Inactivation of PPARβ/δ adversely affects satellite cells and reduces postnatal myogenesis.

Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a ubiquitously expressed gene with higher levels observed in skeletal muscle. Recently, our laboratory showed (Bonala S, Lokireddy S, Arigela H, Teng S, Wahli W, Sharma M, McFarlane C, Kambadur R. J Biol Chem 287: 12935-12951, 2012) that PPARβ/δ modulates myostatin activity to induce myogenesis in skeletal muscle. In the present study, we show that PPARβ/δ-null mice display reduced body weight, skeletal muscle weight, and myofiber atrophy during postnatal development. In addition, a significant reduction in satellite cell number was observed in PPARβ/δ-null mice, suggesting a role for PPARβ/δ in muscle regeneration. To investigate this, tibialis anterior muscles were injured with notexin, and muscle regeneration was monitored on days 3, 5, 7, and 28 postinjury. Immunohistochemical analysis revealed an increased inflammatory response and reduced myoblast proliferation in regenerating muscle from PPARβ/δ-null mice. Histological analysis confirmed that the regenerated muscle fibers of PPARβ/δ-null mice maintained an atrophy phenotype with reduced numbers of centrally placed nuclei. Even though satellite cell numbers were reduced before injury, satellite cell self-renewal was found to be unaffected in PPARβ/δ-null mice after regeneration. Previously, our laboratory had showed (Bonala S, Lokireddy S, Arigela H, Teng S, Wahli W, Sharma M, McFarlane C, Kambadur R. J Biol Chem 287: 12935-12951, 2012) that inactivation of PPARβ/δ increases myostatin signaling and inhibits myogenesis. Our results here indeed confirm that inactivation of myostatin signaling rescues the atrophy phenotype and improves muscle fiber cross-sectional area in both uninjured and regenerated tibialis anterior muscle from PPARβ/δ-null mice. Taken together, these data suggest that absence of PPARβ/δ leads to loss of satellite cells, impaired skeletal muscle regeneration, and postnatal myogenesis. Furthermore, our results also demonstrate that functional antagonism of myostatin has utility in rescuing these effects.

Postmortem serum protein growth arrest-specific 6 levels in sepsis-related deaths.

Growth arrest-specific 6 (Gas6) is widely expressed in leukocytes, platelets, endothelial cells, and monocytes. It regulates various processes including granulocyte adhesion to the endothelium, cell migration, thrombus stabilization, and cytokine release. In humans, increased plasma Gas6 levels have been described in patients with sepsis and septic shock. In this study, Gas6 concentrations were measured in postmortem serum from femoral blood in a series of sepsis-related fatalities and control cases. The aims were twofold: first, to determine whether Gas6 can be reliably determined in postmortem serum; and second, to assess its diagnostic potential in identifying sepsis-related deaths. Two study groups were prospectively formed, a sepsis-related fatalities group (24 cases) and a control group (24 cases) including cases of deep vein thrombosis and fatal pulmonary embolism, cases of systemic inflammatory response syndrome in severe trauma, cases of end-stage renal failure, and cases of hanging (non-septic, non-SIRS, non-end stage renal failure cases). The preliminary results of this study seem to indicate that Gas6 can be effectively measured in postmortem serum. However, Gas6 levels in sepsis-related fatalities do not appear to be clearly distinguishable from concentrations in pulmonary embolism, severe trauma, and end-stage renal failure cases. These findings tend to support previous reports that indicated that Gas6 behaves as an acute phase reactant and can be considered a general marker of inflammation rather than a specific biomarker of sepsis.

A Novel Approach to Major Surgery: Tracking Its Pathophysiologic Footprints.

