Electroencephalographic and Behavioral Effects of Intranasal Administration of a Na+, K+-ATPase-Activating Antibody after Status Epilepticus.

Status epilepticus (SE) is a medical emergency associated with high mortality and morbidity. Na+, K+-ATPase, is a promising therapeutic target for SE, given its critical role in regulation of neuron excitability and cellular homeostasis. We investigated the effects of a Na+, K+-ATPase-activating antibody (DRRSAb) on short-term electrophysiological and behavioral consequences of pilocarpine-induced SE. Rats were submitted to pilocarpine-induced SE, followed by intranasal administration (2 µg/nostril). The antibody increased EEG activity following SE, namely, EEG power in theta, beta, and gamma frequency bands, assessed by quantitative analysis of EEG power spectra. One week later, DRRSAb-treated animals displayed less behavioral hyperreactivity in pick-up tests and better performance in novel object recognition tests, indicating that the intranasal administration of this Na+, K+-ATPase activator immediately after SE improves behavioral outcomes at a later time point. These results suggest that Na+, K+-ATPase activation warrants further investigation as an adjunctive therapeutic strategy for SE.

Preparation and characterization of carboxymethyl cashew gum grafted with immobilized antibody for potential biosensor application.

This report details the design of carboxymethylated cashew gum (CG) as a platform for antibody (Ab) immobilization, which can then be used as a biosensor for bacteria detection. The CG was isolated and characterized, followed by conversion to carboxymethyl cashew gum (CMCG). The CMCG film was a viable support for antibody immobilization; it was electrodeposited on gold surface using the cyclic voltammetry technique, applying a potential sweep from -1.0 V to 1.3 V with a scan rate of 50 mV s-1 and 10 scans. The COOH groups on the surface of the film were critical in promoting Ab bonding. The immobilization of the Ab was mediated by protein A (PrA) for recognition of the antigen. Voltammetry studies were used to monitor the antibody immobilization. Finally, the analytical response of the CMCG-PrA-Ab system was evaluated with the chronoamperometry technique and was found to detect Salmonella Typhimurium bacteria rapidly and efficiently.

LAG-3 antagonists by cancer treatment: a patent review.

Introduction: LAG-3 is checkpoint inhibitor in cancer that coordinates the downregulation of the proliferation of antigen-specific lymphocytes. There is a great need to discover and develop new therapies focused on inhibiting the action of LAG-3 and consequently improving the immune response in the various types of cancer. Areas covered: The patent literature reveals novel therapies, which provide information on cancer therapies. The authors used the patent databases of the six main patent offices of the world: United States Patent and Trademark Office, European Patent Office, World Intellectual Property Organization, Japan Patent Office, State Intellectual Property Office of China and Korean Intellectual Property Office, to generate a detailed landscape of patents and patent applications of active companies related to LAG-3 inhibitors. Specific patents have been grouped into innovative patents and adopting patents. Expert opinion: There is a continuing development of LAG-3 inhibitors, and these inhibitors are being used in combination with other cancer treatment schemes, for example, antibodies against PD-1, PD-L1, and CTLA-4. Immutep and IO Therapeutics were the leaders in generating innovator patents, followed by Gustave Roussy Institute, and Applied Research Systems ARS. Dana-Farber Cancer Institute was the leader in the generation of adopter patents, followed by Novartis .

Toxoplasmosis, leptospirosis, and brucellosis seroepidemiology in veterinary medical students and their relation with unique health / Soroepidemiologia da toxoplasmose, leptospirose e brucelose em acadêmicos de medicina veterinária e sua relação com a saúde única

