The process is characterized by multiple steps: inflammation, fibrosis, and calcification. Numerous studies focalized on its physiopathology highlighting different “actors” for the multiple “acts.” This paper focuses on the role of the tumor necrosis factor superfamily (TNFSF) members in the pathogenesis of CAVD. In particular, we discuss the clinical and experimental studies providing evidence of the involvement of tumor necrosis factor-alpha (TNF- Calcific aortic valve disease (CAVD) represents a slowly progressive pathologic process extending from mild thickening of the aortic valve without obstruction of blood flow, named aortic valve sclerosis, to a severe calcification of valvular leaflets, reduction of valve motion, and obstruction of blood flow, named aortic stenosis (AS) . AS is tβ1 [β [α [In vitro studies of cultured explants of stenotic valves have identified cells with osteoblastic characteristics that undergo phenotypic differentiation and spontaneous calcification [Inflammation is a prominent feature of aortic valve calcification, and it is present in both early and advanced aortic valvular lesions , 10. Hisβ1 , IL-1β [β1 [β , TNF-α [β1 [β [α , and matβ1 [β [α , which cβ1 [β [α . Severalβ1 [β [α , 19. In fication . These ofication , 22 and fication . The inification , 17 or bfication . α), receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL), its membrane receptor RANK and its decoy receptor osteoprotegerin (OPG), and TNF-related apoptosis-inducing ligand (TRAIL) in valvular calcification.This paper focuses on the role of the tumor necrosis factor superfamily (TNFSF) members in the pathogenesis of CAVD. The TNFSF is composed of 19 ligands and 29 receptors and plays highly diversified roles in the body . In partα maps to chromosome 6p21.3 and is primarily produced as a 212-amino-acid-long type II transmembrane protein arranged in stable homotrimers [α-converting enzyme (TACE) [Tumor necrosis factor-alpha or TNF-otrimers , 25. Froe (TACE) .α is produced by different kinds of cells, including activated macrophages, monocytes, T-cells, smooth muscle cells, adipocytes, and fibroblasts. The cytokine is involved in acute and/or chronic inflammation. Whereas, in acute inflammation, TNF-α protects against bacterial endotoxin, viruses, and parasites, provides increased nutrients for immune cells, and favors a proper host response, in chronic inflammation, TNF-α activates pathways responsible for numerous pathological conditions, such as arthritis. In fact, molecules neutralizing it are beneficial in the treatment of diseases. TNF-α was aptly named when it was discovered to induce tumor cell apoptosis [α promotes several cell functions related to immune cell proliferation and adhesion and apoptosis [TNF-poptosis , or progpoptosis , 28.α can induce biological reactions by either TNF receptor 1 (TNFR1) or TNFR2: the first, which contains a death domain (DD), is highly promiscuous and is expressed on every cell type in the body, whereas the expression of the second receptor is limited to cells of the immune system, endothelial cells, and nerve cells. Each receptor can mediate distinct intracellular signals. In particular, TNF-α induces at least 5 different types of signals that include activation of NF-κB, apoptosis pathways, extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK), and c-Jun N-terminal kinase (JNK). When TNF-α binds to TNFR1, it recruits a protein called TNFR-associated death domain (TRADD) through its DD [α can activate mitochondria to sequentially release ROS, cytochrome C, and Bax, leading to activation of caspase-9, caspase-3, and, thus, apoptosis [TNF-h its DD . TRADD th its DD . Alternapoptosis .α has also been shown to activate NF-κB, which, in turn, regulates the expression of proteins associated with cell survival and proliferation [κB activation, the intracellular domain of TNFR1 is bound by an adaptor protein, TNF receptor-associated death domain (TRADD), which mobilizes additional adaptor protein receptor interacting protein-1 (RIP-1), and TRAF2 [β-activated kinase (TAK1) activates the IκB kinase (IKK) complex. The IKK complex phosphorylates IκBα that ubiquitinates and degrades IκBα. This subsequently releases NF-κB subunits, which translocate into the nucleus and promote gene transcription [α is mediated through NF-κB-regulated proteins, such as IL-6, IL-8, IL-18, chemokines, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and 5-lipoxygenase (5-LOX), all major mediators of inflammation. Indeed, TNF-α can induce expression of TNF-α itself through activation of NF-κB [TNF-feration .   For Nnd TRAF2 . Subsequcription –36. The of NF-κB .α can also activate cellular proliferation through activation of another transcription factor, activator protein-1 (AP-1) [α through sequential recruitment of TNFR1, TRADD, TRAF2, MAP/ERK kinase kinase-1 (MEKK1), MAP kinase kinase-7 (MKK7), and JNK. The activation of p38MAPK by TNF-α is mediated through TRADD-TRAF2-MKK3. How TNFR2, which lacks a DD, activates cell signaling is much less clear than how TNFR1 activates cell signaling. Since TNFR2 can directly bind to TRAF2, it can activate both NF-κB and MAPK signaling.TNF-1 (AP-1) , which iα and its family members have now been linked to an array of pathophysiologies, including cancer, neurologic, pulmonary, autoimmune, metabolic, and cardiovascular diseases [Although initially discovered as an anticancer agent, TNF-diseases –47.α in CAVD. TNF-Demer first identified that TNF-α may participate in vascular calcification, upregulating alkaline phosphate (ALP) activity as a necessary component of calcifying vascular cell mineralization in vitro [α in the pathogenesis of aortic valvular calcification has been gradually elucidated; TNF-α is a pleiotropic cytokine which induces ECM remodeling [α is expressed by macrophages in calcific aortic valves and it stimulates in vitro proliferation of human valvular myofibroblasts as well as their expression of MMP-1 [α, MMP-1; conversely, calcific aortic valves present inflammatory infiltrate and colocalized expression of TNF-α, MMP-1 [α. Upon stimulation with TNF-α, human aortic valve myofibroblasts cultured under mineralizing conditions showed increased formation of calcified, ALP-enriched cell nodules, ALP activity, concentration of the bone-type ALP isoenzyme, and concentration of osteocalcin (OCN), all of which are markers of an osteoblast-like cellular phenotype [α increases the gene expression of the osteogenic makers ALP and BMP-2 and induces calcification of VICs obtained from the patients with AS [α-induced calcification, ALP activation, and NF-κB and BMP-2 gene expression are inhibited in the presence of inhibitors of NF-κB signalling, showing that TNF-α activates the NF-κB signalling pathway and translocates NF-κB p65 subunit into the nucleus for upregulation of the BMP-2 and NF-κB genes [α, as well as an increased tissue remodeling [α in CAVD [−/−) mice spontaneously develop AS, and T-cells from IL-1Ra−/− produce much higher levels of TNF-α after anti-CD3 antibody stimulation compared to wild-type mice; furthermore, TNF-α deficiency completely suppressed AS development in IL-1Ra−/− mice, suggesting that TNF-α actively participates in AS development in IL-1Ra−/− mice [α are elevated in patients with severe AS and correlate with the severity of the hemodynamic pressure overload; moreover, the peripheral TNF-α and TNF receptor levels increase in direct relation to deteriorating NYHA functional classification [α levels reduce progressively, returning to normal 6 months after surgical aortic valve replacement (AVR) [in vitro . Thereafmodeling , cell prmodeling , and calmodeling . Kaden sof MMP-1 ; normal α, MMP-1 . Aortic henotype ; by elechenotype . TNF-α i with AS ; TNF-α-iκB genes . OxidizeκB genes ; valves modeling . Additio in CAVD . IL-1 re−/− mice . Circulafication . Circulant (AVR) .The RANKL/RANK/OPG pathway was initially described in the context of bone mass regulation, but now its prominent role in cardiovascular disease is emerging .−/− mice were severely growth retarded due to poor nutrition secondary to lack of tooth eruption and displayed shortened long bones with club-shaped ends, thinning of the calvariae, generalized increase in bone density with very little marrow space, marked chondrodysplasia with thick, irregular growth plates, and relative increase in hypertrophic chondrocytes. Moreover, RANKL−/− mice displayed defects in the immunological compartment: reduced thymus size, spleen enlargement, complete lack of lymph nodes, and smaller Peyer's patches [RANKL is encoded by a single gene at human chromosome 13q14. Alternative splicing of RANKL mRNA allows expression as a type II transmembrane glycoprotein of either 316 or 270 amino acids or as a soluble ligand of 243 amino acids , 57. In  patches , 70, 71.RANK is a type I transmembrane glycoprotein encoded on human chromosome 18q22.1 and is expressed on the surface of osteoclasts and osteoclast precursors as well as bone-marrow-derived dendritic cells, activated T-cells, vascular endothelia, chondrocytes, bone marrow fibroblasts, and mammary gland epithelia. Each RANKL trimer engages three molecules of RANK. Trimerization triggers a conformational change in the cytoplasmic domain of RANK that allows recruitment of TNFR-associated factors (TRAFs). In particular, TRAF2 and TRAF6 are the most critical for RANK signalling –74. TRAFOPG, encoded by a single gene on chromosome 8q24, is a soluble, 110 kDa, disulfide-linked, homodimeric glycoprotein that functions as a decoy receptor for RANKL. Thus, OPG modulates osteoclast formation by inhibiting RANK activation . OPG als−/− mice develop arterial calcification, suggesting that OPG plays a role in the maintenance of VSMCs homeostasis [Transgenic mice expressing OPG exhibited increased bone density, which was explained histologically by a marked decrease in osteoclast number that was presumably due to reduced osteoclast formation . In animeostasis . OPG coueostasis .RANKL/RANK/OPG in CAVD. Kaden et al. first showed by immunohistochemistry that RANKL and OPG are differentially expressed in calcific AS. RANKL is present in aortic valves from patients with AS, while it is not expressed at relevant levels in normal valves; conversely, OPG expression is marked in normal valves but significantly lower in AS. Additionally, areas containing focal calcification exhibit significantly less OPG-positive cells as compared to noncalcified regions [κB is increased in sclerotic valves compared with stenotic valves, whereas the frequency of RANKL is higher in stenotic compared to sclerotic valves. As a consequence, the OPG/RANKL ratio is decreased in stenotic compared to sclerotic valves [Ldlr−/−Apob100/100 mice, which are prone to develop calcific AS. OPG profoundly attenuates valve calcification by inhibition of osteogenic transformation, but it does not prevent valve fibrosis or lipid deposition; in particular, OPG strongly suppresses levels of osterix, OCN, and monocyte-chemoattractant protein-1 [ regions . Furtherc valves . Other sc valves . In cultc valves . RANKL ic valves . Experimrotein-1 . In patirotein-1 . In patirotein-1 . Interesrotein-1 . Tumor necrosis factor- (TNF-) related apoptosis-inducing ligand (TRAIL/Apo2L), located on chromosome 3, as a member of the TNF superfamily of proteins, is expressed as a type II transmembrane protein. Cleavage of its C-terminal part (extracellular domain) allows for a soluble form of TRAIL –107.TRAIL is mostly expressed by cells of the immune system where it was shown to play a role in the homeostasis of certain T-cells and in NK and T-cell-mediated killing of virally and oncogenically transformed cells –110. TRATRAIL firstly received considerable attention as a molecule showing the ability to induce apoptosis in a wide variety of neoplastic cells . HoweverTRAIL in CAVD. VICs sensitivity to TRAIL apoptotic effect is of paramount importance because apoptosis has been shown to be an initiator of vascular calcification in in vitro studies [ studies ; increas studies . Previou studies , 133.β1 is present in human calcific aortic stenotic cusps and promotes calcification of cultured sheep aortic VICs (SAVICs) through mechanisms involving apoptosis [TGF-poptosis ; in factpoptosis . TRAIL hpoptosis , and TRApoptosis . TRAIL ipoptosis . VICs depoptosis . Additiopoptosis . The exppoptosis . VICs cupoptosis ; moreovepoptosis . Taken tα, key cytokines in CAVD pathogenesis, are having a great success in the treatment of osteoporosis and arthritis, respectively [Although, to date, no medical therapeutic options are able to prevent or reduce the progression of CAVD and the only treatment for severe AS is surgical aortic valve replacement (AVR), the understanding of the underlying pathogenic mechanisms of the disease is mandatory to identify promising therapeutic targets. It is known that the recently available biologic drugs neutralizing RANKL and TNF-ectively , 136. Thectively .