BACKGROUND: To study the 'metabolic profile' of different surgical procedures and correlate it with pertinent surgical details and postoperative complications. METHODS: We conducted a prospective pilot study of 70 patients, ten for each of the seven following groups: (1) laparoscopic cholecystectomy, (2) incisional hernia repair, (3) laparoscopic and (4) open colon surgery, (5) upper gastrointestinal, (6) hepatic, and (7) pancreatic resections. Biochemical assessment included white blood cell count (WBC), C-reactive protein (CRP), glucose, triglycerides (TG), albumin (Alb), and pre-albumin (Pre-Alb), from the day before surgery until 5 days thereafter. Biological markers were compared for major versus minor surgery groups, which were defined on a clinical basis. Univariable analysis was used to identify risk factors for postoperative complications and p < 0.05 was the significance threshold. RESULTS: Common findings in all surgery groups were the acute inflammatory response (↑: WBC, CRP, ↓: TG, Alb, pre-Alb). Using cut-off values of 240 min operative (OR) time and 300 ml estimated blood loss (EBL), laparoscopic cholecystectomy, incisional hernia repair, and laparoscopic colectomy could be distinguished from open colectomy, upper gastrointestinal, liver, and pancreas resections. In a biochemical level, increased CRP and reduced postoperative Alb levels were highly discriminative of all types of 'major surgery.' Significant risk factors for postoperative complications were age, male gender, malignancy, longer OR time, higher blood loss, high CRP, and low Alb levels. CONCLUSIONS: Biochemically, CRP and Alb levels can help quantify the magnitude of the surgical trauma, which is correlated with adverse outcomes.

New cationic nanovesicular systems containing lysine-based surfactants for topical administration: Toxicity assessment using representative skin cell lines

Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Cationic nanovesicles have attracted considerable interest as effective carriers to improve the delivery of biologically active molecules into and through the skin. In this study, lipid-based nanovesicles containing three different cationic lysine-based surfactants were designed for topical administration. We used representative skin cell lines and in vitro assays to assess whether the cationic compounds modulate the toxic responses of these nanocarriers. The nanovesicles were characterized in both water and cell culture medium. In general, significant agglomeration occurred after 24 h incubation under cell culture conditions. We found different cytotoxic responses among the formulations, which depended on the surfactant,cell line (3T3, HaCaT, and THP-1) and endpoint assayed (MTT, NRU, and LDH). Moreover, no potential phototoxicity was detected in fibroblast or keratinocyte cells, whereas only a slight inflammatory response was induced, as detected by IL-1a and IL-8 production in HaCaT and THP-1 cell lines, respectively. A key finding of our research was that the cationic charge position and the alkyl chain length of the surfactants determine the nanovesicles resulting toxicity. The charge on the a-amino group of lysine increased the depletion of cell metabolic activity, as determined by the MTT assay, while a higher hydrophobicity tends to enhance the toxic responses of the nanovesicles. The insights provided here using different cell lines and assays offer a comprehensive toxicological evaluation of this group of new nanomaterials.

Mir-21-Sox2 Axis Delineates Glioblastoma Subtypes with Prognostic Impact.

UNLABELLED: Glioblastoma (GBM) is the most aggressive human brain tumor. Although several molecular subtypes of GBM are recognized, a robust molecular prognostic marker has yet to be identified. Here, we report that the stemness regulator Sox2 is a new, clinically important target of microRNA-21 (miR-21) in GBM, with implications for prognosis. Using the MiR-21-Sox2 regulatory axis, approximately half of all GBM tumors present in the Cancer Genome Atlas (TCGA) and in-house patient databases can be mathematically classified into high miR-21/low Sox2 (Class A) or low miR-21/high Sox2 (Class B) subtypes. This classification reflects phenotypically and molecularly distinct characteristics and is not captured by existing classifications. Supporting the distinct nature of the subtypes, gene set enrichment analysis of the TCGA dataset predicted that Class A and Class B tumors were significantly involved in immune/inflammatory response and in chromosome organization and nervous system development, respectively. Patients with Class B tumors had longer overall survival than those with Class A tumors. Analysis of both databases indicated that the Class A/Class B classification is a better predictor of patient survival than currently used parameters. Further, manipulation of MiR-21-Sox2 levels in orthotopic mouse models supported the longer survival of the Class B subtype. The MiR-21-Sox2 association was also found in mouse neural stem cells and in the mouse brain at different developmental stages, suggesting a role in normal development. Therefore, this mechanism-based classification suggests the presence of two distinct populations of GBM patients with distinguishable phenotypic characteristics and clinical outcomes. SIGNIFICANCE STATEMENT: Molecular profiling-based classification of glioblastoma (GBM) into four subtypes has substantially increased our understanding of the biology of the disease and has pointed to the heterogeneous nature of GBM. However, this classification is not mechanism based and its prognostic value is limited. Here, we identify a new mechanism in GBM (the miR-21-Sox2 axis) that can classify ∼50% of patients into two subtypes with distinct molecular, radiological, and pathological characteristics. Importantly, this classification can predict patient survival better than the currently used parameters. Further, analysis of the miR-21-Sox2 relationship in mouse neural stem cells and in the mouse brain at different developmental stages indicates that miR-21 and Sox2 are predominantly expressed in mutually exclusive patterns, suggesting a role in normal neural development.