Toxoplasmosis, leptospirosis, and brucellosis are global zoonoses, with humans as accidental participants in their transmission cycles. The can also be considered occupational diseases, because certain professionals are at greater risk of contact and infection by such zoonoses. These three diseases have different epidemiological characteristics because of the distinct environmental, social, cultural, and economic conditions where these pathogens circulate. Because of the importance of these diseases and their associations with specific occupations, we performed a seroepidemiological survey of Toxoplasma, Leptospira, and Brucella antibodies, with an analysis of the association between positive serum and certain occupational and environmental variables, in students of a veterinary medicine course (1st, 2nd, 3rd, 4th, and 5th year) in a university in the northwestern region in the state of Paraná, Brazil. From May to November 2014, blood samples were collected from 157 volunteers by professionals trained in nursing and biomedicine from the same university as the veterinary students. At the time of blood collection, the students did not present any clinical signs of the three diseases of interest. To detect antiToxoplasma gondii, anti-Leptospira spp., and anti-Brucella antibodies, indirect immunofluorescence test (IIF), microscopic agglutination test (MAT), fast agglutination test (a screening test), and enzymelinked immunosorbent assay (ELISA) were performed. To determine variables associated with these infections, the students were interviewed to complete an epidemiological questionnaire with environmental, behavioral, and occupational information. The associations between these variables and infections were assess by chi-square or Fischer’s exact tests, with a 5% significance level (α)...

A toxoplasmose, leptospirose e a brucelose são zoonoses de ampla distribuição mundial, tendo o homem como participante acidental da sua cadeia epidemiológica, entretanto, podem se apresentar como doenças ocupacionais, em diferentes categorias profissionais, despertando grande preocupação, por estarem constantemente expostos ao risco de contato e contagio com essas zoonoses. Estas três enfermidades possuem características epidemiológicas distintas, como reflexo das diferenças ambientais, sociais, culturais e econômicas encontradas em cada localidade. Considerando a importâncias destas três enfermidades e sua relação com o aspecto ocupacional o objetivo deste trabalho foi realizar um levantamento soroepidemiológico para toxoplasmose, leptospirose e brucelose e identificar variáveis ocupacionais e ambientais relacionadas a estas três enfermidades em acadêmicos do curso de medicina veterinária de uma universidade da região noroeste do estado do Paraná, Brasil. No período de maio a novembro de 2014, foram coletadas amostras de sangue de forma voluntária de 157 acadêmicos do curso de Medicina Veterinária (1°, 2°, 3°, 4° e 5° ano). A coleta de sangue foi realizada por profissionais habilitados da área de enfermagem e da biomedicina da respectiva universidade. No momento da coleta de sangue, os acadêmicos não apresentavam manifestação de qualquer sinal clínico das enfermidades deste estudo. Para detectar anticorpos anti-Toxoplasma gondii, anti-Leptospira spp. e anti-Brucella foram realizadas as técnicas de imunofluorescência indireta (RIFI), aglutinação microscópica (SAM), aglutinação rápida (triagem) e ensaio imunoenzimático (ELISA) respectivamente e para a detecção de variáveis associadas às infecções, os acadêmicos foram entrevistados, respondendo a um questionário epidemiológico com informações ambientais, comportamentais e ocupacionais relacionadas às enfermidades...

Toxoplasmosis, leptospirosis, and brucellosis seroepidemiology in veterinary medical students and their relation with unique health / Soroepidemiologia da toxoplasmose, leptospirose e brucelose em acadêmicos de medicina veterinária e sua relação com a saúde única

Toxoplasmosis, leptospirosis, and brucellosis are global zoonoses, with humans as accidental participants in their transmission cycles. The can also be considered occupational diseases, because certain professionals are at greater risk of contact and infection by such zoonoses. These three diseases have different epidemiological characteristics because of the distinct environmental, social, cultural, and economic conditions where these pathogens circulate. Because of the importance of these diseases and their associations with specific occupations, we performed a seroepidemiological survey of Toxoplasma, Leptospira, and Brucella antibodies, with an analysis of the association between positive serum and certain occupational and environmental variables, in students of a veterinary medicine course (1st, 2nd, 3rd, 4th, and 5th year) in a university in the northwestern region in the state of Paraná, Brazil. From May to November 2014, blood samples were collected from 157 volunteers by professionals trained in nursing and biomedicine from the same university as the veterinary students. At the time of blood collection, the students did not present any clinical signs of the three diseases of interest. To detect antiToxoplasma gondii, anti-Leptospira spp., and anti-Brucella antibodies, indirect immunofluorescence test (IIF), microscopic agglutination test (MAT), fast agglutination test (a screening test), and enzymelinked immunosorbent assay (ELISA) were performed. To determine variables associated with these infections, the students were interviewed to complete an epidemiological questionnaire with environmental, behavioral, and occupational information. The associations between these variables and infections were assess by chi-square or Fischers exact tests, with a 5% significance level (α)...(AU)

