The reactive site loop of the serpin SCCA1 is essential for cysteine proteinase inhibition

The high-molecular-weight serine proteinase inhibitors (serpins) are restricted, generally, to inhibiting proteinases of the serine mechanistic class. However, the viral serpin, cytokine response modifier A, and the human serpins, antichymotrypsin and squamous cell carcinoma antigen 1 (SCCA1), inhibit different members of the cysteine proteinase class. Although serpins employ a mobile reactive site loop (RSL) to bait and trap their target serine proteinases, the mechanism by which they inactivate cysteine proteinases is unknown. Our previous studies suggest that SCCA1 inhibits papain-like cysteine proteinases in a manner similar to that observed for serpin–serine proteinase interactions. However, we could not preclude the possibility of an inhibitory mechanism that did not require the serpin RSL. To test this possibility, we employed site-directed mutagenesis to alter the different residues within the RSL. Mutations to either the hinge or the variable region of the RSL abolished inhibitory activity. Moreover, RSL swaps between SCCA1 and the nearly identical serpin, SCCA2 (an inhibitor of chymotrypsin-like serine proteinases), reversed their target specificities. Thus, there were no unique motifs within the framework of SCCA1 that independently accounted for cysteine proteinase inhibitory activity. Collectively, these data suggested that the sequence and mobility of the RSL of SCCA1 are essential for cysteine proteinase inhibition and that serpins are likely to utilize a common RSL-dependent mechanism to inhibit both serine and cysteine proteinases.

T helper type 2 inflammatory disease in the absence of interleukin 4 and transcription factor STAT6

An important signaling pathway for the differentiation of T helper type 2 (TH2) cells from uncommitted CD4 T cell precursors is activation of the STAT6 transcription factor by interleukin 4 (IL-4). The protooncogene BCL-6 is also involved in TH2 differentiation, as BCL-6 −/− mice develop an inflammation of the heart and lungs associated with an overproduction of TH2 cells. Surprisingly, IL-4 −/− BCL-6 −/− and STAT6 −/− BCL-6 −/− double-mutant mice developed the same TH2-type inflammation of the heart and lungs as is characteristic of BCL-6 −/− mice. Furthermore, a TH2 cytokine response developed in STAT6 −/− BCL-6 −/− and IL-4 −/− BCL-6 −/− mice after immunization with a conventional antigen in adjuvant. In contrast to these in vivo findings, STAT6 was required for the in vitro differentiation of BCL-6 −/− T cells into TH2 cells. BCL-6, a transcriptional repressor that can bind to the same DNA binding motifs as STAT transcription factors, seems to regulate TH2 responses in vivo by a pathway independent of IL-4 and STAT6.

Rôle des cellules endothéliales dans l’immunité innée précoce induite lors d’infections par des coronavirus murins

Les cellules endothéliales (EC) constituent une première barrière physique à la dissémination de virus pléiotropiques circulant par voie hématogène mais leur contribution à la défense innée anti-virale est peu connue. Des dysfonctions des EC de la barrière hémato-encéphalique (BMEC) et des sinusoïdes hépatiques (LSEC) ont été rapportées dans des neuropathologies et des hépatites aiguës ou chroniques d’origine virale, suggérant que des atteintes à leur intégrité contribuent à la pathogenèse. Les sérotypes de coronavirus de l’hépatite murine (MHV), se différenciant par leur capacité à induire des hépatites et des maladies neurologiques de sévérité variable et/ou leur tropisme pour les EC, représentent des modèles viraux privilégiés pour déterminer les conséquences de l’infection des EC sur la pathogenèse virale. Lors d’infection par voie hématogène, le sérotype MHV3, le plus virulent des MHV, induit une hépatite fulminante, caractérisée par une réponse inflammatoire sévère, et des lésions neurologiques secondaires alors que le sérotype moins virulent, MHV-A59, induit une hépatite modérée sans atteintes secondaires du système nerveux central (SNC). Par ailleurs, le sérotype MHV3, à la différence du MHV-A59, démontre une capacité à stimuler la production de cytokines par la voie TLR2. Les variants atténués du MHV3, les virus 51.6-MHV3 et YAC-MHV3, sont caractérisés par un faible tropisme pour les LSEC et induisent respectivement une hépatite modérée et subclinique. Compte tenu de l’importance des LSEC dans le maintien de la tolérance hépatique et de l’élimination des pathogènes circulants, il a été postulé que la sévérité de l’hépatite et de la réponse inflammatoire lors d’infections par les MHV est associée à la réplication virale et à l’altération des propriétés tolérogéniques et vasculaires des LSEC. Les désordres inflammatoires hépatiques pourraient résulter d’une activation différentielle du TLR2, plutôt que des autres TLR et des hélicases, selon les sérotypes. D’autre part, compte tenu du rôle des BMEC dans la prévention des infections du SNC, il a été postulé que l’invasion cérébrale secondaire par les coronavirus est reliée à l’infection des BMEC et le bris subséquent de la barrière hémato-encéphalique (BHE). À l’aide d’infections in vivo et in vitro par les différents sérotypes MHV, chez des souris ou des cultures de BMEC et de LSEC, nous avons démontré, d’une part, que l’infection in vitro des LSEC par le sétotype MHV3, à la différence des variants 51.6- et YAC-MHV3, altérait la production du facteur vasodilatant NO et renversait leur phénotype tolérogénique en favorisant la production de cytokines et de chimiokines inflammatoires. Ces dysfonctions se traduisaient in vivo par une réponse inflammatoire incontrôlée et une dérégulation du recrutement intrahépatique de leucocytes, favorisant la réplication virale et les dommages hépatiques. Nous avons aussi démontré, à l’aide de souris TLR2 KO et de LSEC dont l’expression du TLR2 a été abrogée par des siRNA, que la sévérité de l’hépatite et de la réponse inflammatoire induite par le sérotype MHV3, dépendait en partie de l’induction et de l’activation préférentielle du TLR2 par le virus dans le foie. D’autre part, la sévérité de la réplication virale au foie et des désordres dans le recrutement leucocytaire intrahépatique induits par le MHV3, et non par le MHV-A59 et le 51.6-MHV3, corrélaient avec une invasion virale subséquente du SNC, au niveau de la BHE. Nous avons démontré que l’invasion cérébrale du MHV3 était associée à une infection productive des BMEC et l’altération subséquente des protéines de jonctions serrées occludine, VE-cadhérine et ZO-1 se traduisant par une augmentation de la perméabilité de la BHE et l’entrée consécutive du virus dans le cerveau. Dans l’ensemble, les résultats de cette étude mettent en lumière l’importance du maintien de l’intégrité structurale et fonctionnelle des LSEC et des BMEC lors d’infections virales aigües par des MHV afin de limiter les dommages hépatiques associés à l’induction d’une réponse inflammatoire exagérée et de prévenir le passage des virus au cerveau suite à une dissémination par voie hématogène. Ils révèlent en outre un nouveau rôle aggravant pour le TLR2 dans l’évolution de l’hépatite virale aigüe ouvrant la voie à de nouvelles avenues thérapeutiques visant à moduler l’activité inflammatoire du TLR2.