Intracellular ATP Decrease Mediates NLRP3 Inflammasome Activation upon Nigericin and Crystal Stimulation.

Activation of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome initiates an inflammatory response, which is associated with host defense against pathogens and the progression of chronic inflammatory diseases such as gout and atherosclerosis. The NLRP3 inflammasome mediates caspase-1 activation and subsequent IL-1β processing in response to various stimuli, including extracellular ATP, although the roles of intracellular ATP (iATP) in NLRP3 activation remain unclear. In this study, we found that in activated macrophages artificial reduction of iATP by 2-deoxyglucose, a glycolysis inhibitor, caused mitochondrial membrane depolarization, leading to IL-1β secretion via NLRP3 and caspase-1 activation. Additionally, the NLRP3 activators nigericin and monosodium urate crystals lowered iATP through K(+)- and Ca(2+)-mediated mitochondrial dysfunction, suggesting a feedback loop between iATP loss and lowering of mitochondrial membrane potential. These results demonstrate the fundamental roles of iATP in the maintenance of mitochondrial function and regulation of IL-1β secretion, and they suggest that maintenance of the intracellular ATP pools could be a strategy for countering NLRP3-mediated inflammation.

High expression levels of macrophage migration inhibitory factor sustain the innate immune responses of neonates.

The vulnerability to infection of newborns is associated with a limited ability to mount efficient immune responses. High concentrations of adenosine and prostaglandins in the fetal and neonatal circulation hamper the antimicrobial responses of newborn immune cells. However, the existence of mechanisms counterbalancing neonatal immunosuppression has not been investigated. Remarkably, circulating levels of macrophage migration inhibitory factor (MIF), a proinflammatory immunoregulatory cytokine expressed constitutively, were 10-fold higher in newborns than in children and adults. Newborn monocytes expressed high levels of MIF and released MIF upon stimulation with Escherichia coli and group B Streptococcus, the leading pathogens of early-onset neonatal sepsis. Inhibition of MIF activity or MIF expression reduced microbial product-induced phosphorylation of p38 and ERK1/2 mitogen-activated protein kinases and secretion of cytokines. Recombinant MIF used at newborn, but not adult, concentrations counterregulated adenosine and prostaglandin E2-mediated inhibition of ERK1/2 activation and TNF production in newborn monocytes exposed to E. coli. In agreement with the concept that once infection is established high levels of MIF are detrimental to the host, treatment with a small molecule inhibitor of MIF reduced systemic inflammatory response, bacterial proliferation, and mortality of septic newborn mice. Altogether, these data provide a mechanistic explanation for how newborns may cope with an immunosuppressive environment to maintain a certain threshold of innate defenses. However, the same defense mechanisms may be at the expense of the host in conditions of severe infection, suggesting that MIF could represent a potential attractive target for immune-modulating adjunctive therapies for neonatal sepsis.

Netting neutrophils in skin wounds recruit and activate plasmacytoid dentritic cells