A toxoplasmose, leptospirose e a brucelose são zoonoses de ampla distribuição mundial, tendo o homem como participante acidental da sua cadeia epidemiológica, entretanto, podem se apresentar como doenças ocupacionais, em diferentes categorias profissionais, despertando grande preocupação, por estarem constantemente expostos ao risco de contato e contagio com essas zoonoses. Estas três enfermidades possuem características epidemiológicas distintas, como reflexo das diferenças ambientais, sociais, culturais e econômicas encontradas em cada localidade. Considerando a importâncias destas três enfermidades e sua relação com o aspecto ocupacional o objetivo deste trabalho foi realizar um levantamento soroepidemiológico para toxoplasmose, leptospirose e brucelose e identificar variáveis ocupacionais e ambientais relacionadas a estas três enfermidades em acadêmicos do curso de medicina veterinária de uma universidade da região noroeste do estado do Paraná, Brasil. No período de maio a novembro de 2014, foram coletadas amostras de sangue de forma voluntária de 157 acadêmicos do curso de Medicina Veterinária (1°, 2°, 3°, 4° e 5° ano). A coleta de sangue foi realizada por profissionais habilitados da área de enfermagem e da biomedicina da respectiva universidade. No momento da coleta de sangue, os acadêmicos não apresentavam manifestação de qualquer sinal clínico das enfermidades deste estudo. Para detectar anticorpos anti-Toxoplasma gondii, anti-Leptospira spp. e anti-Brucella foram realizadas as técnicas de imunofluorescência indireta (RIFI), aglutinação microscópica (SAM), aglutinação rápida (triagem) e ensaio imunoenzimático (ELISA) respectivamente e para a detecção de variáveis associadas às infecções, os acadêmicos foram entrevistados, respondendo a um questionário epidemiológico com informações ambientais, comportamentais e ocupacionais relacionadas às enfermidades...(AU)

Microbiota-Induced Antibodies Are Essential for Host Inflammatory Responsiveness to Sterile and Infectious Stimuli.

The indigenous intestinal microbiota is frequently considered an additional major organ of the human body and exerts profound immunomodulating activities. Germ-free (GF) mice display a significantly different inflammatory responsiveness pattern compared with conventional (CV) mice, and this was dubbed a "hyporesponsive phenotype." Taking into account that the deposition of immune complexes is a major event in acute inflammation and that GF mice have a distinct Ig repertoire and B cell activity, we aimed to evaluate whether this altered Ig repertoire interferes with the inflammatory responsiveness of GF mice. We found that serum transfer from CV naive mice was capable of reversing the inflammatory hyporesponsiveness of GF mice in sterile inflammatory injury induced by intestinal ischemia and reperfusion, as well as in a model of lung infection by Klebsiella pneumoniae Transferring serum from Ig-deficient mice to GF animals did not alter their response to inflammatory insult; however, injecting purified Abs from CV animals restored inflammatory responsiveness in GF mice, suggesting that natural Abs present in serum were responsible for altering GF responsiveness. Mechanistically, injection of serum and Ig from CV mice into GF animals restored IgG deposition, leukocyte influx, NF-κB activation, and proinflammatory gene expression in inflamed tissues and concomitantly downregulated annexin-1 and IL-10 production. Thus, our data show that microbiota-induced natural Abs are pivotal for host inflammatory responsiveness to sterile and infectious insults.

Down regulation of protective genes is associated with cellular and antibody- mediated rejection

Despite advances in immunosuppressive therapy, rejection still remains the main obstacleto a successful transplant. This study aims to explore the gene expression profileof the rejection process in order to decrease the number of unnecessary endomyocardialbiopsies in stable patients. Methods: A total of 300 formalin- fixed and paraffin- embedded (FFPE) endomyocardialbiopsies sampled from 63 heart allograft recipients were included in this study. Acute cellular rejection (ACR) and antibody- mediated rejection (AMR) were diagnosedby histological analysis and immunohistochemical C4d staining, respectively. Analysisof gene expression was performed by quantitative real- time polymerase chain reaction.The samples were grouped according to the ISHLT rejection classification, aimingthe statistical analysis. Results: There was a significant decrease in the HMOX1, AIF1, and CCL2 transcriptover the post- transplantation period in non- rejection group (P<.001). Furthermore, the ADIPOR1, ADIPOR2, BCL2L1, and VEGFA protective genes were significantly downregulatedin the ACR group (P<.05). ADIPOR2, BCL2L1, IL6, and NOS2 genes were alsosignificantly downregulated in the AMR group than in the non- rejection group (P<.05). Conclusion: The downregulations of the protective genes contribute to the allograftrejection, and the archived FFPE samples are useful for the gene expression analysisaiming the allograft rejection surveillance.

