Cysteine cathepsins B, L and S are endosomal/lysosomal proteases that participate in numerous physiological systems. Cathepsin expression and activity are altered during various inflammatory diseases, including rheumatoid arthritis, atherosclerosis, neurodegenerative diseases and cancers. Early immune responses to microbial pathogens are mediated by pattern-recognition receptors, including Toll-like receptors (TLR). Signaling through TLR causes cell activation and release of inflammatory mediators, which can contribute to the severity of chronic inflammatory diseases. The impact of TLR cell activation on cathepsins B, L and S activities was investigated using live-cell enzymatic assays. Individual ligands of TLR4, TLR2 and TLR3 increased intracellular activities of the three cathepsins indicating the involvement of both MyD88-dependent and -independent pathways. To investigate the role of inflammatory cytokines in regulating these proteases, a lipopolysaccharide (LPS) non-responsive cell line was utilized. LPS non-responsive cells co-cultured with LPS responsive macrophages upregulated cathepsin activities. Furthermore, culture supernatants from LPS-stimulated macrophages increased cathepsin activities in LPS non-responsive cells, which could be reduced by neutralizing antibodies to TNF-α or IL-1β. These findings indicate cytokines regulate cathepsin activities during macrophage responses to TLR stimulation. Using LPS as a model for inflammation, the ability of the cannabinoids, delta9-tetrahydrocannabinol (THC), and CP55940 to suppress cysteine cathepsins during an inflammatory response was investigated. Cannabinoids, including the major psychoactive component of marijuana THC, modulate a variety of immune responses and have been proposed as possible therapeutics to control chronic inflammation. Cannabinoids may mediate their effects through receptor-dependent or independent mechanisms. Cannabinoid receptor subtype 1 (CB1) and receptor subtype 2 (CB2) have differential expression in leukocytes. Dose response studies showed that 1 nM THC was sufficient to inhibit cathepsin enhancement in LPS-stimulated cells. P388D1 macrophages expressed CB2 mRNA, but had no detectable CB1 mRNA indicating a role for the CB2 receptor. Utilizing a CB2-/- macrophage cell line, the role of CB2 receptor participation in THC inhibition of cysteine cathepsin upregulation was explored. THC did not affect cathepsin activity in LPS-stimulated cells lacking CB2 expression. These findings support the possibility of receptor selective agonists as therapeutic treatment during inflammatory diseases to prevent cathepsin involvement in pathological tissue destruction.

Este trabalho apresenta alguns aspectos específicos sobre o tema “proteção de estruturas contra descargas atmosféricas”, principalmente no que se refere ao uso de partes estruturais das edificações como integrantes do sistema de proteção. São discutidos neste trabalho diversos pontos polêmicos referentes ao tema, tais como: captores especiais, o sistema dissipativo e o uso das ferragens das colunas de concreto armado como parte dos subsistemas de descida e de aterramento. São abordados, também, os principais danos que podem ocorrer nas edificações devido às descargas atmosféricas, principalmente quando a proteção utilizar partes naturais da estrutura, tais como: os telhados metálicos e as emendas das ferragens das colunas de concreto armado. Após uma verificação do estado da arte no assunto, onde são estudados diversos trabalhos, normas e livros sobre as características das descargas, sobre os danos oriundos das descargas, blindagens, medições de campos eletromagnéticos, distribuição de correntes e sobre a normalização e ensaios referentes à proteção de estruturas contra descargas atmosféricas, o assunto foi estudado em três partes que compõem os subsistemas do SPDA. No subsistema de captação, os danos em telhas metálicas e de concreto protendido foram estudados através de ensaios e verificações teóricas e, principalmente, experimentais. No subsistema de descida, o estudo experimental deu enfoque às emendas das ferragens e ao uso destas como descidas naturais. Finalmente, no subsistema de aterramento, as fundações das edificações foram estudadas através de verificações experimentais como parte do aterramento. Este trabalho acrescenta ao conhecimento atual sobre o assunto, diversas particularidades não contempladas nas normas e nas publicações mais recentes