Les mécanismes qui régulent le processus de guérison de la peau lésée ne sont pas entièrement compris. Nous avons précédemment montré que les cellules dendritiques plasmocytoïdes (pDCs) sont normalement absentes de la peau saine mais infiltrent rapidement la peau humaine ainsi que celle des souris après une blessure cutanée. Après avoir infiltré la peau, ces pDCs sont capables de détecter les acides nucléiques par l'expression des récepteurs de type Toll 7 et 9 ce qui les active à produire de 1' interféron (IFN) de type I. Ce processus est primordial pour la re- épithélisation des blessures cutanées. Cependant, les mécanismes conduisant à l'infiltration et à 1'activation des pDCs restent inconnus. Dans notre projet, nous montrons que la chimiokine CxcllO est responsable de l'infiltration des pDCs. De façon importante, nous démontrons que les neutrophiles qui infiltrent également la peau lésée sont la source majeure de cette chimiokine. La déplétion des neutrophiles abolit d'ailleurs le recrutement des pDCs confirmant ainsi que CxcllO produit par les neutrophiles est responsable de l'infiltration des pDCs dans la peau endommagée. De façon intéressante, nous avons trouvé que CxcllO en plus de son activité chimiotactique, est capable de former des complexes avec l'ADN et d'activer ainsi les pDCs à produire de l'IFN de type I. De plus, nous avons observé que les neutrophiles qui infiltrent la peau forment des Neutrophil Extracellular Traps (NETs). Ces NETs sont constitués de filaments extracellulaires d'ADN recouverts par de nombreuses protéines principalement d'origine granulaire. D'une manière frappante, le blocage de la NETose ou l'utilisation de souris déficientes pour la formation de NETs altère le recrutement et l'activation des pDCs ainsi que la réponse inflammatoire qui en découle ainsi que le processus de re-epithélisation qui s'ensuit. En prenant en compte toutes ces données, nos résultats démontrent que suite à une blessure de la peau, les neutrophiles par la production de CxcllO contrôlent l'infiltration des pDCs dans la peau lésée et par la formation de NETs, promeuvent l'activation des pDCs. Notre étude fournit donc de nouvelles informations sur les mécanismes de guérison de la peau et ouvre de nouvelles perspectives thérapeutiques quant à la réparation tissulaire de la peau soit dans le but de l'amplifier ou de l'inhiber. -- The mechanisms that regulate healing of the injured skin are not well understood. We have previously shown that plasmacytoid dendritic cells (pDCs) are normally absent from the healthy skin, but rapidly infiltrate both murine and human skin upon injury. Upon skin infiltration, pDCs sense nucleic acids via TLR7/TLR9 and are activated to produce type I interferon (IFN), a process that is crucial for re-epithelialisation of skin wounds. However, the mechanisms that drive pDCs recruitment and activation in injured skin remain unclear. We show that CxcllO is responsible for pDCs infiltration. Importantly, we demonstrate that skin infiltrating neutrophils are the major source of this chemokine. Neutrophils depletion completely abrogated pDCs recruitment confirming that CxcllO- driven pDCs recruitment is controlled by neutrophils. Interestingly, CxcllO was also found to form complexes with DNA and to activate pDCs to produce Type I IFN in addition to its chemotactic activity. Moreover, we observed that infiltrating neutrophils release Neutrophils Extracellular Traps (NETs) composed of DNA filaments decorated with neutrophils-derived proteins. Strikingly, blocking NETosis or using mice deficient for NETs production impaired pDCs recruitment and activation as well as the subsequent inflammatory response and the re-epithelialisation process. Altogether, these data demonstrate that upon skin injury, neutrophils control pDCs infiltration into the injured skin by the release of CxcllO and via the production of NETs, they allow complex formation between CxcllO and NET-DNA leading to pDCs activation. Our findings provide new insights into the mechanisms of wound healing and open new avenues for potential therapeutic interventions to boost or inhibit wound repair in the skin.

The lipopolysaccharide of Aeromonas spp: structure-activity relationships.