Aptamers: current challenges and future prospects.

INTRODUCTION: Aptamers are oligonucleotide molecules raised in vitro from large combinatorial libraries of nucleic acids and developed to bind to targets with high affinity and specificity. Whereas novel target molecules are proposed for therapeutic intervention and diagnostic, aptamer technology has a great potential to become a source of lead compounds. AREAS COVERED: In this review, the authors address the current status of the technology and highlight the recent progress in aptamer-based technologies. They also discuss the current major technical limitations of aptamer technology and propose original solutions based on existing technologies that could result in a solid aptamer-discovery platform. EXPERT OPINION: Whereas aptamers have shown to bind to targets with similar affinities and specificities to those of antibodies, aptamers have several advantages that could outweigh antibody technology and open new opportunities for better medical and diagnostic solutions. However, the current status of the aptamer technology suffers from several technical limitations that slowdown the progression of novel aptamers into the clinic and makes the business around aptamers challenging.

Hippocampal--prefrontal BDNF and memory for fear extinction.

Infusing brain-derived neurotrophic factor (BDNF) into the infralimbic (IL) prefrontal cortex is capable of inducing extinction. Little is known, however, about the circuits mediating BDNF effects on extinction or the extent to which extinction requires BDNF in IL. Using local pharmacological infusion of BDNF protein, or an antibody against BDNF, we found that BDNF in the IL, but not prelimbic (PL) prefrontal cortex, is both necessary and sufficient for fear extinction. Furthermore, we report that BDNF in IL can induce extinction of older fear memories (14 days) as well as recent fear memories (1 day). Using immunocytochemistry, we show that BDNF is increased in the ventral hippocampus (vHPC), but not IL or PL, following extinction training. Finally, we observed that infusing BDNF into the vHPC increased the firing rate of IL, but not PL neurons in fear conditioned rats. These findings indicate that an extinction-induced increase in BDNF within the vHPC enhances excitability in IL targets, thereby supporting extinction memories.

Evaluation of histological damage of solid organs after donor preconditioning with thymoglobulin in an experimental rat model.

Rabbit anti-rat thymoglobulin (rATG) administered to donors with brain death (BD) may improve organs quality. We explored the effects of rATG administered to BD donors in the histology of heart, lungs and small bowel in a rat experimental model. Animals were randomly assigned to 3 groups: V (n=5) no BD, 2h ventilation; BD (n=5) BD and 2h ventilation; BD and rATG: BD, 2h ventilation, rATG (10mg/kg) after BD diagnosis. Histopathological damage scores were based on neutrophil infiltration, airway epithelial cell damage, interstitial edema, hyaline membrane formation, and pulmonary hemorrhage (lungs); neutrophil infiltration and interstitial edema (heart); Park score (bowel). Lung damage was significantly lower in BD+rATG group: V 5 ± 1.6; BD 11.25 ± 0.5, BD+rATG 6.5 ± 1.9 (p<0.01). Heart: V 2.0 ± 0.81; BD 4.75 ± 1.25 and BD+rATG 3.5 ± 1.7 (p>0.05). Small bowel: BD 2.25 ± 0.96 vs. BD+rATG 1.00 ± 1.15 (n.s.). Histological damage amelioration in lung and attenuation tendency in heart and small bowel encourages research of cytoprotective strategies to improve organ viability.

The REMEDEE trial: a randomized comparison of a combination sirolimus-eluting endothelial progenitor cell capture stent with a paclitaxel-eluting stent.