no que tange à proteção de estruturas contra as descargas atmosféricas utilizando partes naturais das edificações. / This work presents some specific aspects on the subject “lightning protection of structures“, mainly when the use of structural parts of the buildings as part of the protection systems is concerned. Several polemical issues are discussed, such as: special air-termination, the dissipation array system and the use of steel-reinforcing rods of the reinforced concrete columns as part of the down-conductor system and of the earth-termination system. Also treated is the main damage that can occur to the buildings due to lightning, mainly when the protection system uses the natural components of the structures, such as: the metallic rooftops and the joints of the steel-reinforcing rods of the reinforced concrete columns. After examining the state-of-the-art in the subject, which analyses several papers, standards and books about lightning characteristics, damage resulting from lightning, shieldings, measurements of electromagnetic fields, distribution of currents and about standardisation and tests related to the protection of structures against lightning, the subject was studied in the three parts that compose the lightning protection systems. In the air-termination system, the damage to metallic rooftops and to pre-stressed concrete rooftop tiles was studied by means of tests and theoretical and mainly experimental analyses. In the down-conductor system, the experimental study focused on the joints of the steel-reinforcing rods and on the use of these as natural down-conductors. Finally, in the earth-termination system, the foundations of the buildings were studied (as part of the grounding) through experimental analyses. This work adds to the current knowledge about the subject several particularities which are not observed in the standards or in the most recent publications concerning the lightning protection systems in buildings that use their natural components for this purpose.

In both the legume symbiont Sinorhizobium meliloti and the mammalian pathogen Brucella abortus, the inner membrane BacA protein is essential for host persistence. In free-living S. meliloti and B. abortus loss of the BacA protein also results in an increased resistance to the glycopeptide bleomycin and a ~ 50% decrease in the lipopolysaccharide (LPS) very-long-chain-fatty-acid (VLCFA) content. Consequently, it was proposed that BacA may be involved in transport of peptides into the cell and/or that BacA may be involved in the VLCFA modification of the LPS. During this work it was determined that the increased resistance observed in an S. meliloti DbacA mutant to bleomycin and to the truncated eukaryotic peptide Bac7(1-16), is independent of the VLCFA modification. These data support a model for BacA having multiple non-overlapping functions. Using flow cytometry studies with fluorescently labelled forms of bleomycin and Bac7(1-16) it was found that the BacA protein plays a role in the uptake of bleomycin. However, BacA was shown to be essential for the uptake of Bac7(1-16). Additionally, it was determined that two symbiotically defective bacA site directed mutants with known reductions in their VLCFA could still take up Bac7, suggesting that the BacA function that leads to the VLCFA modification could also play a key role in host persistence. To investigate further the role of BacA in the VLCFA modification and where in the cell envelope the lipid A is modified with the VLCFA, the role of the putative lipid trafficking protein MsbA2 was investigated. Interestingly, it was discovered that S. meliloti lacking the MsbA2 protein, is unable to enter host cells and induces a plant defence response more characteristic of a pathogen. To investigate the importance of the VLCFA modification during the symbiosis S. meliloti mutants lacking either the AcpXL (VLCFA acyl carrier protein) or LpxXL (VLCFA acyl transferase protein) were characterized in the host. Although not essential for host persistence, loss of each of the proteins did result in distinct defects, suggesting the VLCFA modification is important during the symbiosis. Since there are hundreds of nodule specific cysteine-rich peptides produced by the host plant Medicago truncatula, the BacA mediated uptake of one of these peptides combined with the VLCFA modification may account for the essential role of the BacA protein in the legume symbiosis.