The effect of U50,488H administration during chronic hypoxia was investigated. Administration of U50,488H significantly decreased mPAP and right ventricular hypertrophy as evidenced by reduction in RVP and RVHI. These effects were mediated by κ-opioid receptor. In the meantime, treatment with U50,488H significantly improved endothelial function as evidenced by enhanced relaxation in response to ACh. Moreover, U50,488H resulted in a significant increase in eNOS phosphorylation, NO content in serum, and T-AOC in pulmonary artery of HPH rats. In addition, the activity of eNOS was enhanced, but the activity of iNOS was attenuated in the pulmonary artery of chronic hypoxic rats treated with U50,488H. On the other hand, U50,488H markedly blunted HPH-induced elevation of gp91phox expression and nitrotyrosine content in pulmonary artery, and these effects were blocked by nor-BNI, a selective κ-opioid receptor antagonist. These data suggest that κ-opioid receptor stimulation with U50,488H improves endothelial function in rats with HPH. The mechanism of action might be attributed to the preservation of eNOS activity, enhancement of eNOS phosphorylation, downregulation of iNOS activity and its antioxidative/nitrative effect.The present study was designed to investigate the effect of κ-opioid receptor stimulation with U50,488H on endothelial function and underlying mechanism in rats with hypoxic pulmonary hypertension (HPH). Chronic hypoxia-induced HPH was simulated by exposing the rats to 10% oxygen for 2 wk. After hypoxia, mean pulmonary arterial pressure (mPAP), right ventricular pressure (RVP) and right ventricular hypertrophy index (RVHI) were measured. Relaxation of pulmonary artery in response to acetylcholine (ACh) was determined. Expression and activity of endothelial nitric oxide (NO) synthase (eNOS) and inducible NO synthase (iNOS) with NO production, total antioxidant capacity (T-AOC), gp91 Pulmonary hypertension (PH) is a chronic disease characterized by progressively increased pulmonary vascular resistance and vascular remodeling and it has been recognized as ‘cancer of cardiovascular diseases’ because of its high mortality and morbidity. So far, there has been no magic cure for pulmonary hypertension, and the goal of treatment is to delay or prevent the progression of this disease. Among various types of PH, hypoxic pulmonary hypertension (HPH) which occurs in patients with cardiopulmonary disease and residents at high altitude has aroused great interest from researchers. Although many investigators have demonstrated that the pathogenesis of the hypoxic pulmonary vasoconstriction has been associated with Kv channel, endothelin-1, serotonin and so on 2−) to form the toxical oxidant peroxynitrite (ONOO−) which can result in oxidative damage, nitration, and S-nitrosylation of biomolecules Hypoxia induced endothelium injury is believed to play a great role in the initiation and development of HPH. When the vascular endothelium was impaired, the balance between a variety of vasomotor factors such as nitric oxide (NO), endothelin (ET) and angiotensin II (AngII) was disturbed, leading to pulmonary vascular vasoconstriction and ultimately pulmonary vascular remodeling in endstage. Impaired production of NO, which exhibits vasorelaxtion effect Therefore, the aims of the present study were 1) to investigate whether U50,488H might improve endothelial function of HPH rats and, if so, 2) to investigate the mechanisms involved.Male Sprague-Dawley rats (200±10 g) from the animal center of the Fourth Military Medical University on Animal Care were used. This study conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health, NIH Publication No. 85–23, revised 1996. Ethical approval for this study was also granted by the University Ethics Committee.Rat HPH model was produced as described previously Adult male SD rats were anesthetized by intraperitoneal injection of 60 mg/kg pentobarbital sodium. Supplemental doses of sodium pentobarbital were given when needed. The mean pulmonary arterial pressure (mPAP) and right ventricular pressure (RVP) were measured by inserting a polyethylene micro-catheter from right external jugular vein into right ventricle and pulmonary artery as described previously 2, we may come to a conclusion that endothelial function is impaired. By comparing different responses to two types of vasodilators, we investigate whether U50,488H treatment improves endothelial function.The pulmonary artery was separated gentlely and carefully, and quickly placed in the low temperature Kerb's solution (0∼4°C) in order to maintain its vasoactive activity. After the perivascular fat connective tissues were carefully removed, the pulmonary arteries were cut into 3 mm segments, and quickly placed in the low temperature Kerb's solution (0∼4°C) for further treatment. Pulmonary artery segment was subjected to isolated vascular perfusion system and connected to the tension sensor and the pulmonary artery tension was recorded by multi-channel physiological recording system (RM6280). If the relaxation activity of pulmonary artery in response to ACh (endothelium dependent vasodilators) decreases but has normal relaxation activity in response to NaNOBlood samples were drawn at the end of 2 wk of hypoxia period. After centrifuging 20 min at 3000 rpm, the supernants were collected. Nitric oxide (NO) can be easily oxidated, leading to the formation of nitrate/nitrite. Thus, nitrate/nitrite levels are accepted as an indicator of NO production. Nitrate/nitrite levels were measured in serum of the rats with a commercially available colorimetric assay kit (nitrate reductase), as previously described phox (abcam), p-eNOS Ser1177, overnight at 4°C. Then, the membranes were washed with PBS and incubated with horseradish peroxidase-conjugated IgG antibody for 1 h at room temperature. The immunoblotting was detected using an enhanced chemiluminescence detection kit (Millipore) with ChemiDocXRS , and the blot densities were analyzed with Quantity One Software.Pulmonary artery tissues were lysed in buffer , 1% sodium dodecyl sulphate (SDS), and 5 mM ethylene diamine tetraacetic acid (EDTA)). Equal amounts of protein (40 g protein/lane) were electrophoresed on an 8% SDS-polyacrylamide gel and electrophoretically transferred to nitrocellulose membranes . After blocking with 3% BSA (3% wt/vol) in Tris-buffered saline at room temperature for 1 h, the membranes were incubated with an antibody against eNOS(abcam), iNOS (abcam), or gp912O2 (4.5%) to quench/inhibit endogenous peroxidase. After blocking, the sections were reacted with anti-3-NT antibody  for 1 h at room temperature. After extensive washing with PBS, the sections were incubated with antibody peroxidase conjugated for 1 h and finally incubated with diaminobenzidine for 30 min. Quantitative image analysis was performed with image analysis software . The software determines densitometry mean values of selected tissue regions. Thus, 10 fields/rats were randomly selected, and the intensity of the 3-NT immunostaining was determined.Immunofluorescence detection of 3-nitrotyrosine  in right lung frozen section was conducted as previously described Pulmonary artery tissues were minced and homogenized in 0.9% NaCl solution  with a Heidolph DIA900 tissue homogenizer . The homogenate was centrifuged (3000 rpm at 4°C for 10 min), the supernatant was decanted, and total NOS activity and inducible NOS (iNOS) activity were determined using an NOS activity assay kit  as previously described After rinsed, the pulmonary artery was homogenized in 0.9% NaCl solution , and the mixture was subjected to centrifuge at 3000 rpm for 10 min. Then the pellet was discarded and the supernatant was decanted. T-AOC was measured with a spectrophotometric assay kit (Nanjing Jiancheng Bioengineering Institute), according to the manufacture’s instructions.P<0.05 was considered to be statistically significant.Data were presented as mean±SE. The results were analyzed by one way ANOVA followed by Tukey post hoc testing. A value of After chronic hypoxia for 2 weeks, mPAP was significantly increased by 57%, and this change was almost abolished by the peritoneal injection of U50,488H, a selective κ-opioid receptor agonist during chronic hypoxia. As shown in Figure1, chronic hypoxia also elicited significantly RV hypertrophy as evidenced by increased RVP, RV/(LV+S) and RV/BW. These changes were significantly attenuated by the peritoneal injection of U50,488H. The effect of U50,488H was abolished by nor-BNI, a selective κ-opioid receptor antagonist, which itself had no effect on these parameters, indicating that U50,488H exerted depressive effect on HPH and RV hypertrophy and this effect was mediated by κ-opioid receptor.2) was not altererd  in different treatment groups were collected. As shown in altererd . These rTo investigate the precise mechanism of the improvement of endothelial function, NO content was measured. As demonstrated in We next investigated whether U50,488H-induced increase of NO generation was caused by enhanced eNOS or iNOS protein expression level. As demonstrated in As shown in The mechanism of U50,488H-induced elevation of NO production in HPH rats remains unclear, we, therefore, sought to investigate whether U50,488H exerted this effect via activating eNOS/NO pathway. As shown in In order to elucidate the precise mechanism of the stimulative role of U50,488H on NO release, eNOS and iNOS activity were measured respectively. As demonstrated in phox, a major source of superoxide, was detected by Western blotting. As showed in phox expression was significantly increased in pulmonary arteries of HPH rats. Administration of U50,488H caused a moderate, yet statistically significant, reduction in gp91phox expression. Moreover, this effect was abolished by nor-BNI, indicating that the reducing effect of U50,488H on gp91phox expression was mediated by κ-opioid receptor.The data presented above indicate that U50,488H can stimulate bioactive NO production by regulation of eNOS and iNOS activity. However, it remains unclear whether U50,488H can prevent NO destruction. As we all know, the decrease of total antioxidant capacity (T-AOC) may lead to NO destruction. As summarized in −, an index of nitrative stress, which was the biradical reaction product of superoxide and NO, was determined by measurement of nitrotyrosine. As shown in To obtain more evidence to support this notion that U50,488H prevent NO destruction, ONOO2 was markedly ameliorated by U50,488H, 2) U50,488H stimulated bioactive NO production via preservation of eNOS activity and enhancement of eNOS phosphorylation and 3) U50,488H prevented NO destruction by elevating total antioxidant capacity, decreasing gp91phox and ONOO− formation of pulmonary artery.In the present study, chronic treatment with U50,488H was demonstrated to ameliorate endothelial dysfunction in chronic hypoxia. This conclusion is based on the following observations:1) relaxation response of pulmonary artery to ACh but not to NaNOOur study provides the first evidence that U50,488H, a κ-opioid receptor agonist, significantly attenuated chronic hypoxia-induced endothelial dysfunction. This result holds potential promises that therapeutic application of U50,488H may represent a useful treatment of cardiovascular diseases with vascular disorders. Consistent with previous studies HPH is a severe clinical disease, which is characterized by hypoxic pulmonary vasoconstriction and pulmonary vascular remodeling NO is generated from L-arginine through enzymatic conversion by NOS synthase 2+]i and altered eNOS phosphorylation 2+]i, the latter may also account for the changes of eNOS activity. Further study is needed to confirm this speculation.Xu et al has demonstrated that κ-opioid receptor and caveolin-1 are coexpressed in lipid rafts In this study, we found that neither HPH nor U50,488H altered iNOS expression but they significantly changed iNOS activity. Study by Sharon et al 2−) reacts with NO to form peroxynitrite (ONOO−) and other reactive nitrogen species, effectively reducing NO bioavailability. ONOO− is a highly reactive species and can initiate both nitrosative and oxidative reactions in vitro and in vivo. A characteristic reaction of ONOO− is the nitration of protein-bound tyrosine residues to generate 3-nitrotyrosine-positive proteins −− formation in pulmonary artery of HPH rats was significantly attenuated after U50,488H treatment. Second, the present study has demonstrated that chronic treatment with U50,488H significantly reduced gp91phox (an important component of NADPH oxidase) expression in pulmonary artery of HPH rats. Collectively, our study has obtained evidence that U50,488H ameliorates endothelial dysfunction induced by chronic hypoxia, at least in part by its antioxidative and antinitrative property that have never been previously identified.Accumulating evidence now suggests that NADPH oxidase is the major source of superoxide in vascular tissues In summary, we have demonstrated for the first time that U50,488H may confer endothelium protection, at least partly via its promotion on NO production and its antioxidant/nitrative properties which prevent NO destruction. The results not only provide new ideas regarding the relationship between opioid receptor and HPH, but also highlight U50,488H as a potential agent against HPH in clinical practice.