OBJECTIVES: This study sought to compare the efficacy and safety results after coronary implantation of a combined sirolimus-eluting CD34 antibody coated Combo stent (OrbusNeich Medical, Ft. Lauderdale, Florida) with the paclitaxel-eluting Taxus Liberté stent (PES) (Boston Scientific, Natick, Massachusetts). This report summarizes the first-in-man randomized, controlled multicenter REMEDEE trial (Randomized study to Evaluate the safety and effectiveness of an abluMinal sirolimus coatED bio-Engineered StEnt) angiographic, intravascular ultrasound, and clinical results up to 12 months. BACKGROUND: Drug-eluting stents have limited restenosis and reintervention but are complicated by especially late and very late stent thrombosis and accelerated neoatherosclerosis. Alternative or adjunct technologies should address these limitations. METHODS: One hundred eighty-three patients with de novo native coronary artery stenoses were randomized 2:1 to Combo stent or PES implantation. The primary endpoint is the angiographic in-stent late lumen loss at 9 months, which was tested for noninferiority between the 2 stent groups. Secondary endpoints include the occurrence of major adverse cardiac events. RESULTS: The Combo stent was found to be noninferior to the PES in 9-month angiographic in-stent late lumen loss with 0.39 ± 0.45 mm versus 0.44 ± 0.56 mm (pnoninferiority = 0.0012). At 12 months, the occurrence of major adverse cardiac events was 8.9% in the Combo group and 10.2% in the PES group (p = 0.80) with no difference in mortality, occurrence of myocardial infarction, or target lesion revascularization. No stent thrombosis was reported in either group. CONCLUSIONS: In the REMEDEE trial the Combo stent has shown to be effective by meeting the primary noninferiority angiographic endpoint and safe, with an overall low rate of clinical events in both stent groups, including no stent thrombosis up to 12 months.

Controle da eficácia de bacterinas antileptospirose: relação entre os resultados dos testes de inibição de crescimento de leptospiras in vitro com os do desafio in vivo em hamsters / Efficacy of anti-leptospirosis bacterines: relation between the results of the in vitro growth inhibition test and the potency test, in vivo, with challenge in hamsters

Foi efetuada a comparação em hamsters da proteção conferida e dos níveis de anticorpos induzidos por duas bacterinas comerciais antileptospirose. Os ensaios empregados foram o teste oficial de potência com desafio (TP), o ensaio proposto, teste de inibição de crescimento de leptospiras in vitro (ICLIV) e a soroaglutinação microscópica (SAM). O protocolo de imunização foi representado por duas aplicações individuais de 0,25 mL das bacterinas, puras ou de suas diluições geométricas de razão dois variando de 200 a 51.200 para a bacterina A e de 200 a 3.200 para a bacterina B, por via subcutânea com o intervalo de 15 dias. Decorridos 15 dias da segunda aplicação de vacina, um grupo de animais foi desafiado com 0,2 mL de cultivos de leptospiras, por indivíduo, respectivamente dos sorovares Canicola (bacterinas A e B) ou Kennewicki (bacterina A). Os números de doses infectantes empregados nos desafios foram de 100 e 631 respectivamente, para os sorovares Canicola e Kennewicki. Decorridos 21 dias do desafio, os grupos de animais utilizados nos testes de ICLIV e SAM foram sangrados e os seus soros foram reunidos em pools (n = 5). No TP, adotando-se os critérios internacionais, as bacterinas foram aprovadas. A comparação do desempenho das bacterinas para os sorovares adotados, segundo sua concentração, por meio das proporções de animais sobreviventes ao TP e a média dos títulos de anticorpos identificados no teste de ICLIV, indicou que um título igual ou superior a 0,77 log corresponde ao nível de aprovação da bacterina no TP.

It was performed a comparison between the protection afforded in hamsters and the antibody levels induced by two commercial vaccines against leptospirosis. The assays used were the official challenge test (TP), the in vitro leptospires growth inhibition test (ICLIV) and microscopic agglutination test (MAT). The immunization protocol consisted of two single applications, 15 days from each other, of 0.25 mL of the bacterins, pure or its two-fold serial dilutions: 200 to 51,200 for bacterin A and 200 to 3.200 bacterin B, both of them administered subcutaneously. A group of animals was challenged, after 15 days from the second vaccine application, with 0.2 mL/animal of live leptospire cultures, with Canicola (bacterin A and B) or Kennewicki (bacterin A) serovars. The numbers of infective doses employed in the challenges were 100 and 631 for Canicola and Kennewicki serovars, respectively. After 15 days from the second vaccine dose the groups of animals used in ICLIV and SAM tests were bled and their sera were collected in pools (n = 5). In TP, adopting the criteria established by the Code Federal Regulation, both bacterins were approved. The comparison of the performance of the tested bacterins with the adopted serovars, according to its concentration, by the proportions of surviving animals to the challenge assay and the average of the neutralizing antibodies titers, established a neutralizing antibodies titer equal or higher than 0.77 log corresponding with the bacterin level of approval in the potency assay.