Este trabalho apresenta alguns aspectos específicos sobre o tema “proteção de estruturas contra descargas atmosféricas", principalmente no que se refere ao uso de partes estruturais das edificações como integrantes do sistema de proteção. São discutidos neste trabalho diversos pontos polêmicos referentes ao tema, tais como: captores especiais, o sistema dissipativo e o uso das ferragens das colunas de concreto armado como parte dos subsistemas de descida e de aterramento. São abordados, também, os principais danos que podem ocorrer nas edificações devido às descargas atmosféricas, principalmente quando a proteção utilizar partes naturais da estrutura, tais como: os telhados metálicos e as emendas das ferragens das colunas de concreto armado. Após uma verificação do estado da arte no assunto, onde são estudados diversos trabalhos, normas e livros sobre as características das descargas, sobre os danos oriundos das descargas, blindagens, medições de campos eletromagnéticos, distribuição de correntes e sobre a normalização e ensaios referentes à proteção de estruturas contra descargas atmosféricas, o assunto foi estudado em três partes que compõem os subsistemas do SPDA. No subsistema de captação, os danos em telhas metálicas e de concreto protendido foram estudados através de ensaios e verificações teóricas e, principalmente, experimentais. No subsistema de descida, o estudo experimental deu enfoque às emendas das ferragens e ao uso destas como descidas naturais. Finalmente, no subsistema de aterramento, as fundações das edificações foram estudadas através de verificações experimentais como parte do aterramento. Este trabalho acrescenta ao conhecimento atual sobre o assunto, diversas particularidades não contempladas nas normas e nas publicações mais recentes no que tange à proteção de estruturas contra as descargas atmosféricas utilizando partes naturais das edificações. / This work presents some specific aspects on the subject “lightning protection of structures“, mainly when the use of structural parts of the buildings as part of the protection systems is concerned. Several polemical issues are discussed, such as: special air-termination, the dissipation array system and the use of steel-reinforcing rods of the reinforced concrete columns as part of the down-conductor system and of the earth-termination system. Also treated is the main damage that can occur to the buildings due to lightning, mainly when the protection system uses the natural components of the structures, such as: the metallic rooftops and the joints of the steel-reinforcing rods of the reinforced concrete columns. After examining the state-of-the-art in the subject, which analyses several papers, standards and books about lightning characteristics, damage resulting from lightning, shieldings, measurements of electromagnetic fields, distribution of currents and about standardisation and tests related to the protection of structures against lightning, the subject was studied in the three parts that compose the lightning protection systems. In the air-termination system, the damage to metallic rooftops and to pre-stressed concrete rooftop tiles was studied by means of tests and theoretical and mainly experimental analyses. In the down-conductor system, the experimental study focused on the joints of the steel-reinforcing rods and on the use of these as natural down-conductors. Finally, in the earth-termination system, the foundations of the buildings were studied (as part of the grounding) through experimental analyses. This work adds to the current knowledge about the subject several particularities which are not observed in the standards or in the most recent publications concerning the lightning protection systems in buildings that use their natural components for this purpose.

Sepsis is the body’s response to an overwhelming infection and is a serious consequence of critical illness. It can cause tissue damage, organ failure, and death. Sepsis continues to have an unacceptably high mortality rate, due to the lack of effective treatments. Specific therapeutic targets for sepsis remain elusive since the complex functional changes that result in a septic state remain poorly understood. Macrophage Scavenger Receptor A (SRA, CD204) is a surface receptor that binds negatively charged, endogenous and exogenous ligands. We have discovered that SRA plays a significant role in the pathophysiology of sepsis. We have shown that mice with SRA have increased inflammation, decreased survival, and increased bacterial burden compared to SRA deficient mice. We have also found an increase in the expression of SRA on monocytes and macrophages in septic wild type mice. To determine the mechanism responsible for increased SRA expression in sepsis we treated a mouse macrophage cell line, (J774a.1), with mediators that stimulate toll like receptors (TLRs), innate immune receptors which are activated in sepsis. The cells were cultured with ultra pure LPS (a TLR 4 ligand), PAM3CSK4 (a TLR 2 ligand), glucan (a Dectin-1 ligand), ultra pure LPS and PAM3CSK4, or ultra pure LPS and glucan for 24 hours. The cells were stained with an SRA antibody, and flow cytometry was used to measure the SRA expression for each treatment group. LPS treatment alone resulted in a significant increase in SRA expression when compared to control cells. Specifically, LPS increased SRA expression by 53.4% compared to media alone (p