To produce a thorough review of the existing state of knowledge on sample size calculations for cluster randomised trials (CRT’s) and to identify gaps in the knowledge.A systematic review is being conducted of sample size methodology for cluster randomised trials. The sources for the search include electronic databases PubMed and Web of Science, key text books on cluster randomised trials and discussions with experts in the field.The search strategy involves a compliment of Medical Subject Headings and free text terms to aid a comprehensive search. The references of papers eligible for the review will also be searched and a search on the first author conducted. This process will continue until no more additional papers are located.This work forms the beginning of a PhD research project.Of 8697 citations obtained from PubMed and Web of Science, the majority have currently been assessed for eligibility into the review and 57 papers so far identified for inclusion.The majority of papers discuss sample size for continuous or binary outcomes, with four papers discussing time to event outcomes. In terms of the analysis method used, most assume a random effects analysis (cluster specific approach) or a cluster level analysis, with fewer papers assuming a generalized estimating Equation (population averaged approach) methodology.An emerging theme, discussed in six papers, is sample size methodology for 3-level cluster randomised trials, where we may randomise clinics (level 3) and each clinic will treat multiple subjects (level 2 units) who in turn are measured on repeated occasions (level 1 units).Eight papers consider sample size calculations for trials with varying cluster sizes. These papers account for the loss in power due to varying cluster sizes through an examination of the relative efficiency of unequal versus equal cluster sizes or by proposing an appropriate design effect to account for this loss for both continuous and binary outcomes.Sample size for alternative trial designs such as cross-over trials, stepped wedge designs, testing for non-inferiority, stratified, and matched designs were identified. Papers covering adjustments to sample size for dealing with non-compliance or attrition, accounting for the use of cluster or person level covariates and dealing with imprecision in the estimate of the intracluster correlation coefficient (ICC) were identified.We will provide the results of the search and preliminary insight into potential gaps in the knowledge.


Background. Methicillin-resistant Staphylococcus aureus (MRSA) can stimulate massive cytokine release. Ketamine suppresses tumor necrosis factor (TNF) secretion by MRSA-stimulated RAW264.7 macrophages, and the mechanism likely involves N-methyl-D-aspartic acid (NMDA) receptor antagonism. The downstream effects of NMDA-mediated TNF suppression, specifically the PI3K/Akt and mTOR modulation, have not been described. Methods. RAW264.7 cells were stimulated for 18 hrs with 105 to 107 CFU/mL inocula of either of two prototypical community-acquired- (CA-) MRSA isolates, USA300 strain LAC and USA400 strain MW2. Then we added the NMDA inhibitors ketamine or 2R-amino-5-phosphonopentanoate (AP5), NMDA substrate, LY294002, and rapamycin in various combinations. Results. NMDA inhibition suppressed TNF secretion by almost a third compared to the no-ketamine control. When NMDA substrate was added, the TNF secretion increased by 10%. Addition of LY294002 suppressed TNF production by macrophages by 20%. Rapamycin exhibited a concentration-dependent TNF induction-suppression response: induction at doses of 0.1 and 1 ng/mL and suppression at 10 and 100 ng/mL. Induction of TNF was abolished when LY294002 was added and the suppression became uniform. Ketamine-induced suppression of TNF secretion was intensified 10–15% when rapamycin was added, but not when LY294002 was added. Conclusion. These findings suggest that NMDA-induced TNF suppression can be augmented by concurrent mTOR inhibition. Staphylococcal infections are the leading cause of sepsis . Bacterimulation , 5. TNF mulation . The infmulation , 10. We mulation . Tumor nStreptomyces hygroscopicus [Staphylococcus aureus- (MRSA-) stimulated RAW264.7 murine macrophage model. It assesses whether the NMDA-induced TNF inhibition can be augmented with rapamycin, according to the observed ketamine rapamycin antidepression model [Ketamine's action begins with antagonism of the N-methyl-D-aspartate (NMDA) receptor. Its effects on the downstream pathway are not known. It has been shown recently that ketamine has a rapid antidepressant effect that is attributed to activation of the mammalian target of rapamycin (mTOR) . Calcinescopicus , 22. It scopicus . Rapamycscopicus . The FKBscopicus , 26. Thescopicus –29, whicscopicus . Recent scopicus . Rapamycscopicus  Some stuscopicus . The preon model . The comFor these studies, we utilized two well-characterized clinical isolates, LAC (Los Angeles County), representative of the USA300 group of organisms, closely related to the dominant CA-MRSA clone associated with soft tissue infections and serious invasive disease in the Memphis area and MW2, another clinical isolate from a Midwestern child with fatal CA-MRSA sepsis, representative of the USA400 group of organisms that constitute the other main lineage of CA-MRSA isolates in the United States , 35.δ-toxin and generously provided these strains for our studies: in this experiment we used the Δ-PSM-α-LAC-MRSA, a stable strain with a well-described diminished TNF response in comparison to the isogenic wild-type strain [Otto and DeLeo and colleagues recently generated isogenic strains of LAC and MW2 engineered to lack expression of PSMs or e strain , 36.5–107 CFU/mL) was studied, based upon our previously published data with other CA-MRSA strains and our preliminary experiments [E-test method in the microbiology laboratory at Le Bonheur Children's Hospital (LBCH). Both strains were fully susceptible to vancomycin . Beside bacteria for macrophage stimulation, we used bacterial products such as lipopolysaccharide (LPS), a component of Gram-negative bacteria cell walls, and lipoteichoic acid (LTA), a component of Gram-positive bacteria cell walls, in the experiments with rapamycin. This was done to confirm that the rapamycin effects are independent of any rapamycin-MRSA interaction. Bacteria were grown to late logarithmic phase at 37°C in tryptic soy broth  and washed three times in endotoxin-free phosphate-buffered saline. Concentrations were determined by colony counts. A range of concentrations of bacteria  supplemented with 10% fetal bovine serum  and 2 mM glutamine . Experiments were done in 24-well tissue culture plates  targeting 1 × 10μg/mL, was added to the cell cultures immediately before the addition of live staphylococci (105–107 CFU/mL). Cells were then incubated for 18 hours [Vancomycin, at a clinically achievable concentration of 20 μM), AP5 (3 mM), LY-294002 (500 nM), and NMDA substrate (30 μM). Rapamycin was added at concentrations of 0.1, 1, 10, and 100 ng/mL: the relevance of the concentrations selected have been described previously [μM, and the selected concentration (100 μM) is based on the achievable anesthetic concentrations, what is reported in the existing literature and our own work [These experiments were repeated in parallel and in various combinations in the presence of ketamine . These findings were consistent with our previous studies [After incubation, cell-free supernatants were collected and assayed for TNF concentrations by using a solid-phase sandwich enzyme-linked immunoabsorbent assay as specified by the manufacturer . TNF is a key cytokine produced by macrophages during MRSA stimulation. We measured secretion of other cytokines and found that IL-6 and IL-12 secretions were strongly correlated with TNF secretion in response to these bacteria -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) according to the manufacturer's instructions (Promega). The MTS reagent is reduced by metabolically active cells into a colored formazan product whose absorbance is then measured. In brief, MTS solution was added to wells of a 96-well microtiter plate, and the cells were incubated for 2 h. The absorbance at 490 nm was then measured.α LAC, and for MW2 wild-type MRSA strains. We confirmed uniform MRSA strain response in all of the experiments. There is an intrinsic experimental variation of absolute maximum values of TNF production in different cell culture flasks due to the unique cell culture and endogenous macrophage differences, a finding consistent in all our previous studies [For the comparison experiments, TNF secretion measurements were validated with an average of at least three well replicates and each of the experiments was repeated at least three times . The experiments were performed separately for LAC wild-type, Δ-PSM- studies –11. To m studies –48. The a priori) contrasts; that is, we set all our comparisons in advance of multiple setting experiments. Significant differences were presumed at a probability value of P < 0.05. When post hoc tests were used we applied the Bonferroni correction. The results were graphed as fold of increase compared to the control using error bars with 95% confidence intervals.The results were analyzed with R 2.13.1 and the ggplot2 graph package. They were validated by SPSS19-IBM. The experimental design consisted of factorial multiple measurements. The normality was assessed with the Kolmogorov-Smirnov test and a plot. The ANOVAs are between independent groups, and we tested the homogeneity of the variance with Levene's test. We set preplanned , LY-294002, or NMDA substrate served as the control. The control TNF production was contrasted with stimulation in the presence of the PI3 K inhibitor LY294002 (500 nM), NMDA substrate (30 μM), and a combination of both NMDA substrate and LY294002. The same sequence of exposure  was repeated in the presence of NMDA inhibitors ketamine (100 μM) or AP5 (3 mM). Our goal was to test the NMDA pathway downstream inhibition (PI3 K) or NMDA induction (NMDA substrate) alone or concurrently with NMDA inhibition (ketamine and AP5). RAW264.7 murine macrophages were stimulated for 18 hrs with 10P < 0.05). Adding NMDA substrate increased TNF production by 20% (P < 0.05). LY294002 in the presence of NMDA substrate decreased TNF secretion by only 8%—a difference not significantly different than the control. The addition of ketamine decreased TNF production by 35% (P < 0.05). When ketamine and LY294002 were added together, the reduction in TNF was significantly (33%) different than the control (P < 0.05) but not significantly different from the concentration induced