plays essential roles in particle assembly and polyprotein processing. It harbors an N-terminal membrane domain comprising three putative transmembrane s egments ( amino acids [aa] 1-93) a nd a C-terminal cysteine protease domain (aa 94-217). Given that the latter has been predicted to be membrane-associated, we aimed to identify molecular determinants for membrane association of the NS2 protease domain. Methods: A comprehensive panel of NS2 deletion constructs was analyzed by fluorescence microscopy, selective membrane extraction, and m embrane flotation assays. Candidate aa r esidues involved in membrane association were substituted by site-directed mutagenesis. Results: The NS2 protease domain alone was found to associate with membranes. Two N-terminal α-helices comprising aa 102-114 and aa 123-136 were found to m ediate this a ssociation, w ith c onserved hydrophobic and positively charged aa residues representing the key determinants. I nterestingly, m utagenesis analyses r evealed that electrostatic interactions involving a positively charged aa residue in α-helix aa 123-136 are required for membrane association. Mono- and bicistronic (i.e. NS2 c leavage-independent) HCV constructs were prepared to i nvestigate the effect o f these substitutions on RNA replication and infectious viral particle formation. Conclusions: T he NS2 protease d omain itself harbors m olecular determinants for membrane association within α-helices aa 102-114 and aa 1 23-136 which may contribute to p roper p ositioning of t he active site. These results provide new insights i nto the membrane topology and t he p oorly understood f unction of t his essential viral protease.

Brain-derived neurotrophic factor enhances the expression of the monocarboxylate transporter 2 through translational activation in mouse cultured cortical neurons.

MCT2 is the predominant neuronal monocarboxylate transporter allowing lactate use as an alternative energy substrate. It is suggested that MCT2 is upregulated to meet enhanced energy demands after modifications in synaptic transmission. Brain-derived neurotrophic factor (BDNF), a promoter of synaptic plasticity, significantly increased MCT2 protein expression in cultured cortical neurons (as shown by immunocytochemistry and western blot) through a translational regulation at the synaptic level. Brain-derived neurotrophic factor can cause translational activation through different signaling pathways. Western blot analyses showed that p44/p42 mitogen-activated protein kinase (MAPK), Akt, and S6 were strongly phosphorylated on BDNF treatment. To determine by which signal transduction pathway(s) BDNF mediates its upregulation of MCT2 protein expression, the effect of specific inhibitors for p38 MAPK, phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK), p44/p42 MAPK (ERK), and Janus kinase 2 (JAK2) was evaluated. It could be observed that the BDNF-induced increase in MCT2 protein expression was almost completely blocked by all inhibitors, except for JAK2. These data indicate that BDNF induces an increase in neuronal MCT2 protein expression by a mechanism involving a concomitant stimulation of PI3K/Akt/mTOR/S6, p38 MAPK, and p44/p42 MAPK. Moreover, our observations suggest that changes in MCT2 expression could participate in the process of synaptic plasticity induced by BDNF.

Towards standardization of UV eye protection: what can be learned from photodermatology?

While knowledge about standardization of skin protection against ultraviolet radiation (UVR) has progressed over the past few decades, there is no uniform and generally accepted standardized measurement for UV eye protection. The literature provides solid evidence that UV can induce considerable damage to structures of the eye. As well as damaging the eyelids and periorbital skin, chronic UV exposure may also affect the conjunctiva and lens. Clinically, this damage can manifest as skin cancer and premature skin ageing as well as the development of pterygia and premature cortical cataracts. Modern eye protection, used daily, offers the opportunity to prevent these adverse sequelae of lifelong UV exposure. A standardized, reliable and comprehensive label for consumers and professionals is currently lacking. In this review we (i) summarize the existing literature about UV radiation-induced damage to the eye and surrounding skin; (ii) review the recent technological advances in UV protection by means of lenses; (iii) review the definition of the Eye-Sun Protection Factor (E-SPF®), which describes the intrinsic UV protection properties of lenses and lens coating materials based on their capacity to absorb or reflect UV radiation; and (iv) propose a strategy for establishing the biological relevance of the E-SPF.

Copeptin, a stable peptide derived from the vasopressin precursor, correlates with the individual stress level.