Marine microorganisms, including Aeromonas, are a source of compounds for drug development that have generated great expectations in the last decades. Aeromonas infections produce septicaemia, and ulcerative and haemorrhagic diseases in fish. Among the pathogenic factors associated with Aeromonas, the lipopolysaccharides (LPS), a surface glyconconjugate unique to Gram-negative bacteria consisting of lipid A (lipid anchor of the molecule), core oligosaccharide and O-specific polysaccharide (O antigen), are key elicitors of innate immune responses. The chemical structure of these three parts has been characterized in Aeromonas. Based on the high variability of repeated units of O-polysaccharides, a total of 97 O-serogroups have been described in Aeromonas species, of which four of them (O:11; O:16; O:18 and O:34) account for more than 60% of the septicemia cases. The core of LPS is subdivided into two regions, the inner (highly conserved) and the outer core. The inner core of Aeromonas LPS is characterized by the presence of 3-deoxy-D-manno-oct-2-ulosonic (ketodeoxyoctonic) acid (Kdo) and L-glycero-D-manno-Heptoses (L,D-Hep), which are linked to the outer core, characterized by the presence of Glc, GlcN, Gal, and GalNAc (in Aeromonas salmonicida), D,D-Hep (in Aeromonas salmonicida), and L,D-Hep (in Aeromonas hydrophila). The biological relevance of these differences in the distal part of the outer core among these species has not been fully assessed to date. The inner core is attached to the lipid A, a highly conserved structure that confers endotoxic properties to the LPS when the molecule is released in blood from lysed bacteria, thus inducing a major systemic inflammatory response known as septic or endotoxic shock. In Aeromonas salmonicida subsp. salmonicida the Lipid A components contain three major lipid A molecules, differing in acylation patterns corresponding to tetra-, penta- and hexaacylated lipid A species and comprising of 4′-monophosphorylated β-2-amino-2-deoxy-D-glucopyranose-(1→6)-2-amino-2-deoxy-D-glucopyranose disaccharide. In the present review, we discuss the structure-activity relationships of Aeromonas LPS, focusing on its role in bacterial pathogenesis and its possible applications.

The role of endothelial enzymes NOS, VAP-1 and CD73 in acute lung injury

Acute lung injury (ALI) is a syndrome of acute hypoxemic respiratory failure with bilateral pulmonary infiltrates that is not caused by left atrial hypertension. Since there is no effective treatment available, this frequent clinical syndrome significantly contributes to mortality of both medical and surgical patients. Great majority of the patients with the syndrome suffers from indirect ALI caused by systemic inflammatory response syndrome (SIRS). Sepsis, trauma, major surgery and severe burns, which represent the most common triggers of SIRS, often induce an overwhelming inflammatory reaction leading to dysfunction of several vital organs. Studies of indirect ALI due to SIRS revealed that respiratory dysfunction results from increased permeability of endothelium. Disruption of endothelial barrier allows extravasation of protein-rich liquid and neutrophils to pulmonary parenchyma. Both under normal conditions and in inflammation, endothelial barrier function is regulated by numerous mechanisms. Endothelial enzymes represent one of the critical control points of vascular permeability and leukocyte trafficking. Some endothelial enzymes prevent disruption of endothelial barrier by production of anti-inflammatory substances. For instance, nitric oxide synthase (NOS) down-regulates leukocyte extravasation in inflammation by generation of nitric oxide. CD73 decreases vascular leakage and neutrophil emigration to inflamed tissues by generation of adenosine. On the other hand, vascular adhesion protein-1 (VAP-1) mediates leukocyte trafficking to the sites of inflammation both by generation of pro-inflammatory substances and by physically acting as an adhesion molecule. The aims of this study were to define the role of endothelial enzymes NOS, CD73 and VAP-1 in acute lung injury. Our data suggest that increasing substrate availability for NOS reduces both lung edema and neutrophil infiltration and this effect is not enhanced by concomitant administration of antioxidants. CD73 protects from vascular leakage in ALI and its up-regulation by interferon-β represents a novel therapeutic strategy for treatment of this syndrome. Enzymatic activity of VAP-1 mediates neutrophil infiltration in ALI and its inhibition represents an attractive approach to treat ALI.