by ketamine alone. NMDA and ketamine induced 7% suppression, which was not significantly different from the control response. The ketamine-NMDA-LY294002 response was decreased 17% and was significantly different from the control (P < 0.05). The results are depicted in The presence of LY294002 suppressed TNF production by 18% (P < 0.05. The level of TNF secretion in the presences of a combination of NMDA and LY294002 (8% decrease) was not significantly different from the control. AP5 caused 25% suppression of TNF production and AP5 plus LY294002 caused 33% suppression, both significant differences at P < 0.05. AP5 with NMDA and AP5 with NMDA and LY294002 (10% increase and 7% decrease) were not significantly different from the control. The responses were similar for both CA-MRSA strains MW2 and LAC (not shown). The results were similar when AP5 (APV) was used instead of ketamine in the same sequence of experimental settings (P < 0.05), and suppression with ketamine treatment of 25% and 22% of the control (P < 0.05). The control response for IL-6 ± standard error (SE) was 19 ± 0.6 pg/mL, and the response for IL-12 was 127 ± 3.2 pg/mL.IL-6 and IL-12 production demonstrated similar patterns of response when substrate NMDA was added—an increase of 13% and 11%, respectively  [Exposing unstimulated macrophages to rapamycin had no effect. Visual inspection of the monolayer, low-power microscopic inspection, and exclusion of 0.2% trypan blue dye confirmed that at least 90% of macrophages were viable after 18–24 hours of exposure. Thus, rapamycin did not affect the viability of macrophages during the experimental period. Neither the interaction of rapamycin with bacterially stimulated macrophages nor dose responses have been described. These had to be established before we considered manipulations of the NMDA-PI3 K-mTOR pathway. We tested several models of stimulation, including MW2 MRSA (wild-type), LAC MRSA (wild-type), Δ-PSM-Macrophages stimulated by MW2 (wild-type CA-MRSA) in the presence of 0.1 and 1 ng/mL of rapamycin exhibited an increase in production of TNF of 13% and 8%, respectively, but in the presence of 10 and 100 ng/mL they exhibited a decrease in TNF production ; the results are depicted in α-LAC-MRSA stimulation also caused a biphasic response: 7% and 20% increase with 0.1 and 1 ng/mL of rapamycin, and 17% and 11% decrease with 10 and 100 ng/mL of rapamycin . The responses in the presence of 0.1 and 1 ng/mL rapamycin for both IL-6 and IL-12 were not significantly different from the control. Suppression was noticed also with MRSA-LAC stimulation at 100 ng/mL rapamycin . In these experiments, the control response for IL-6 was 21 ± 0.8 pg/mL and the control response for IL-12 was 131 pg/mL ± 4.1 pg/mL.The analysis of specimens using IL-6 and IL-12 in the MRSA-MW2 stimulation model revealed suppression at the 10 ng/mL and 100 ng/mL rapamycin doses: 10% and 11% for IL-6 and 11% and 13% for IL-12, respectively . When ketamine was combined with rapamycin at 0.1 and 1 ng/mL, we noticed a decrease in TNF secretion of 22% and 18%, respectively, compared to the untreated control  in comparison to the control—a stronger suppression than that achieved with ketamine alone. In the presence of ketamine and rapamycin at 10 and 100 ng/mL, we noticed a decrease of TNF production of 43% and 47% (P < 0.05), 32% for ketamine + LY294002 + rapamycin at 0.1 ng/mL (P < 0.05), 37% for ketamine + LY294002 + rapamycin at 1 ng/mL (P < 0.05), 44% for ketamine + LY294002 + rapamycin at 10 ng/mL (P < 0.05), and 47% for ketamine + LY294002 + rapamycin at 100 ng/mL (P < 0.05). The results are depicted in Subsequently, we repeated the exposure in the presence of LY294002. TNF suppression became more uniform, that is, 28% for ketamine alone  of rapamycin were added, but not with LY294002. LY294002 is a PI3 K inhibitor, and although it did not exhibit an additive effect with ketamine, it suppressed TNF production in the CA-MRSA macrophage stimulation model by 20%. We attribute this finding to a common pathway inhibition (PI3 K) of ketamine and LY294002, which of course cannot be augmented by synergy.α-LAC, LPS, or LTA. Thus, there is an intrinsic system response to rapamycin. Adding ketamine (100 μM) to the CA-MRSA monocyte stimulation model pretreated with the concentration gradient of rapamycin caused 20% TNF suppression at 0.1 and 1 ng/mL of rapamycin and 40–50% TNF suppression at 10 and 100 ng/mL. The concentration-based biphasic response was abolished when the PI3 K/Akt inhibitor LY294002 was added and suppression became uniform  and approximately 10% suppression at higher concentrations (10 and 100 ng/mL). This biphasic response to rapamycin was observed after stimulation by MRSA-MW2 wild type and LAC wild type, Δ-PSM-mTORC2 is an inductive part of PI3 K/Akt. In the past, the rapamycin FKBP 12 complex was considered to be unable to modify mTORC2, but recent evidence shows the interaction to be time, cell, and dose dependent , 52. PosOur data suggest that a very desirable moderation of the TNF produced by CA-MRSA-stimulated RAW264.7 macrophages can be achieved with a combination of NMDA and mTOR inhibition, possibly with “higher” rapamycin doses, although more work is necessary to help us understand these relationships.NMDA inhibitors mediate an anti-inflammatory action on bacterially stimulated macrophages. The anti-inflammatory response can be augmented by treatment of cells with rapamycin at doses of 10 and 100 ng/mL. Such inhibition may be related to activation of the mTOR1/mTOR2 pathway. Such findings may have a significant impact on the immunological treatment of sepsis.


Understanding the pathogenesis of the chiari-like malformation in the Cavalier King Charles Spaniel (CKCS) is incomplete, and current hypotheses do not fully explain the development of syringomyelia (SM) in the spinal cords of affected dogs. This study investigates an unconventional pathogenetic theory for the development of cerebrospinal fluid (CSF) pressure waves in the subarachnoid space in CKCS with SM, by analogy with human diseases. In children with achondroplasia the shortening of the skull base can lead to a narrowing of the jugular foramina (JF) between the cranial base synchondroses. This in turn has been reported to cause a congestion of the major venous outflow tracts of the skull and consequently to an increase in the intracranial pressure (ICP). Amongst brachycephalic dog breeds the CKCS has been identified as having an extremely short and wide braincase. A stenosis of the JF and a consequential vascular compromise in this opening could contribute to venous hypertension, raising ICP and causing CSF jets in the spinal subarachnoid space of the CKCS. In this study, JF volumes in CKCSs with and without SM were compared to assess a possible role of this pathologic mechanism in the development of SM in this breed.3; median right JF: 0.0703 cm3; p < 0.0001) when compared with CKCSs with SM . There was no significant difference between the left and right JF within each group. Bland-Altman analysis revealed excellent reproducibility of all volume measurements.Computed tomography (CT) scans of 40 CKCSs > 4 years of age were used to create three-dimensional (3D) models of the skull and the JF. Weight matched groups (7–10 kg) of 20 CKCSs with SM and 20 CKCSs without SM were compared. CKCSs without SM presented significantly larger JF -volumes (median left JF: 0.0633 cmA stenosis of the JF and consecutive venous congestion may explain the aetiology of CSF pressure waves in the subarachnoid space, independent of cerebellar herniation, as an additional pathogenetic factor for the development of SM in this breed. Extensive studies have been carried out to explain a possible relationship between cranial and cervical dimensions and the development of the chiari-like malformation (CLM) and syringomyelia (SM) in the Cavalier King Charles Spaniel (CKCS). In an effort to identify pathogenetic alterations of the skull in this possible canine analogue to the human chiari malformation type 1, work has focused on a mismatch of caudal fossa capacity and its contents as hypothesized in man. ResultsIn the human literature there is debate as to whether obliteration of the foramen magnum is the only pathogenetic factor responsible for the development of CSF pressure waves, because herniation of less than 5 mm or even absent herniation can still be accompanied by classic Chiari symptomatology-10.More than one or even multiple factors might be etiologically important and should be considered in the pathogenesis of CLM/SM in the CKCS.Recently the CKCS has been shown to have an extremely high braincase length-width ratio  compared to other brachycephalic dogs. As onlyThe longitudinal extension of the whole braincase is strongly dependent on the growth of the skull base. The synAs determinations of the diameter of skull foramina can be variable depending on the imaging plane we decidMagnetic Resonance (MR)-images were used to identify SM in the cervical spinal cord . T1 and T2 weighted sagittal and transverse images of the head were obtained. T2 weighted and FLAIR sagittal images of the spinal cord up to the level of the fifth thoracic vertebra were acquired as well as a transverse fast field echo (FFE)-sequence of the spinal cord that was added if SM was present. All imaging studies were performed for breeding selection on the owner`s request. All dogs showed cerebellar indentation or deviation into the foramen magnum and obliteration of the CSF space around the caudal contour of the cerebellum (CLM). None of the dogs had a history of scratching or cervical hyperaesthesia; however, this was not an inclusion criterion. Dogs were selected according to the following criteria: Only adult dogs were chosen, as skull growth is incomplete in dogs <1 year. As SM cImage processing for volume determination of the JF was with done using graphical software . The internal boundaries of the JF were segmented using hand tracing slice by slice in sagittal, dorsal and transverse CT images FigureB. The doThe dorsal boundary was set along a line following the straight contour of the basioccipital bone from its midline laterally. The ventral boundary was set along a line perpendicular to the first line through the ventro-medial margin of the JF FigureC. AlthouMultiplanar image reconstruction Figure D and voVolumes of left and right JFs were determined for CKCSs with and without SM and compared. Volume determinations were carried out blinded to the MRI findings and the order of the datasets was randomised. All volume determinations were performed twice by the same examiner (MS) in two different sittings.All data was analysed using