BACKGROUND: During stress, vasopressin is a potent synergistic factor of CRH as a hypothalamic stimulator of the HPA axis. The measurements of CRH and vasopressin levels are cumbersome because of their instability and short half-life. Copeptin is a more stable peptide stoichiometrically released from the same precursor molecule. The aim of our study was to compare copeptin and cortisol levels in different stress situations. METHODS: Three groups of patients with increasing stress levels were investigated: a) healthy controls without apparent stress (n=20), b) hospitalized medical patients with moderate stress (n=25) and c) surgical patients 30 minutes after extubation, with maximal stress (n=29). In all patients we assessed cortisol and copeptin levels. Copeptin levels were measured with a new sandwich immunoassay. RESULTS: Cortisol levels in controls were (median, IQ range, 486 [397-588] nmol/L), not significantly different as compared to medical patients (438 [371-612] nmol/L, p=0.69). Cortisol levels in surgical patients after extubation were higher (744 [645-1062] nmol/L p<0.01 vs controls and medical patients). Copeptin levels in controls were 4.3 [3.2-5.5] pmol/L, which was lower as compared to medical patients (17.5 [6.4-24.1], p<0.001) and surgical patients after extubation (67.5 [37.8-110.0] pmol/L, p<0.001). The correlation between copeptin levels and cortisol was r=0.46, p<0.001. CONCLUSION: Copeptin is a novel marker of the individual stress level. It more subtly mirrors moderate stress as compared to cortisol values.

Effects of colostrum feeding and glucocorticoid administration on insulin-dependent glucose metabolism in neonatal calves

Colostrum feeding and glucocorticoid administration affect glucose metabolism and insulin release in calves. We have tested the hypothesis that dexamethasone as well as colostrum feeding influence insulin-dependent glucose metabolism in neonatal calves using the euglycemic-hyperinsulinemic clamp technique. Newborn calves were fed either colostrum or a milk-based formula (n=14 per group) and in each feeding group, half of the calves were treated with dexamethasone (30 microg/[kg body weight per day]). Preprandial blood samples were taken on days 1, 2, and 4. On day 5, insulin was infused for 3h and plasma glucose concentrations were kept at 5 mmol/L+/-10%. Clamps were combined with [(13)C]-bicarbonate and [6,6-(2)H]-glucose infusions for 5.5h (i.e., from -150 to 180 min, relative to insulin infusion) to determine glucose turnover, glucose appearance rate (Ra), endogenous glucose production (eGP), and gluconeogenesis before and at the end of the clamp. After the clamp liver biopsies were taken to measure mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC). Dexamethasone increased plasma glucose, insulin, and glucagon concentrations in the pre-clamp period thus necessitating a reduction in the rate of glucose infusion to maintain euglycemia during the clamp. Glucose turnover and Ra increased during the clamp and were lower at the end of the clamp in dexamethasone-treated calves. Dexamethasone treatment did not affect basal gluconeogenesis or eGP. At the end of the clamp, dexamethasone reduced eGP and PC mRNA levels, whereas mitochondrial PEPCK mRNA levels increased. In conclusion, insulin increased glucose turnover and dexamethasone impaired insulin-dependent glucose metabolism, and this was independent of different feeding.

Effect of malaria and fever on energy metabolism in Gambian children.

The aim of the present study was to measure the changes in resting energy expenditure (REE) induced by malaria and to assess to what extent they are related to fever and nutritional status. The REE of 19 Gambian children (mean age +/- SEM, 9 +/- 1 y; weight, 24 +/- 2 kg; expected weight for height 86 +/- 1%) were measured with a hood system at repeated intervals at the onset of malaria crisis (test A), 3 to 4 d after therapy (test B), and 14 to 21 d later (test C). Axillary temperature averaged 39.2 +/- 0.1, 36.6 +/- 0.1, and 36.7 +/- 0.1 degrees C in the tests A, B, and C, respectively. REE in test A was significantly higher than REE in test B (223 +/- 10 versus 174 +/- 8 kJ/kg.d, p less than 0.0001), but in test C (169 +/- 8 kJ/kg.d), it did not differ from that observed in test B. The percentage of increase in REE was significantly correlated with the difference in axillary temperature (r = 0.46, p less than 0.05); the slope of the regression line indicated an increase of 6.9% in REE/degree C of fever. Furthermore, the individual increase in REE/degree C was correlated to the percentage of weight for height of the children (r = 0.54, p less than 0.05), indicating that the child's nutritional status influences the magnitude of the hypermetabolism due to fever. We concluded that Gambian children suffering from an acute episode of malaria have an increase in REE averaging 30%; however, REE promptly returns to baseline value a few days after the beginning of therapy.