The lipopolysaccharide of Aeromonas spp: structure-activity relationships

Marine microorganisms, including Aeromonas, are a source of compds. for drug development that have generated great expectations in the last decades. Aeromonas infections produce septicemia, and ulcerative and haemorrhagic diseases in fish. Among the pathogenic factors assocd. with Aeromonas, the lipopolysaccharides (LPS)​, a surface glyconconjugate unique to Gram-​neg. bacteria consisting of lipid A (lipid anchor of the mol.)​, core oligosaccharide and O-​specific polysaccharide (O antigen)​, are key elicitors of innate immune responses. The chem. structure of these three parts has been characterized in Aeromonas. Based on the high variability of repeated units of O-​polysaccharides, a total of 97 O-​serogroups have been described in Aeromonas species, of which four of them (O:11; O:16; O:18 and O:34) account for more than 60​% of the septicemia cases. The core of LPS is subdivided into two regions, the inner (highly conserved) and the outer core. The inner core of Aeromonas LPS is characterized by the presence of 3-​deoxy-​d-​manno-​oct-​2-​ulosonic (ketodeoxyoctonic) acid (Kdo) and l-​glycero-​d-​manno-​Heptoses (l,​d-​Hep)​, which are linked to the outer core, characterized by the presence of Glc, GlcN, Gal, and GalNAc (in Aeromonas salmonicida)​, d,​d-​Hep (in Aeromonas salmonicida)​, and l,​d-​Hep (in Aeromonas hydrophila)​. The biol. relevance of these differences in the distal part of the outer core among these species has not been fully assessed to date. The inner core is attached to the lipid A, a highly conserved structure that confers endotoxic properties to the LPS when the mol. is released in blood from lysed bacteria, thus inducing a major systemic inflammatory response known as septic or endotoxic shock. In Aeromonas salmonicida subsp. salmonicida the Lipid A components contain three major lipid A mols., differing in acylation patterns corresponding to tetra-​, penta- and hexa-​acylated lipid A species and comprising of 4'-​monophosphorylated β-​2-​amino-​2-​deoxy-​d-​glucopyranose-​(1→6)​-​2-​amino-​2-​deoxy-​d-​glucopyranose disaccharide. In the present review, we discuss the structure-​activity relationships of Aeromonas LPS, focusing on its role in bacterial pathogenesis and its possible applications.

The lipopolysaccharide of Aeromonas spp: structure-activity relationships

Marine microorganisms, including Aeromonas, are a source of compds. for drug development that have generated great expectations in the last decades. Aeromonas infections produce septicemia, and ulcerative and haemorrhagic diseases in fish. Among the pathogenic factors assocd. with Aeromonas, the lipopolysaccharides (LPS)​, a surface glyconconjugate unique to Gram-​neg. bacteria consisting of lipid A (lipid anchor of the mol.)​, core oligosaccharide and O-​specific polysaccharide (O antigen)​, are key elicitors of innate immune responses. The chem. structure of these three parts has been characterized in Aeromonas. Based on the high variability of repeated units of O-​polysaccharides, a total of 97 O-​serogroups have been described in Aeromonas species, of which four of them (O:11; O:16; O:18 and O:34) account for more than 60​% of the septicemia cases. The core of LPS is subdivided into two regions, the inner (highly conserved) and the outer core. The inner core of Aeromonas LPS is characterized by the presence of 3-​deoxy-​d-​manno-​oct-​2-​ulosonic (ketodeoxyoctonic) acid (Kdo) and l-​glycero-​d-​manno-​Heptoses (l,​d-​Hep)​, which are linked to the outer core, characterized by the presence of Glc, GlcN, Gal, and GalNAc (in Aeromonas salmonicida)​, d,​d-​Hep (in Aeromonas salmonicida)​, and l,​d-​Hep (in Aeromonas hydrophila)​. The biol. relevance of these differences in the distal part of the outer core among these species has not been fully assessed to date. The inner core is attached to the lipid A, a highly conserved structure that confers endotoxic properties to the LPS when the mol. is released in blood from lysed bacteria, thus inducing a major systemic inflammatory response known as septic or endotoxic shock. In Aeromonas salmonicida subsp. salmonicida the Lipid A components contain three major lipid A mols., differing in acylation patterns corresponding to tetra-​, penta- and hexa-​acylated lipid A species and comprising of 4'-​monophosphorylated β-​2-​amino-​2-​deoxy-​d-​glucopyranose-​(1→6)​-​2-​amino-​2-​deoxy-​d-​glucopyranose disaccharide. In the present review, we discuss the structure-​activity relationships of Aeromonas LPS, focusing on its role in bacterial pathogenesis and its possible applications.