a statistical program package . Data was analysed for normality using a Kolmogorow-Smirnov test.We tested for significant differences in left and right JF volumes between group 1 and group 2 using a student`s t-test FigureA/C. As vFurthermore, to assess the precision of the manual segmentation process and the determined volumes, the reproducibility of the volume calculation was measured using a Bland-Altman analysis, which compares the differences between the first and second volume measurement of each dog. The differences between the two measurements were then plotted against the average (mean) of the two measurements. In the graphic presentation of this analysis Figure A-D, a fThereafter, a student`s t-test was again used to establish if there were statistically significant differences between the groups using the data of the second volume determination FigureB/D. P-vaThe volumes of the JF could be clearly determined. Figure3, and the median right JF volume of CKCSs without SM was 0.07 cm3. The median left JF volume of CKCSs with SM was 0.038 cm3, and the median right JF volume of CKCSs with SM was 0.043 cm3. Comparing the volumes of both the left and right JF of the CKCSs with SM to the CKCSs without SM revealed a clear statistically significant difference with p < 0.0001.Comparisons between JF volumes from the first measurement are presented in FigureThere was no significant difference between the volumes of the left and right JF within either group 1 (p = 0.145) or group 2 (p = 0.084).3, and the median right JF volume of CKCSs without SM was 0.07 cm3. The median left JF volume of CKCSs with SM was 0.045 cm3, and the median right JF volume of CKCSs with SM was 0.044 cm3. Comparing the volumes of both the left and right JF of CKCSs with SM to CKCSs without SM again demonstrated a clear statistically significant difference, with p < 0.0001.The results from the second measurement of left and right JF volumes are presented in FigureAlso in the second measurement there was no significant difference between the left and right JF volumes within either group 1 (p = 0.12) or group 2 (p = 0.096).The assessment of reproducibility is shown in FigureThese figures indicate there was little variation between measurements. The data shows that both the left and right JF of CKCSs with SM have significantly reduced volumes in comparison to CKCSs without SM.The cranial base is of crucial importance in skull development. Unlike most of the skull bones, it is formed through endochondral ossification of its cartilaginous anlages. During the ontogenesis of the skull, ossification centres form within these cartilages and the segments separating these centres are referred to as synchondroses. In the The volume of the endocranial cavity is reduced less than one would expect from the degree of shortening of the neurocranium. HoweverIn this study we focused on examination of the JF, because the other exit foramen for cerebral veins, the postglenoid foramen, has been found to be very variable. In canids the postglenoid foramen can even be divided so some dogs can have multiple postglenoid foramina. Its diameter is only 30% of the dimension of the JF in dogs. In contrast the JF is singular and characterized by minor variability in its morphology. We use The results presented in this study can explain turbulent flow of CSF in the subarachnoid space as a consequence of raised ICP. This concept is independent of possible disproportionate volumes of the caudal skull compartment and the cerebellum. The increase in ICP could further exacerbate the CSF pulse pressure created by the decreased compliance in the subarachnoid space, which is considered to be the driving force of SM. It can Although the findings of this study seem to present a plausible explanation for an increase in subarachnoid CSF pressure, it must be emphasized that this study merely presents morphological findings and definite data about a functional impairment of venous outflow needs to be obtained. Angiographic presentation of a collateral network of emissary veins trying to compensate for the constrained venous drainage would give further evidence of an existing functional impairment of blood flow.Sagittal sinus blood flow has been reported to be decreased in children with acondroplasia and constrained venous drainage from smaller JFs. DetermiFurthermore, treatment of raised ICP in these dogs might be directed at improving intracranial venous drainage rather than at vault expansion or CSF shunting procedures. Relief of raised ICP by surgical decompression of JF stenosis has been reported in children with achondroplasia and craniosynostosis, and ICPA stenosis of the JF has been identified in CKCSs with SM. A consecutive venous congestion may explain the aetiology of CSF pressure waves in the subarachnoid space independent from cerebellar herniation or as an additional pathogenetic factor for the development of SM in this breed.CLM: Chiari-like malformation; CKCS: Cavalier King Charles Spaniel; CSF: Cerebrospinal fluid; CT: Computed tomography; FLAIR: Fluid attenuated inversion recovery; FFE: Fast field echo; ICP: Intracranial pressure; JF: Jugular foramen; MR: Magnetic Resonance; SD: Standard deviation; SM: Syringomyelia; 3D: Three-dimensional.The authors declare that they have no competing interests.HV and MS conceived the study and planned the volume determinations, MK participated in the design and coordination of the study. NO performed all diagnostic imaging procedures and selected appropriate image sets. Maren Sauerbrey and CR performed the statistics. MS drafted the manuscript and NO and HV helped to draft the manuscript. All authors have approved the final manuscript.


Matrix metalloproteinase-9 (MMP-9) has been shown to be induced by cytokines including TNF-α and may contribute to bone inflammatory diseases. However, the mechanisms underlying MMP-9 expression induced by TNF-α in MC3T3-E1 cells remain unclear.We applied gelatin zymography, Western blot, RT-PCR, real-time PCR, selective pharmacological inhibitors of transcription , translation , c-Src (PP1), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), and NF-κB (Bay11-7082), respective siRNAs transfection, promoter assay, immunofluorescence staining, and ELISA to investigate the MMP-9 expression and soluble ICAM-1 (sICAM-1) release induced by TNF-α in MC3T3-E1 cells. Here we demonstrated that TNF-α-induced MMP-9 expression was attenuated by Act.D, CHI, PP1, U0126, SB202190, SP600125, and Bay11-7082, and by the transfection with siRNAs for ERK2, p38 MAPK, and JNK2. TNF-α-stimulated TNFR1, TRAF2, and c-Src complex formation was revealed by immunoprecipitation and Western blot. Furthermore, TNF-α-stimulated NF-κB phosphorylation and translocation were blocked by Bay11-7082, but not by PP1, U0126, SB202190, or SP600125. TNF-α time-dependently induced MMP-9 promoter activity which was also inhibited by PP1, U0126, SB202190, SP600125, or Bay11-7082. Up-regulation of MMP-9 was associated with the release of sICAM-1 into the cultured medium, which was attenuated by the pretreatment with MMP-2/9i, an MMP-9 inhibitor.In this study, we demonstrated that TNF-α up-regulates MMP-9 expression via c-Src, MAPKs, and NF-κB pathways. In addition, TNF-α-induced MMP-9 expression may contribute to the production of sICAM-1 by MC3T3-E1 cells. The interplay between MMP-9 expression and sICAM-1 release may exert an important role in the regulation of bone inflammatory diseases. Matrix metalloproteinases (MMPs) are a family of extracellular matrix (ECM)-degrading enzymes and induced by different stimuli including growth factors, cytokines, and tumor promoters . MMPs plSeveral proinflammatory mediators, including tumor necrosis factor-α (TNF-α) have been reported to be associated with many bone functions such as resorption and inflammation. The expression of MMPs has been shown to be regulated by several extracellular stimuli such as TNF-α and IL-1β in various cell types ,10. Numein vitro. In. In25]. in vitro. TNF-α sin vitro. The levin vitro,31. Howei.e. ERK1/2, p38 MAPK, and JNK1/2) and NF-κB is essential for the induction of the MMP-9 gene expression in these cells. Moreover, the induction and activation of MMP-9 are important for sICAM-1 release from MC3T3-E1 cells. These results provide new insights into the mechanisms of TNF-α action that the c-Src-dependent MAPKs and IKK/NF-κB may be associated with the MMP-9 up-regulation and the sICAM-1 release from osteoblasts-like MC3T3-E1 cells.In this study, the mechanisms underlying TNF-α-induced MMP-9 expression and the effects of increased MMP-9 on MC3T3-E1 cells were investigated. We found that the activation of three MAPKs (536), anti-phospho-c-Src, anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, and anti-phospho-IκB-α antibodies were from Cell Signaling . anti-NF-κB (p65), anti-lamin A, anti-TRAF2, anti-TNFR1, anti-c-Src, anti-ERK2, anti-p38, anti-JNK2, anti-IκB-α, and anti-sICAM-1 antibodies were from Santa Cruz . The anti-GAPDH antibody was from Biogenesis . Actinomycin D (Act.D), cycloheximide (CHI), PP1, U0126, SB202190, SP600125, GM6001, MMP2/9 inhibitor, and Bay11-7082 were from Biomol . Enzymes and other chemicals were from Sigma .Minimal essential medium-alpha (α-MEM), fetal bovine serum (FBS), and TRIzol were purchased from Invitrogen . Hybond C membrane and ECL Western blotting detection system were from Amersham Biosciences . Recombinant human TNF-α and the anti-TNFR1 neutralizing antibody were from R&D System . Luciferase assay kit was from Promega . Metafectene transfection reagent was from Biontex Lab . SMARTpool RNA duplexes corresponding to Src, TRAF2, ERK2, p38 MAPK, JNK2, and scrambled #2 siRNA were from Dharmacon Research Inc . Anti-phospho-IKKα/β, anti-phospho-NF-κB p65 (Ser2 atmosphere. When the cultures reach confluence (4 days), cells were treated with 0.05% (w/v) trypsin/0.53 mM EDTA for 5 min at 37°C. The cell suspension was diluted with α-MEM containing 10% (v/v) FBS to a concentration of 2 × 105 cells/ml. The cell suspension was plated onto (1 ml/well) 12-well culture plates and (10 ml/dish) 10-cm culture dishes for the measurement of protein expression and mRNA accumulation, respectively. Culture medium was changed after 24 h and then every 3 days.Murine osteoblastic cell line, MC3T3-E1, a homogeneous source of non-transformed cell, was a gift from Dr. Hyun-Mo Ryoo  . MC3T3-EMC3T3-E1 cells were plated onto 12-well culture plates and made quiescent at confluence by incubation in serum-free α-MEM for 24 h. Growth-arrested cells were incubated with TNF-α at 37°C for the indicated time intervals. When inhibitors were used, they were added 1 h prior to the application of TNF-α. The