Total energy expenditure and patterns of activity in 8-10-year-old obese and nonobese children.

Total energy expenditure (TEE) and patterns of activity were measured by means of a heart rate (HR)-monitoring method in a group of 8-10-year-old children including 13 obese children (weight, 46 +/- 10 kg; fat mass: 32 +/- 9%) and 16 nonobese children (weight, 31 +/- 5 kg; fat mass, 18 +/- 5%). Time for sleeping was not statistically different in the two groups of children (596 +/- 33 vs. 582 +/- 43 min; p = NS). Obese children spent more time doing sedentary activities (400 +/- 129 vs. 295 +/- 127 min; p < 0.05) and less time in nonsedentary activities (449 +/- 126 vs. 563 +/- 135 min; p < 0.05) than nonobese children. Time spent in moderate or vigorous activity-i.e., time spent at a HR between 50% of the maximal O2 uptake (peak VO2) and 70% peak VO2 (moderate) and at a HR > or = 70% peak VO2 (vigorous)-was not statistically different in obese and nonobese children (88 +/- 69 vs. 52 +/- 35 min and 20 +/- 21 vs. 16 +/- 13 min, respectively; p = NS). TEE was significantly higher in the obese group than in the nonobese group (9.46 +/- 1.40 vs. 7.51 +/- 1.67 MJ/day; p < 0.01). The energy expenditure for physical activity (plus thermogenesis) was significantly higher in the obese children (3.98 +/- 1.30 vs. 2.94 +/- 1.39 MJ/day; p < 0.05). The proportion of TEE daily devoted to physical activity (plus thermogenesis) was not significantly different in the two groups, as shown by the ratio between TEE and the postabsorptive metabolic rate (PMR): 1.72 +/- 0.25 obese vs 1.61 +/- 0.28 non-obese. In conclusion, in free-living conditions obese children have a higher TEE than do nonobese children, despite the greater time devoted to sedentary activities. The higher energy cost to perform weight-bearing activities as well as the higher absolute PMR of obese children help explain this apparent paradox.

p.L1612P, a novel voltage-gated sodium channel Nav1.7 mutation inducing a cold sensitive paroxysmal extreme pain disorder.

BACKGROUND: Mutations in the SCN9A gene cause chronic pain and pain insensitivity syndromes. We aimed to study clinical, genetic, and electrophysiological features of paroxysmal extreme pain disorder (PEPD) caused by a novel SCN9A mutation. METHODS: Description of a 4-generation family suffering from PEPD with clinical, genetic and electrophysiological studies including patch clamp experiments assessing response to drug and temperature. RESULTS: The family was clinically comparable to those reported previously with the exception of a favorable effect of cold exposure and a lack of drug efficacy including with carbamazepine, a proposed treatment for PEPD. A novel p.L1612P mutation in the Nav1.7 voltage-gated sodium channel was found in the four affected family members tested. Electrophysiologically the mutation substantially depolarized the steady-state inactivation curve (V1/2 from -61.8 ± 4.5 mV to -30.9 ± 2.2 mV, n = 4 and 7, P < 0.001), significantly increased ramp current (from 1.8% to 3.4%, n = 10 and 12) and shortened recovery from inactivation (from 7.2 ± 5.6 ms to 2.2 ± 1.5 ms, n = 11 and 10). However, there was no persistent current. Cold exposure reduced peak current and prolonged recovery from inactivation in wild-type and mutated channels. Amitriptyline only slightly corrected the steady-state inactivation shift of the mutated channel, which is consistent with the lack of clinical benefit. CONCLUSIONS: The novel p.L1612P Nav1.7 mutation expands the PEPD spectrum with a unique combination of clinical symptoms and electrophysiological properties. Symptoms are partially responsive to temperature but not to drug therapy. In vitro trials of sodium channel blockers or temperature dependence might help predict treatment efficacy in PEPD.