Kainic acid-induced seizures: inflammation and excitotoxic neuronal damage in the developing rat hippocampus

Epileptic seizures are harmful to the developing brain. During epileptic seizures, overactivation of glutamate receptors (GluR) leads to neuronal degeneration, defined as excitotoxicity. The hippocampus is especially vulnerable to excitotoxic neuronal death, but its mechanism has remained incompletely known in the developing brain. Recently, signs of activation of inflammatory processes after epileptic seizures have been detected in the hippocampus. The purpose of this thesis was to study the inflammatory reaction and death mechanisms in excitoxic neurodegeneration induced by the glutamate analogue kainic acid (KA) in the developing hippocampus. Organotypic hippocampal slice cultures (OHCs), prepared from 6-7-day-old rats (P6-7) and treated with KA, served as an in vitro model. KA-induced status epilepticus in P9 and P21 rats was used as an in vivo model. The results showed that the pyramidal cell layers of the hippocampus were the most susceptible to irreversible and age-specific neurodegeneration, which occurred in the juvenile (P21), but not in the immature (P9), rat hippocampus. The primary death mechanism was necrosis as there were no significant changes in the expression of selected apoptosis markers and morphological cellular features of necrosis were found. Inflammatory response was similarly age-dependent after KA treatment as a rapid, fulminant and wide response was detected in the juvenile, but not in the immature, rat brain. An anti-inflammatory drug treatment, given before KA, was not neuroprotective in OHCs, possibly because of the timing of the treatment. In summary, the results suggest that KA induces an age-dependent inflammatory response and necrotic neurodegeneration, which may cause disturbances in hippocampal connectivity and promote epileptogenesis.

Leukocyte trafficking: a special focus on VAP-1 and CLEVER-1

It is crucial that lymphocytes patrol the body against foreign intruders and that leukocytes invade inflamed tissues to ameliorate the infection or injury. The adhesion molecules in leukocytes and endothelial cells play an essential role in the immune response by directing the traffic of leukocytes. However, the same molecules that guide leukocyte traffic under physiological conditions are also involved in pathological situations, when an overly excessive or harmful inflammatory response leads to tissue destruction and organ dysfunction or tumor growth. Vascular adhesion protein-1 (VAP-1) and Common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1) are endothelial molecules that participate in the adhesion of leukocytes to the endothelia. This study was designed to elucidate, using different inflammation models, the role of VAP-1 and CLEVER-1 in leukocyte migration to the inflamed tissue, and to evaluate the use of antibodies against these molecules as an anti-adhesive therapy. Also, the role of CLEVER-1 during tumorigenesis was studied. Blocking the function of VAP-1 with antibodies significantly decreased the accumulation of leukocytes in the inflamed tissue. Targeting CLEVER-1 prevented cell migration via lymphatic vessels, as well as leukocyte traffic during inflammation. Following the anti-CLEVER-1 antibody treatment the number of immune regulating leukocytes in tumors was reduced, which led to a decrease in tumor growth. However, the normal immune response towards immunization or bacterial infection was not compromised. Thus, VAP-1 and CLEVER-1 are both potential targets for antiinflammatory therapies for preventing the harmful accumulation of leukocytes in inflamed areas. Targeting CLEVER-1 may also inhibit tumor growth by reducing immunosuppressive leukocytes in tumors

The effect of timing temporary cements to treat induced pulp necrosis in the teeth of dogs

During endodontic therapy (pulpectomy, root canal debridement and root canal filling) microbiological management is a major concern. Bacteria present in dentine tubules, apical foramina and apical delta are causally related to failure of the procedure. Studies have shown that during single session endodontic treatment bacteria remain within dental structures. The aim of the present study was to evaluate endodontic treatment performed as two sessions, using temporary endodontic dressing materials for different periods in four groups of experimental dogs. A total of 80 roots of second and third upper premolar teeth and second, third and fourth lower premolar teeth were divided into four groups. The pulp chamber was opened with burrs and the pulp exposed for 60 days to induce pulpal inflammation and necrosis. Groups II, III and IV were treated with calcium hydroxide plus camphorated paramono-chlorophenol (PMCC) for 7, 15 and 30 days, respectively. In all groups, the root canals were filled with zinc oxide-eugenol and gutta-percha cones. Clinical and radiographical measurements were performed every 2 weeks. After 60 days a small block section containing the teeth, surrounding periapical tissues and the periodontium was removed for histological and microbiological study. Histological analysis revealed intense inflammatory response in all groups. Microbiological analysis showed microbial reduction inversely proportional to the period of time that the intracanal temporary medicament was left in place.