culture medium was collected and centrifuged at 14000 rpm for 5 min at 4°C to remove cells and debris, then each sample was mixed with equal amounts of (×2) non-reducible sample buffer and electrophoresed on 10% SDS-PAGE containing 1 mg/ml gelatin as the protease substrate, as previously described .g at 4°C for 1 h to yield the whole cell extract [Growth-arrested MC3T3-E1 cells were incubated with TNF-α at 37°C for the indicated time intervals. The cells were washed, scraped, collected, lysed with ice-cold lysis buffer, and centrifuged at 45,000  extract . SamplesTotal RNA was isolated from MC3T3-E1 cells treated with TNF-α for the indicated time intervals with TRIzol according to the protocol of the manufacturer. RNA concentration was spectrophotometrically determined at 260 nm. First-strand cDNA synthesis was performed with 2 μg of total RNA using random hexamers as primers in a final volume of 20 μl . The reaction was carried out at 37°C for 60 min. cDNAs encoding β-actin and MMP-9 were amplified from 3 to 5 μl of the cDNA reaction mixture using specific gene primers. Oligonucleotide primers for β-actin and MMP-9 were follow as: β-actin (514 bp): 5′-GAACCCTAAGGCCAACCGTG-3′ (sense), 5′-TGGCATAGAGGTCTTTACGG-3′ (antisense); MMP-9 (458 bp): 5′-TGTTCAGCAAGGGGCGTGTC-3′ (sense) 5′-AAACAGTCCAACAAGAAAGG-3′ (anti-sense). The amplification was performed in 35 cycles at 55°C, 1 min; 72°C, 1 min; 94°C, 1 min. After the last cycle, all samples were incubated for an additional 5 min at 72°C. The expression of β-actin was used as an internal control for the assay of a constitutively expressed gene.Real-time PCR was performed with the TaqMan gene expression assay system, using primers and probe mixes for MMP-9 and endogenous GAPDH control genes. PCRs were performed using a 7500 Real-Time PCR System . The primers were: 5′-(TGATGCCATTGCTGATATCCA)-3′ (sense), 5′-(CGGATCCTCAAAGGCTGAGT)-3′ (anti-sense) for MMP-9; 5′-(AACTTTGGCATCGTGGAAGG)-3′ (sense), 5′-(GTGGATGCAGGGATGATGTTC)-3′ (anti-sense) for GAPDH. Relative gene expression was determined by the ΔΔCt method, where Ct meant threshold cycle. All experiments were performed in triplicate (n = 3).Cell lysates containing 1 mg of protein were incubated with 2 μg of anti-TNFR1 antibody at 4°C for 24 h, and then 10 μl of 50% protein A-agarose beads was added and mixed at 4°C for 24 h. The immunoprecipitates were collected and washed thrice with a lysis buffer without Triton X-100. 5X Laemmli buffer was added and subjected to electrophoresis on 12% SDS-PAGE, and then blotted using the anti-TRAF2, anti-c-Src or anti-TNFR1 antibody.A 710-bp (−720 to −11) segment from the 5′-promoter region of the MMP-9 gene was cloned as described . Briefly6 cells/ml (1 ml/well) in 12-well culture plates for 24 h, reaching about 80% confluence. Cells were replaced with 0.4 ml of α-MEM containing 10% FBS. The DNA Metafectene reagent complex was prepared according to the instructions of the manufacturer . The amount of siRNA directed against, ERK2 , JNK2 , p38 , c-Src , TRAF2  or control siRNA was kept at 100 nM for each well. The DNA Metafectene complex (0.1 ml) was added to each well and then incubated at 37°C for 24 h. The cells were washed twice with PBS and maintained in α-MEM containing 1% FBS for 72 h before treatment with TNF-α for the indicated time intervals.MC3T3-E1 cells were plated at 1 × 10MC3T3-E1 cells were seeded in a 10-cm dish. After they reached 90% confluence, cells were starved for 24 h in serum-free α-MEM medium. After stimulation with 15 ng/ml TNF-α for various time intervals, and when inhibitors were used, they were added 1 h prior to the application of TNF-α. As previously described , the celMC3T3-E1 cells were plated on 6-well culture plates with coverslips. Cells were further cultured in serum-free α-MEM for 24 h and treated with 15 ng/ml TNF-α for various time intervals. When inhibitors were used, they were added 1 h prior to the application of TNF-α. After washing twice with ice-cold PBS, the cells were fixed with 4% (w/v) paraformaldehyde in PBS for 30 min, and then permeabilized with 0.3% Triton X-100 in PBS for 15 min. The staining was performed by incubating with 10% normal goat serum in PBS for 30 min followed by incubating with the primary NF-κB (p65) antibody (1:100 dilution) for 1 h in PBS with 1% BSA, washing thrice with PBS, incubating for 1 h with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibody (1:100 dilution) in PBS with 1% BSA, washing thrice with PBS, and finally mounting with aqueous mounting medium. The cell nucleus was stained by DAPI. The images were observed under a fluorescence microscope .sICAM-1 released into the culture media of MC3T3-E1 cells was collected and detected by using an ELISA kit according to the manufacturer’s instructions .P < 0.05 was considered significant.Concentration-effect curves were fitted and estimated by using the GraphPad Prism Program . Data were expressed as mean ± S.E.M. and analyzed by one-way ANOVA followed with Tukey’s post-hoc test. TNF-α has been shown to induce the expression of MMP-9 in human osteoblasts, osteoprogenitors, and mesenchymal stem cells ,36. To dTNF receptor l (TNFR1) and TNF receptor-associated factor 2 (TRAF2) generate distinct signals by TNF-α for the induction of differently biological responses in many cell types. Recent evidences suggest that MMP-9 expression was markedly suppressed in TNFR1 KO mice as compared to wild-type mice . PreviouMAPKs, including ERK1/2, p38 MAPK, and JNK1/2, can regulate expression of several genes through activation of downstream kinases or nuclear proteins. Previous study has demonstrated that TNF-α induces MMP-9 expression via p42/p44 MAPK and JNK1/2 in A549 cells . Here, tTo determine whether p38 MAPK is involved in TNF-α-induced MMP-9 expression, a p38 MAPK inhibitor (SB202190) was used. As shown in Figures In addition, to determine whether the activation of JNK1/2 is also involved in TNF-α-induced MMP-9 expression, a pharmacological inhibitor of JNK1/2 SP600125 was used. As shown in Figures Inflammatory responses following stimulation by TNF-α are highly dependent on activation of the transcription factor NF-κB. Moreover, NF-κB is one of the major mediators of the intracellular functions of TNF-α. Therefore, we investigated whether the involvement of NF-κB activation in TNF-α-induced MMP-9 expression in MC3T3-E1 cells, an NF-κB pharmacological inhibitor Bay11-7082 was used. As shown in Figure A significant response was obtained within 5–15 min and declined to the basal level within 30 min. To investigate whether TNF-α stimulates translocation of p65 NF-κB, the cytosolic and nuclear fractions were used to determine the p65 translocation by Western blotting using an anti-p65 antibody. The data showed that TNF-α time-dependently induced translocation of the p65 subunit of NF-κB into nucleus with a significant increase within 15–30 min , MEK1/2 (U0126), p38 MAPK (SB202190), or JNK1/2 (SP600125) for 1 h and then stimulated with TNF-α for the indicated time intervals. Phosphorylation of ERK1/2, p38 MAPK, JNK1/2, IKKα/β and p65 was assayed by Western blotting. As shown in Figure Several studies have shown that up-regulation of MMP-9 mRNA is mediated through an NF-κB-dependent pathway . MMP-9 pPrevious report has shown that TNF-α induces membrane and soluble forms of ICAM-1 release by MMP-9 activity in human osteoblast-like cells . Thus, wi.e. ERK1/2, p38 MAPK, and JNK1/2) and c-Src-independent IKK-NF-κB signaling pathways involved in TNF-α-induced MMP-9 expression and s-ICAM-1 release from MC3T3-E1 cells.MMP-9 is highly expressed in osteoclasts and plays an important role in degradation of ECM. Given the role of osteoblasts in building the bone, it is somewhat counter-intuitive that they also express many matrix degrading MMPs including MMP-2, MMP-3, MMP-8, MMP-9, MMP-13, and MMP-14 . It is gSeveral reports have indicated that most known responses to TNF-α are triggered by binding to one of two distinct receptors, TNFRl and TNFR2, which are differentially regulated on various cell types in normal and diseased tissues. In osteoblasts, TNF-α stimulates osteoblast differentiation through its TNFR1 receptor . Recent Several groups of investigators have reported that TNF-α released during acute and chronic diseases activates multiple intracellular signaling cascades including the MAPKs and NF-κB signaling pathways in various cell types. Previous reports have shown that aggregation of TNFR1/TRAF2 protein complex transducer activates downstream IKKα/β-NF-κB cascade and JNK1/2 and p38 MAPK in skeletal pathologies . TNF-α, i.e. ERK1/2, p38 MAPK, and JNK1/2) and c-Src-independent IKK/NF-κB pathways. MAPKs are serine/threonine protein kinases, which contribute to several cellular pathophysiological responses through regulation of their downstream molecules including transcription factors. Previous studies have indicated that TNF-α induces MMP-9 expression via a MAPK-dependent activation of NF-κB or AP-1 in several cell types [In addition, NF-κB is an inducible transcription factor that plays a key role in the expression of inflammatory response genes. NF-κB plays a pivotal role in bone remodeling cycle . TNF-α bll types ,39,49. HIn bone metabolism, ICAM-1 importantly mediates cell-cell adhesion of osteoblasts and osteoclast precursors, thereby facilitating osteoclast differentiation and bone resorption ,50. OsteOur study provides new insights into the mechanisms by which TNFR1/TRAF2 activates both IKKα/β-NF-κB and c-Src-ERK1/2, p38 MAPK, and JNK1/2 pathways may be associated with MMP-9 expression in osteoblasts-like MC3T3-E1 cells (Figure TNF-α: Tumor necrosis factor-α; MMP-9: Matrix metalloproteinase-9; MC3T3 cells: Mouse osteoblast-like cells; α-MEM: Minimal essential medium-alpha; FBS: Fetal bovine serum; ECL: Enhanced chemiluminescence; BCA: Bicinchoninic acid; PBS: Phosphate-buffered saline; BCA: Bicinchoninic acid; TNFR1: TNF receptor l; TRAF2: TNF receptor-associated factor 2; MAPKs: Mitogen-activated protein kinases; ERK: Extracellular-regulated protein kinase; JNK: c-Jun-N-terminal kinase; sICAM-1: Soluble intercellular adhesion molecule-1; IP: Immunoprecipitation; siRNA: Small interfering RNA; RT-PCR: Reverse transcription-polymerase chain reaction; NF-κB: Nuclear factor-kappa B.The authors declare that they have no competing interests related to this work.Conceived and designed the experiments: CLT, HLH, WCC, and CMY. Acquisition of data: CLT, PLC, and WCC. Analysis and interpretation of data: CMY. Drafting the article: CLT, HLH, WCC and LDH. Revising the article for important intellectual content: HLH and CMY. Final approval of the version to be published: CLT, HLH, PLC, WCC, LDH, and CMY.