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Bovine mastitis remains one of the costliest diseases affecting the dairy industry. Individual susceptibility to mastitis and severity of infection varies between animals and can only be partially explained by genetics. As such, understanding how genetic predisposition coordinately interacts with epigenetic modifications and environmental exposures is necessary to bridge the gap in missing heritability. The role of DNA methylation in regulating the response to bacterial lipopolysaccharide (LPS) was first determined by performing reduced representation bisulfite sequencing on fibroblasts isolated from heifers at 5- and 16-months of age that exhibit an age-dependent up-regulation in LPS-responsiveness. More than 14,000 differentially methylated sites were identified between the two sets of cultures with a trend towards decreased methylation with age. Young cultures were also hyper-methylated in gene promoters regulated by NF-κB and exhibited lower expression in genes that regulate the innate immune response, suggesting that methylation contributes to gene regulation in fibroblast innate response. Previously, TLR4 expression was shown to differ in the age-dependent fibroblast model, however, it was not known if variation in TLR4 expression would affect mastitis severity. Therefore, fibroblasts were isolated from sixty lactating, adult Holstein cows and their expression of TLR4, along with LPS-induced production of IL-8 and IL-6, was used to rank the animals from high to low. Six high responders and six low responders were then experimentally infected in one mammary gland with E. coli. Overall, severity of mastitis was quite variable, with a few notable differences between high and low responders. High responding animals had an earlier increase in somatic cell count and febrile response that coincided with more efficient bacterial clearance. However, tissue damage and milk production did not differ between the two groups, indicating that while rapid up-regulation of the innate response addresses bacterial clearance, subsequent down-regulation is required to alleviate damage within the mammary gland. Finally, one-week old bull calves were subjected to treatment with either saline or LPS to determine if neonatal exposure to endotoxin would make calves less responsive to a second LPS challenge at 32-days of age. The initial treatment showed a large effect of LPS as measured by higher plasma IL-6 and TNF-α concentrations in calves treated with LPS over saline. Subsequent treatment of all 10 calves with LPS showed a very similar response between the two treatment groups and significant inter-animal variability in clinical response. Fibroblasts and monocyte-derived-macrophages (MDMs) were also isolated following initial treatment to determine if any changes occurred at the cellular level as a result of LPS exposure. Fibroblasts isolated from calves at 20-days of age had a very low response to LPS that did not differ between the early life treatments. MDMs isolated from calves at 28-days of age were more responsive to LPS, but again no differences were detected between the early life treatments. In summary, our results suggest that DNA methylation likely plays a role in the cellular response to LPS and may partially contribute to differences between animals in severity of E. coli mastitis, however, the appropriate in vitro phenotype to detect susceptible animals still needs to be characterized before epigenetic biomarkers can be identified, and perhaps modified by environmental interventions.