Controle da eficácia de bacterinas antileptospirose: relação entre os resultados dos testes de inibição de crescimento de leptospiras in vitro com os do desafio in vivo em hamsters / Efficacy of anti-leptospirosis bacterines. Relation between the results of the in vitro growth inhibition test and the potency test, in vivo, with challenge in hamsters

Foi efetuada a comparação em hamsters da proteção conferida e dos níveis de anticorpos induzidos por duas bacterinas comerciais antileptospirose. Os ensaios empregados foram o teste oficial de potência com desafio (TP), o ensaio proposto, teste de inibição de crescimento de leptospiras in vitro (ICLIV) e a soroaglutinação microscópica (SAM). O protocolo de imunização foi representado por duas aplicações individuais de 0,25 mL das bacterinas, puras ou de suas diluições geométricas de razão dois variando de 200 a 51.200 para a bacterina A e de 200 a 3.200 para a bacterina B, por via subcutânea com o intervalo de 15 dias. Decorridos 15 dias da segunda aplicação de vacina, um grupo de animais foi desafiado com 0,2 mL de cultivos de leptospiras, por indivíduo, respectivamente dos sorovares Canicola (bacterinas A e B) ou Kennewicki (bacterina A). Os números de doses infectantes empregados nos desafios foram de 100 e 631 respectivamente, para os sorovares Canicola e Kennewicki. Decorridos 21 dias do desafio, os grupos de animais utilizados nos testes de ICLIV e SAM foram sangrados e os seus soros foram reunidos em pools (n = 5). No TP, adotando-se os critérios internacionais, as bacterinas foram aprovadas. A comparação do desempenho das bacterinas para os sorovares adotados, segundo sua concentração, por meio das proporções de animais sobreviventes ao TP e a média dos títulos de anticorpos identificados no teste de ICLIV, indicou que um título igual ou superior a 0,77 log corresponde ao nível de aprovação da bacterina no TP(AU)

It was performed a comparison between the protection afforded in hamsters and the antibody levels induced by two commercial vaccines against leptospirosis. The assays used were the official challenge test (TP), the in vitro leptospires growth inhibition test (ICLIV) and microscopic agglutination test (MAT). The immunization protocol consisted of two single applications, 15 days from each other, of 0.25 mL of the bacterins, pure or its two-fold serial dilutions: 200 to 51,200 for bacterin A and 200 to 3.200 bacterin B, both of them administered subcutaneously. A group of animals was challenged, after 15 days from the second vaccine application, with 0.2 mL/animal of live leptospire cultures, with Canicola (bacterin A and B) or Kennewicki (bacterin A) serovars. The numbers of infective doses employed in the challenges were 100 and 631 for Canicola and Kennewicki serovars, respectively. After 15 days from the second vaccine dose the groups of animals used in ICLIV and SAM tests were bled and their sera were collected in pools (n = 5). In TP, adopting the criteria established by the Code Federal Regulation, both bacterins were approved. The comparison of the performance of the tested bacterins with the adopted serovars, according to its concentration, by the proportions of surviving animals to the challenge assay and the average of the neutralizing antibodies titers, established a neutralizing antibodies titer equal or higher than 0.77 log corresponding with the bacterin level of approval in the potency assay(AU)

Mechanisms involved in IL-6-induced muscular mechanical hyperalgesia in mice.