Intraventricular injections of the fibroblast growth factor 2 (FGF2) are known to increase the size of the optic tectum in embryonic chicks. Here we show that this increase in tectum size is due to a delay in tectal neurogenesis, which by definition extends the proliferation of tectal progenitors. Specifically, we use cumulative labeling with the thymidine analog EdU to demonstrate that FGF2 treatment on embryonic day 4 (ED4) reduces the proportion and absolute number of unlabeled cells in the rostroventral tectum when EdU infusions are begun on ED5, as one would expect if FGF2 retards tectal neurogenesis. We also examined FGF2′s effect on neurogenesis in the caudodorsal tectum, which is born 2-3 days after the rostroventral tectum, by combining FGF2 treatment on ED4 with EDU infusions beginning on ED8. Again, FGF2 treatment reduced the proportion and number of EdU-negative  cells, consistent with a delay in neurogenesis. Collectively, these data indicate FGF2 in embryonic chicks delays neurogenesis throughout much of the tectum and continues to do so for several days after the FGF2 injection. One effect of this delay in neurogenesis is that tectal cell numbers more than double. In addition, tectal laminae that are born early in development become abnormally thin and cell-sparse after FGF2 treatment, whereas late-born layers remain unaffected. Combined with the results of prior work, these data indicate that FGF2 delays tectal neurogenesis and, thereby, triggers a cascade of changes in tectum size and morphology. Comparative work in evo-devo neurobiology has shown that evolutionary increases in brain region volumes are often due to delays in cell cycle exit of neuronal precursors unlabeled cells must have been born prior to the thymidine injection. By examining birds that were injected with tritiated thymidine at different stages of embryonic development, Lavail and Cowan were able to deduce cellular birthdates across the different parts of the tectum. They reported a rostroventral to caudodorsal gradient, with tectal neurogenesis beginning in the lateral rostroventral region as early as ED3 and ending in the medial caudodorsal region around ED9  rather than tritiated thymidine, because it can be detected more easily. As expected, we confirmed the neurogenetic gradient reported by Lavail and Cowan l tectum . SpecifiIn this study we also analyze FGF2-induced changes in the morphology of tectal laminae, including changes in laminar thickness and the number of cells above a unit area of ventricular zone surface. We focus these analyses on changes in the lateral and caudal areas of the tectum, as these areas correspond to regions where FGF2-induced laminar disturbances had been observed in our previous study What remains unclear from all this work is why the FGF2 effect in birds appears to be limited to the optic tectum, whereas it extends into the telencephalon in mammals Gallus gallus domesticus) were obtained from a commercial supplier and incubated in a rotating egg incubator  at 38° and 50–60% humidity. On embryonic day 4 (ED4), 0.5–1 µl of human recombinant bFGF  and dyed with methylene blue) was injected into the tectal ventricles. The injected FGF2 rapidly diffused throughout the ventricles, regardless of injection site. Control chicks were injected with 0.5–1 µl of dyed 0.1 M PBS. After injection, the eggs were resealed and transferred to the incubator.This study did not require approval of UC Irvine's Institutional Animal Care and Use Committee, because embryos are not covered by the relevant legislation. Fertile chicken eggs , dehydrated, embedded in paraffin, and sectioned at 8 µm. Roughly 20–30 evenly spaced sections from throughout the tectum were mounted onto Superfrost Plus slides (Fisher Scientific), stained with Giemsa , and cover-slipped. Based on examination of these Giemsa-stained sections, we identified specific tectal sectors in individual birds . For simOur EdU processing followed the protocol of Warren et al. All cell counts and laminar thickness measurements were made in ImageJ . Thickness measurements were performed on Giemsa-stained sections in lateral and caudal areas of the tectum . To test this prediction, we measured the number of cells radially above a unit area of ventricular zone surface (8 µm section thickness×100 µm) and found it to be 14% lower in FGF2 treated birds than in controls  expansion of some tectal laminae, which then expands the tectum's surface area. This hypothesis assumes that cell density within a laminae is not affected by FGF2.To test the latter hypothesis, we counted cells within 100 µm-wide sampling regions in individual tectal laminae see . We obseIn the caudal tectum the deep layers exhibited only an 11% decrease in cell numbers per counting box (t(19) = −2.34; P<0.05; n = 10, 11) in FGF2 treated birds relative to controls. There were no significant changes in cell number within the superficial or middle layers (t(19) = −0.24; P = 0.41; n = 10, 11). Thus, FGF2 has less effect on laminar thickness and cell numbers in the caudal tectum, where the expanded tectum folds , than inTo test whether FGF2 changes the neuronal birthdates of tectal laminae we counted the proportion of EdU-unlabeled cells in the major layers. In birds infused with EdU starting on ED5, the proportion of EdU unlabeled cells in the deep layers of the rostroventral tectum was decreased by 32% in FGF2 treated birds relative to controls  are clearly critical players. Much of the relevant work has been performed on mammalian cells Very little is known about the functions of FGF2 in birds. One study showed that FGF2 is present in the embryonic CSF of chicks and greatly enhances midbrain progenitor proliferation To test for an FGF2 induced delay of tectal neurogenesis, we focused our analysis on the early-born rostroventral tectum in embryos that were infused with EdU shortly after the beginning of tectal neurogenesis (ED5), and on the late-born caudodorsal tectum in embryos infused with EdU near the end of tectal neurogenesis (ED8). In both cases, a delay in cell cycle exit should manifest as an increase in the proportion of EdU-unlabeled cells . Indeed,Because a delay in cell cycle exit was evident in both the rostroventral and caudodorsal areas, we conclude that FGF2 is acting on much of the tectum, rather than focally. This finding is important because it shows that FGF2 has an effect throughout the tectum. A previous studies had shown that, of the three FGF receptors likely to interact with FGF2 inside the brain, only FGFR1 is expressed uniformly in the tectum In one set of experiments we injected FGF2 on ED4 and observed a delay in neurogenesis 4 days later, on ED8. This observation implies that FGF2 acted not just immediately after the injection on ED4, but for several days afterwards. Our observation that the FGF2-induced decrease in the number of EdU-unlabeled cells is greater when EdU is infused on ED5 than when it is infused on ED8 suggests that the potency of the injected FGF2 wanes over time, as one might expect, given that the injected FGF2 is never replenished. Alternatively, it may stem from a lower concentration of FGF2-binding receptors in the caudodorsal tectum, where we did our measurements in the embryos injected with EdU on ED8. Additional experiments are needed to disentangle these hypotheses.If FGF2 delays tectal neurogenesis, then one would expect FGF2 to increase the number of cells within the tectum dramatically. Indeed, the cell counts in the present study, in conjunction with the earlier data on tectal surface area expansion Given our findings, one may ask how a delay of tectal neurogenesis affects the birthdates of individual tectal layers, which are normally born at different, though overlapping stages of development Even more interesting is that FGF2 treatment reduces cell density and laminar thickness most drastically in the deep layers, mildly in the superficial layers, and least in the middle layers. This pattern parallels the birthdate data obtained by Lavail and Cowan


Passive blockade of tumor necrosis factor-alpha (TNF-α) has demonstrated high therapeutic efficiency in chronic inflammatory diseases, such as rheumatoid arthritis, although some concerns remain such as occurrence of resistance and high cost. These limitations prompted investigations of an alternative strategy to target TNF-α. This study sought to demonstrate a long-lasting therapeutic effect on established arthritis of an active immunotherapy to human (h) TNF-α and to evaluate the long-term consequences of an endogenous anti-TNF-α response.hTNF-α transgenic mice, which spontaneously develop arthritides from 8 weeks of age, were immunized with a heterocomplex  composed of hTNF-α and keyhole limpet hemocyanin after disease onset. We evaluated arthritides by clinical and histological assessment, and titers of neutralizing anti-hTNF-α antibody by enzyme-linked immunosorbent assay and L929 assay.Arthritides were dramatically improved compared to control mice at week 27. TNF-K-treated mice exhibited high levels of neutralizing anti-hTNF-α antibodies. Between weeks 27 and 45, all immunized mice exhibited symptoms of clinical deterioration and a parallel decrease in anti-hTNF-α neutralizing antibodies. A maintenance dose of TNF-K reversed the clinical deterioration and increased the anti-hTNF-α antibody titer. At 45 weeks, TNF-K long-term efficacy was confirmed by low clinical and mild histological scores for the TNF-K-treated mice. Injections of unmodified hTNF-α did not induce a recall response to hTNF-α in TNF-K immunized mice.Anti-TNF-α immunotherapy with TNF-K has a sustained but reversible therapeutic efficacy in an established disease model, supporting the potential suitability of this approach in treating human disease. Rheumatoid arthritis (RA) is a chronic autoimmune disease with an estimated prevalence of about 0.5% in the adult population. This disease, characterized by synovial membrane hyperplasia and immune cell infiltration, affects multiple peripheral joints and leads to destruction of bone and cartilage, inducing pain and disability. Although its precise etiology is still unknown, the pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-17, and more recently IL-23, have been shown to be critical mediators in the inflammatory process , two claActive immunotherapy is based on the established principles of vaccination. The aim of such a strategy is to use immunization with a protein compound to generate high titers of neutralizing antibodies to a given antigen, which can be either a self-protein or an environmental non-infectious agent. Therapeutic immunization has produced promising results in several fields, and in the case of active immunotherapy against cytokines (AIC), the choice of the target cytokine is informed by the long-term experience with mAbs, receptors, or antagonists in inflammatory and autoimmune diseases . Over thMore recently, with the aim of addressing diseases mediated by human TNF-α (hTNF-α), we developed an anti-hTNF-α compound called TNF kinoid (TNF-K), which is composed of biologically inactive but immunogenic hTNF-α conjugated to a carrier, keyhole limpet hemocyanin (KLH). We have tested TNF-K in hTNF-α transgenic (TTg) mice, which overexpress hTNF-α and develop an erosive polyarthritis that shares many features with RA ,15. ThisSix- to nine-week-old male hemizygous TTg mice (1006-T) were purchased from Taconic Farms  . These mWe obtained hTNF-α kinoid (TNF-K), a protein complex of hTNF-α and KLH, as previously described . DulbeccAll treatments were started after the onset of arthritis, when TTg mice reached an average clinical score of 3 out of 12. The experimental protocol was as follows  . PrecoatThe neutralizing capacity was assessed by using the L929 cytotoxicity assay, reflecting neutralizing antibodies . BrieflyThirty-six TTg mice received three IM injections of TNF-K emulsified in ISA-51 adjuvant at 7, 8, and 11 weeks of age. They were then randomly subdivided in two subgroups of ten and two subgroups of eight TTg mice. Neutralizing anti-hTNF-α antibody titers were monitored every month. When a decrease of 50% of the neutralizing capacity of these antibodies was observed, mice were intraperitoneally injected with native hTNF-α (10 ng), native hTNF-α (100 ng), KLH (10 μg), or PBS  24 weeks after the primary injection. Four weeks later, these mice received IM injections of the same compound with the same doses. The mice were further followed for 10 weeks. The native hTNF-α doses were chosen based on previous results we obtained in a TNF-α-dependent lethal shock experiment, in which we showed that 1 μg of native hTNF-α injections in TTg mice sensibilized with D-galactosamine was enough to kill the mice .Blinded weekly monitoring of body weight and arthritis scores in all four limbs was started from the reception of the animals (9 weeks of age). Clinical severity of arthritis for each paw  was quantified by attributing a score ranging from 0 to 3: 0, normal; 1, slight redness and swelling; 2, pronounced edematous swelling of the entire foot; 3, joint deformity and rigidity . The scoFor histological assessment of arthritis, all animals were sacrificed after 18-week or 36-week follow-up. Left forelimbs and right hind paws were collected, fixed with formol, decalcified, dehydrated, and included in paraffin blocks. Slides of 5 μm in thickness were made using a microtome. At least four serial sections were realized for each paw in order to obtain a reliable spatial evaluation of articular hints. Slides were then stained with hematoxylin and eosin or with safranin-O before microscopic observation . Synovitis and bone erosions were defined on slides stained with hematoxylin and eosin. Lesions were evaluated quantitatively on each slide using a 3-point scale ranging from 0 to 3, where 0 = normal articulation; 1 = slight inflammation and thickening of the synovium; 2 = mild thickening