Anhand einer umfassenden Falldarstellung einer jungen Patientin mit einem lebensbedrohlichen Gesichtsschmerzsyndrom, das nach septischer Thrombose der periorbitalen venösen und arteriellen Gefäße aufgetreten war, wurde die Bedeutung einer medikamentösen Antikoagulation für die erfolgreiche Schmerztherapie herausgearbeitet. An diesem Fallbeispiel konnte aber auch gezeigt werden, dass keine sicheren Parameter für die Indikation einer solchen Gerinnungstherapie vorlagen. Die Bedeutung dieses Falls lag unzweifelhaft in der Erkenntnis, dass in einer anhaltenden Aktivierung des Kontaktsystems der Gerinnung ein bislang unterschätztes Potential für die Entstehung und Unterhaltung ungeklärter Schmerzen liegen könnte und nicht zuletzt auch daran, dass sich diese ätiologische Komponente in der Komplexität der Erkrankung diagnostisch nicht eindeutig sichern ließ. Mit der Translokation von LPS aus der intestinalen Mukosa in endothelial vorgeschädigte Gefäßabschnitte wurde eine Hypothese vorgetragen, die neben einer schwer detektierbaren inflammatorischen Komponente auch das prokoagulatorische Potential der Schmerzentstehung erklären könnte. Die prokoagulatorische Komponente dieses hypothetischen Entstehungs-mechanismus chronischer Schmerzen müsste, so die Arbeitshypothese, umso dominanter sein, wenn prokoagulatorisch wirksame genetische Faktoren bei den Patienten hinzukommen. Unter der Annahme, dass eine solche zusätzliche Diathese nicht nur eine Schrittmacherfunktion haben, sondern auch einen diagnostischen Beitrag liefern könnte, wurde dieses diagnostische Pilotprojekt mit der empirisch begründeten Heparintherapie von 97 Schmerzpatienten verbunden. Alle Pa-tienten wurden mit dem niedermolekularen Heparin Enoxaparin behandelt und nach zehn Behandlungstagen in vier verschiedene Respondergruppen (Gruppe 1 bis 4) eingeteilt. Diese Gruppen wurden auf fünf prothrombotische Parameter untersucht. Dazu wurden die Allelprävalenzen des Plasminogen Aktivator Inhibitor-(PAI-1 4G/5G) Polymorphismus, der Faktor V-Leiden-Mutation, der Prothrombin (G20210A) Genmutation sowie die Prävalenzen der Hyperfibrinogenämie und des Protein S-Mangels ermittelt. Mit Hilfe des exakten Fisher Tests wurden jeweils die Allelprävalenzen und Parameter sowohl der Respondergruppen 1 bis 3 mit einem Kollektiv der Allgemeinbevölkerung als auch mit dem Kollektiv der Non-Responder (Gruppe 4) verglichen. Die Prävalenz des Allels A der Faktor V-Leiden-Mutation G1691A war im Enoxaparin-Kollektiv bei den Respondern der Gruppen 1 bis 3 im Vergleich zur Allgemeinbevölkerung und zur Non-Respondergruppe (Gruppe 4) signifikant erhöht. Die Allelprävalenzen und Parameter der übrigen prokoagulatorischen Faktoren unterschieden sich von denen der Kontrollgruppen nicht. Anhand des Kallikrein-Kinin-Systems als möglichem Effektor des Hämosta-sesystems konnten Hinweise auf die kausale Wirksamkeit des nieder-molekularen Heparins Enoxaparin bei der Behandlung chronischer Schmerzen gegeben werden. / We showed that low molecular heparins (enoxaparin) may help as a remedy in chronic pain syndromes. In our findings the inherited disorder Factor V Leiden was significantly higher in patients with chronic pain that had a benefit from enoxaparin in comparison to non-responders and to common population. The effect was proven by the Kallikrein-kinin-system.

Some male sexually selected traits are sensitive to stressors early in life and provide females with information to discriminate among males with different developmental experiences. Moreover, female early life experiences could also impact which males they choose. Females might either choose honest traits indicative of male quality, no matter their own experiences, or they might choose mates to match or compensate for their own experiences. To determine how developmental stressors alter male sexually-selected traits and female preference thereof, I exposed zebra finches (Taeniopygia guttata, ZEFI) to i) lipopolysaccharide (LPS), an immunogenic, Gram-negative bacterial component, ii) corticosterone (CORT), an avian stress steroid, iii) both challenges (CORT/LPS), or iv) none of the above (control vehicles). Finches were exposed during development (12-28 days post-hatch) and male traits (e.g., body size, bill and cheek coloration) and female behaviors (e.g., general activity, male sampling effort, and male preference) were then measured in adulthood. Control males were predicted to express the most elaborate traits followed by LPS, CORT, and then CORT/LPS males. If female preference was generally driven by male quality, control females were predicted to be most selective followed by LPS, CORT, and CORT/LPS females. Alternatively, if female choice was contingent on her own experience, females might choose males with similar (i.e, matching) or distinct (i.e, complementarity) developmental histories. Of the male characteristics measured, only cheek coloration was impacted by treatment early in life; CORT/LPS males had duller, less orange cheeks than controls. For females, overall activity was reduced in CORT/LPS females. More importantly in regards to mate choice, females exhibited a blend of matching and complimentary behavior; females not exposed to LPS or CORT preferred males also not exposed to LPS or CORT. In general, females avoided LPS males no matter their own experience. Altogether, this study suggests that female mate preference is quite sensitive to early-life experiences and driven by a mix of choice of outright male quality and relative complementarity.