Interleukin-6 (IL-6) is an inflammatory cytokine known to modulate muscle pain. However, the mechanisms underlying this effect still remain unclear. Here we show that the injection of IL-6 into mice gastrocnemius muscle evoked a time- and dose-dependent mechanical hyperalgesia. This effect is in part dependent on the presence of gp130 expression in inflammatory cells in the gastrocnemius muscle as well as in DRG neurons. We also demonstrated an increased inflammatory cell recruitment and cytokines levels, namely TNF-α, IL-1ß and KC. TNFR1(-/-) mice or mice pre-treated with the selective CXCR2 antagonist, SB225002, with the anti-macrophage, anti-TNF-α or anti-KC antibodies or with IL-1 receptor antagonist (IL-1RA) showed decreased IL-6-mediated mechanical hyperalgesia. Furthermore, systemic pre-treatment with the classically used drugs indomethacin, celecoxib, guanetidine, morphine, thalidomide or dexamethasone, also prevented IL-6-induced muscle pain. Likewise, local pre-treatment with inhibitors of phospholipase A2 (PACOCF3), phospholipase C (U73122), protein kinase C (GF109203X), protein kinase A (KT-5720) or with phosphatidylinositol 3-kinase (AS605204) also consistently diminished IL-6-induced muscle hyperalgesia. The intramuscular injection of the selective inhibitors of p38 MAPK (SB203580), ERK (PD98059) or JNK (SP60015) also prevented IL-6-mediated muscular pain. Simultaneous flow cytometry measurements revealed that ERK, p38 MAPK and JNK were phosphorylated as early as 5 min after IL-6 injection. These findings provided new evidence indicating that IL-6 exerts a relevant role in the development and maintenance of muscular hyperalgesia. The IL-6-mediated muscular pain response involves resident cell activation, polymorphonuclear cell infiltration, cytokine production, prostanoids and sympathomimetic amines release and the activation of intracellular pathways, especially MAPKs.

Different regions of the class P-III snake venom metalloproteinasejararhagin are involved in binding to a2b1 integrin and collagen

SVMPs are multi-domain proteolytic enzymes in which disintegrin-like and cysteine-rich domains bind to cell receptors, plasma or ECM proteins. We have recently reported thatjararhagin, a P-III class SVMP, binds to collagen with high affinity through an epitope located within the Da-disintegrin sub-domain. In this study, we evaluated the binding of jararhagin to a2b1 integrin (collagen receptor) using monoclonal antibodies and recombinant jararhagin fragments. In solid phase assays, binding of jararhagin to a2b1 integrin was detectable from concentrations of 20 nM. Using recombinant fragments of jararhagin, only fragment JC76 (residues 344–421), showed a significant binding to recombinant a2b1 integrin. The anti-jararhagin monoclonal antibody MAJar 3 efficiently neutralised binding ofjararhagin to collagen, but not to recombinant a2b1 integrin nor to cell-surface-exposed a2b1 integrin (a2-K562 transfected cells and platelets). The same antibody neutralised collagen-induced platelet aggregation. Our data suggest that jararhagin binding to collagen and a2b1 integrin occurs by two independent motifs, which are located on disintegrin-like and cysteine-rich domains, respectively. Moreover, toxin binding to collagen appears to be sufficient to inhibit collagen-induced platelet aggregation.

Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity.

In animal models of diet-induced obesity, the activation of an inflammatory response in the hypothalamus produces molecular and functional resistance to the anorexigenic hormones insulin and leptin. The primary events triggered by dietary fats that ultimately lead to hypothalamic cytokine expression and inflammatory signaling are unknown. Here, we test the hypothesis that dietary fats act through the activation of toll-like receptors 2/4 and endoplasmic reticulum stress to induce cytokine expression in the hypothalamus of rodents. According to our results, long-chain saturated fatty acids activate predominantly toll-like receptor 4 signaling, which determines not only the induction of local cytokine expression but also promotes endoplasmic reticulum stress. Rats fed on a monounsaturated fat-rich diet do not develop hypothalamic leptin resistance, whereas toll-like receptor 4 loss-of-function mutation and immunopharmacological inhibition of toll-like receptor 4 protects mice from diet-induced obesity. Thus, toll-like receptor 4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response, and determines the resistance to anorexigenic signals.

Intrabursal administration of the antiangiopoietin 1 antibody produces a delay in rat follicular development associated with an increase in ovarian apoptosis mediated by changes in the expression of BCL2 related genes.