of the synovium and mild inflammation with invasion of the subsynovial area by inflammatory cells; 3 = severe inflammation and massive invasion of adjacent tissues by pannus . Other st test) or non-parametric  test was then performed. Post hoc comparisons were performed with the appropriate test according to data distribution (Student-Newman-Keuls for parametric data and Dunn test for non-parametric data). Clinical score time trend was analyzed by Spearman rho, and 95% confidence intervals (CIs) were given. Histological scores were compared with ANOVA or Kruskal-Wallis and their appropriate post hoc analysis according to data distribution. Differences in antibody titer at different time points were analyzed with repeated measures ANOVA due to normal distribution of data. Incidences of arthritis were compared using Fisher exact test with Yates correction. All statistics were performed with MedCalc statistical software version 10.4.8 .Data distribution was preliminarily checked by the Kolmogorov-Smirnov test. Serial measurements of clinical scores, body weight, antibody titers, and antibody neutralizing capacity were analyzed considering the area under the curve for each subject as a summary measure; these measures were then analyzed as raw data . AccordiP < 0.05 versus control group) . Based on a comparison of clinical scores, the TNF-K immunized and infliximab-treated mice showed comparable efficacy, with no statistically significant differences, although the infliximab has a more rapid efficacy than TNF-K immunization. We did not observe significant differences in body weight in any studied group , treatments were started for all of the mice. The control group (eight mice) was injected with PBS emulsified with ISA-51 adjuvant (PBS group) at 15, 16, and 19 weeks of age and developed severe arthritis over a 12-week period. At 27 weeks of age, these mice were euthanized for ethical reasons Figure . Compare) Figure , demonst) Figure , with lop Figure .P < 0.05)  Table . We did 5) Table .P < 0.05), demonstrating the transitory effect of anti-hTNF-α immunization . Histological comparisons were then made between groups of TNF-K immunized mice sacrificed at week 27 and those at week 45. This showed a mild progression of the disease over this 18-week period, with higher inflammation and destruction scores for all of the animals in the week 45 groups  and a deterioration for controls , supporting the efficacy of a maintenance dose of TNF-K in treating the late flare of arthritis  of TNF-K. Therefore, seven TTg mice that had received a primary course of three injections of TNF-K were administered a maintenance dose of TNF-K at 32 weeks of age. As a control, the remaining eight TTg mice that had received the primary course were injected with PBS emulsified in ISA-51 adjuvant. The arthritis clinical score curves decreased for mice that received the maintenance dose and increased for the controls Figure . The difs Figure .Histological inflammation and destruction were assessed at 45 weeks of age Table . All of We also compared the clinical efficacy of TNF-K active immunization with infliximab intermittent treatment on arthritis of TTg mice over this 18-week extension period. No statistically significant difference was detected between the two treatments Figure . HoweverP < 0.05)  hTNF-α after immunization with the TNF-K. We immunized TTg mice with TNF-K; once we observed a clear diminution of the neutralizing anti-hTNF-α antibody titer , it would not be thwarted by an immunization with TNF-K performed a long time before. Taken together, these data are consistent with the transient production and effect of neutralizing anti-hTNF-α antibodies after TNF-K immunization.Finally, we demonstrated that TNF-K and infliximab have comparable efficacy measured by clinical parameters in our model. Moreover, once infliximab weekly injections were discontinued (at 27 weeks of age), infliximab-treated mice exhibited a worsening of arthritides over time following the withdrawal of infliximab. Histopathological scores of these animals were significantly higher than those of TNF-K immunized mice, with or without late maintenance dose.in vivo B-cell memory response to native hTNF-α are all elements supporting a favorable benefit-risk ratio for such a strategy and a transient response against hTNF-α after TNF-K immunization. Further studies should be performed to evaluate the risk of infections or tumors under TNF-K treatment in dedicated models since their occurrences are a matter of debate in patients treated with passive immunotherapies against TNF-α [Our data show that active immunotherapy with TNF-K induced a long-lasting improvement in an RA model. The occurrence of a disease flare in previously immunized mice, the bell-shaped neutralizing anti-hTNF-α antibody curve, the increase of anti-hTNF-α neutralizing antibodies after a maintenance dose, and the absence of evidence of st TNF-α ,29.ANOVA: analysis of variance; CI: confidence interval; ELISA: enzyme-linked immunosorbent assay; hTNF-α: human tumor necrosis factor-alpha; IL: interleukin; IM: intramuscular; IP: intraperitoneal; KLH: keyhole limpet hemocyanin; mAb: monoclonal antibody; OD: optical density; PBS: phosphate-buffered saline; RA: rheumatoid arthritis; TNF-α: tumor necrosis factor-alpha; TNF-K: tumor necrosis factor kinoid; TTg: human tumor necrosis factor-alpha transgenic.GVo and ML are scientists with Neovacs SA , and DZ is a shareholder of Neovacs SA. TNF-K is patented and the patent is held by Neovacs SA. GVu is a scientist with Debiopharm SA . The other authors declare that they have no competing interests.LD and M-CB shared responsibility for the study design and manuscript preparation and helped to interpret the data and to perform the animal experiments. GVo shared responsibility for the study design and helped to interpret the data. GVu and NB shared responsibility for the study design. LS shared responsibility for manuscript preparation and helped to interpret the data and to perform the statistical analysis. DZ shared responsibility for manuscript preparation. EA helped to perform the animal experiments. ML performed the ELISA and L929 cytotoxic assay. All authors read and approved the final manuscript.TNF-K immunization protocol scheme. Long-term follow -up of the experiment is represented by horizontal arrow with time expressed in week . Slashes represent discontinuation of time. A- Control group treated with PBS/ISA-51; B- TNF-K group; C- Intermittent infliximab group. The follow-up for each group  is represented by a larger black line, with vertical black arrows at each time where treatment was given. IP injections, intraperitoneal injections.Click here for fileEvolution of neutralizing anti-hTNF-α antibody titers during time, in TTg mice immunized with TNF-K. 36 TTg mice were immunized with TNFK at days 0, 7 and 28. Bleeding was done every month from day 38 post primo-injection to sacrifice. Results are expressed as mean ± SEM of all the sera of all the 36 immunized mice.Click here for file


Enterovirus 74 (EV74) is a rarely detected viral infection of children. In 2010, EV74 was identified in New Zealand in a 2 year old child with acute flaccid paralysis (AFP) through routine polio AFP surveillance. A further three cases of EV74 were identified in children within six months. These cases are the first report of EV74 in New Zealand. In this study we describe the near complete genome sequence of four EV74 isolates from New Zealand, which shows only limited sequence identity in the non-structural proteins when compared to the other two known EV74 sequences. As is typical of enteroviruses multiple recombination events were evident, particularly in the P2 region and P3 regions. This is the first complete EV74 genome sequenced from a patient with acute flaccid paralysis. Picornaviridae, comprises more than 110 serotypes . Further assembly and annotation was achiev< 1µg) for direct inclusion in the Roche GS FLX sequencer library preparation, so Multiple strand Displacement Amplification (MDA) using the Quantitect Whole Transcriptome Kit  was employed in combination with a SuperScript III First-Stand Synthesis kit, random hexamers and protocol. Two amplification methods were trialled, differing only with the omission of the ligation step in one of the amplification reactions . 500 ng of amplified DNA was nebulized and used to make a barcoded, GS FLX Rapid Library  according to the manufacturer’s instructions. Completed library length was between 500 bp and 900 bp with a concentration of 3.45 x 10ed using Geneious 5.5.6 .All four New Zealand strains were sequenced as described previously . Nucleic acid was reverse transcribed into cDNA using random hexamers and the Superscript III First-Strand Synthesis System for RT-PCR. cDNA was then amplified using Platinum® PCR SuperMix . After an initial activation for 2 minutes at 94°C, a three-step cycling procedure was used consisting of; 40 cycles of denaturation at 94°C for 1 minute, annealing at 45°C for 1 minute and extension at 72°C for 2 minutes. A final extension took place at 72°C for 5 minutes. Samples were run on an E-Gel EX 2% precast agarose gel . DNA bands of interest were extracted and purified using the QIAquick PCR Purification Kit  and sequenced using an ABI 3130XL automated DNA sequencer and ABI Big Dye v3.1 technology (Life Technologies).New Zealand isolate sequences were deposited in the NCBI Genbank database and assigned accession numbers KC568446 (NZ45), KC568447, (NZ234), KC568448 (NZ2205) and KC568449 (NZ2284). These sequences were aligned with complete prototype EV-B genome sequences using Geneious 5.6.5 and CLUSTALW (gap open cost 15 and gap extend cost 6.66). See Aligned sequences were analysed using SimPlot version 3.5.1  to identFour samples from EV74 infected patients were received within a six month period coinciding with the summer in New Zealand. All four cases came from the central North Island, within 400 km (250 miles) of each other . ConsistDe novo assembly of all enterovirus sequence reads provided coverage of 91% of the EV74 genome with an average depth of 65.Deep sequencing of isolate NZ2284 yielded 60,274 sequences ranging between 53 bps and 1200 bps in length before quality trimming. A BLASTN and BLASTX search were performed on the quality-checked dataset which identified 43,824 (73%) of the 60,284 reads as EV and 5,435 reads (9%) specifically as EV74. Closer examination of EV74 BLASTN alignments revealed 39% of sequences aligned with the VP1 region. A sanger–sequencing approach consisting of eight overlapping amplicons was employed to provide full genome sequences for all four New Zealand EV74 samples. All but 30-58 nt of the 5’ NTR was not determined for each EV74 strain with each sequence ranging in length from 7311-7359nt. Complete concordance was observed between deep-sequencing and sanger-sequencing data for isolate NZ2284.Similarity Plot, Bootscan analysis, pairwise sequence comparison and multiple alignments were used to examine the relationship of New Zealand isolates to the two existing full-length EV74 reference sequences available in the public databases. The four New Zealand EV74 isolates showed an average of 92.1% and 92.8% nucleotide sequence identity to the VP1 gene of EV74 reference genomes USA/CA75-10213 (AY556057) and Rikaze-136/XZ/CHN/2010 (JQ397329), respectively, confirming that EV74 was the correct genotype designation for these isolates . Whole gPhylogenetic trees were constructed for each gene and the P1, P2 and P3 coding regions  with allAs a contemporary strain is likely to be a closer ancestor, rather than a reference strain, a BLAST search was performed for each gene region to identify the most similar sequences . UnsurprFour New Zealand EV74 viruses were isolated and sequenced showing 98.7% nucleotide sequence identity. These viruses were isolated during a six month interval within 400km of each other. These genetic and epidemiological indicators suggest a common circulating strain. There was no apparent genetic difference between the NZ2284 strain isolated from a patient with AFP, and other isolates from patients with milder symptoms. This is consistent with previous observations that the severity of symptoms does not necessarily correlate with genomic changes in the viral genome [Full genome sequencing of all four isolates revealed recombination had taken place within the non-structural coding region when compared to either of the two prototypic reference strains. This can be expected, as the California strain, USA/CA75-10213, was isolated in 1975, given the time that has elapsed and frequency of recombination among the EV B species . Work byDuring 2010, an EV74 virus was isolated in China, strain Rikaze-136/XZ/CHN/2010 . DespiteThe determination of the sites of recombination in the NZ EV74 isolates is complexand not the focus of this paper. No recombination was observed within the structural region of EV74  consistent with previous findings that recombination within this region occurs only rarely . SimplotThere is greater sequence homology in the P3 region of the NZ EV74 strains, with a common sequence related to E1, E9, EV84 and EV101 viruses. Again, this situation is complex as when compared to contemporary strains further recombination is evident.The occurrence and clustering of rare enteroviruses in the human population, such as the four EV74 isolates we reported here, reiterates that on-going surveillance is necessary to determine the epidemiology of these viruses. Furthermore, detailed characterisation of the viral genomes is required to understand the evolution of rare enteroviruses and to assist in the assessment of their potential threat as an emergent disease. The genetic data we report in this study support the notion that there was a cluster of EV74 cases in New Zealand, but do not support the hypothesis that AFP could be caused by a distinct genetic lineage of EV74.