The angiopoietin (ANGPT) receptor (TEK) system plays a crucial role in blood vessel development and regression. To date, no reports have addressed the actions of the anti-ANGPT1 antibody on gonadotropin-stimulated follicular development and atresia in the ovary. Therefore, in this study we specifically investigated whether ANGPT1 plays a critical intraovarian survival role for gonadotropin-dependent folliculogenesis. In particular, we examined the effect of local administration of anti-ANGPT1 antibody on follicular development, apoptosis, and expression of BCL2 protein family members (BAX, BCL2, and BCL2L1), TNFRSF6, and FASLG in ovarian follicles from prepubertal eCG-treated rats. The inhibition of ANGPT1 caused an increase in the number of atretic follicles and a decrease in the number of both antral follicles (AFs) and preovulatory follicles in gonadotropin-treated rat ovaries. Taking into account that follicular atresia is mediated by apoptosis, we analyzed the effect of the antibody against ANGPT1 on programmed cell death. The inhibition of the action of ANGPT1 caused an increase both in the number of apoptotic granulosa cells in AFs and in the spontaneous DNA fragmentation of AFs cultured in serum-free medium. Besides, AFs obtained from rats treated with intraovarian antibodies against ANGPT1 showed both a decrease in BCL2 and an increase in BAX protein levels. Moreover, a reduction in the BCL2L1(L)/BCL2L1(S) ratio was observed in this group, with a reduction of BCL2L1(L) greater than that of BCL2L1(S), thus showing that the expression of these antiapoptotic proteins is lower in follicles from treated rats than in those from untreated ones. Our findings suggest that the inhibition of ANGPT1 activity causes an increase in the number of atretic follicles mediated by ovarian apoptosis through an imbalance in the ratio of antiapoptotic to proapoptotic proteins. This could take place through a paracrine effect on granulosa cells mediated by the TEK receptor in theca cells. Therefore, these data clearly indicate that ANGPT1 is necessary for follicular development induced by gonadotropins.

Prolactin in eyes of patients with retinopathy of prematurity: implications for vascular regression.

PURPOSE: Disruption of the anti-angiogenic environment of the retina leads to neovascular eye diseases, including retinopathy of prematurity (ROP). Prolactin (PRL), the hormone originally associated with milk secretion, is proteolytically processed to 16K-PRL, a fragment with potent antiangiogenic, proapoptotic effects. Whether 16K-PRL is produced in eyes of patients with ROP and promotes the regression of intraocular blood vessels associated with this disease was investigated. METHODS: PRL was quantified in the aqueous humor, subretinal fluid, and serum from patients with stage 5 ROP and in patients with non-neovascular eye disorders. Intraocular expression of PRL was evaluated by RT-PCR, in situ hybridization, and Western blot analysis. AntiPRL antibodies were injected intravitreously in neonatal rats, and apoptosis of hyaloid vessels determined by TUNEL and ELISA. RESULTS: PRL was elevated in ocular fluids and serum from ROP patients. There was no correlation between PRL in ocular fluids and its level in serum, whereas PRL in aqueous humor and subretinal fluid were significantly correlated. PRL mRNA was expressed in blood vessels and leukocytes within retrolental fibrovascular membranes of ROP patients, and these membranes contained a 16 kDa immunoreactive PRL. The 16K-PRL isoform was more concentrated in subretinal fluid than in serum and was generated from PRL by subretinal fluid proteases. Intravitreous injection of neutralizing antiPRL antibodies inhibited the apoptosis of hyaloid vessels in neonatal rats. CONCLUSIONS: 16K-PRL derived from PRL internalized from the circulation or synthesized intraocularly can stimulate apoptosis-induced vascular regression and contribute to the development and progression of ROP.

Digoxin antibody prevents cerebral hemorrhage-induced hypertension.

BACKGROUND: Brain injury may induce hypertension. Because serum ouabain-like compound (OLC) has vasoconstrictor activity, digoxin antibody antihypertensive effects were evaluated using an intracerebroventricular (ICV) hemorrhage rat model. METHODS: Four ICV infused Wistar rat groups were studied: control; blood; blood plus digoxin antibody, and cerebrospinal fluid-like solution. Tail-cuff blood pressure, cumulative sodium balance, and serum OLC were measured. RESULTS: The ICV blood infusion increased blood pressure (BP) and OLC without sodium balance change. Digoxin antibody prevented BP and OLC rise. Blood pressure was positively correlated with OLC in blood and blood plus digoxin antibody rats (R = 0.63; P <.05). CONCLUSIONS: Cerebral hemorrhage increased OLC and BP, which were reversed by digoxin antibody administration.