Enhanced clearance of Candida albicans from the oral cavities of mice following oral administration of Lactobacillus acidophilus

Orally administered live Lactobacillus acidophilus was assessed for its capacity to enhance clearance from the oral cavity of DBA/2 mice shown previously to be 'infection prone'. L. acidophilus fed to DBA/2 mice significantly shortened the duration of colonization of the oral cavity compared to controls. Enhanced clearance of Candida albicans correlated with both early mRNA gene expression for interleukin (IL)-4 and interferon (IFN)-gamma and expression of their secreted products in cultures of cervical lymph nodes stimulated with Candida antigen. In addition rapid clearance correlated with higher levels of IFN-gamma and nitric oxide in saliva. Delayed clearance, less pronounced levels of the cytokine response, saliva IFN-gamma and nitric oxide, and later mRNA expression for IL-4 and IFN-gamma relative to feeding with the L. acidophilus isolate were noted in mice fed a different Lactobacillus isolate (L. fermentum). These observations indicate significant variations in individual isolates to activate the common mucosal system.

The roles of exercise-induced immune system disturbances in the pathology of heat stroke - The dual pathway model of heat stroke

Heat stroke is a life-threatening condition that can be fatal if not appropriately managed. Although heat stroke has been recognised as a medical condition for centuries, a universally accepted definition of heat stroke is lacking and the pathology of heat stroke is not fully understood. Information derived from autopsy reports and the clinical presentation of patients with heat stroke indicates that hyperthermia, septicaemia, central nervous system impairment and cardiovascular failure play important roles in the pathology of heat stroke. The current models of heat stroke advocate that heat stroke is triggered by hyperthermia but is driven by endotoxaemia. Endotoxaemia triggers the systemic inflammatory response, which can lead to systemic coagulation and haemorrhage, necrosis, cell death and multi-organ failure. However, the current heat stroke models cannot fully explain the discrepancies in high core temperature (Tc) as a trigger of heat stroke within and between individuals. Research on the concept of critical Tc: as a limitation to endurance exercise implies that a high Tc may function as a signal to trigger the protective mechanisms against heat stroke. Athletes undergoing a period of intense training are subjected to a variety of immune and gastrointestinal (GI) disturbances. The immune disturbances include the suppression of immune cells and their functions, suppression of cell-mediated immunity, translocation of lipopolysaccharide (LPS), suppression of anti-LPS antibodies, increased macrophage activity due to muscle tissue damage, and increased concentration of circulating inflammatory and pyrogenic cytokines. Common symptoms of exercise-induced GI disturbances include diarrhoea, vomiting, gastrointestinal bleeding, and cramps, which may increase gut-related LPS translocation. This article discusses the current evidence that supports the argument that these exercise-induced immune and GI disturbances may contribute to the development of endotoxaemia and heat stroke. When endotoxaemia can be tolerated or prevented, continuing exercise and heat exposure will elevate Tc to a higher level (> 42 degrees C), where heat stroke may occur through the direct thermal effects of heat on organ tissues and cells. We also discuss the evidence suggesting that heat stroke may occur through endotoxaemia (heat sepsis), the primary pathway of heat stroke, or hyperthermia, the secondary pathway of heat stroke. The existence of these two pathways of heat stroke and the contribution of exercise-induced immune and GI disturbances in the primary pathway of heat stroke are illustrated in the dual pathway model of heat stroke. This model of heat stroke suggests that prolonged intense exercise suppresses anti-LPS mechanisms, and promotes inflammatory and pyrogenic activities in the pathway of heat stroke.

The Proinflammatory Environment in Potential Heart and Lung Donors: Prevalence and Impact of Donor Management and Hormonal Therapy

BACKGROUND: Brain stem death can elicit a potentially manipulable cardiotoxic proinflammatory cytokine response. We investigated the prevalence of this response, the impact of donor management with tri-iodothyronine (T3) and methylprednisolone (MP) administration, and the relationship of biomarkers to organ function and transplant suitability. METHODS: In a prospective randomized double-blinded factorially designed study of T3 and MP therapy, we measured serum levels of interleukin-1 and -6 (IL-1 and IL-6), tumor necrosis factor-alpha (TNF-alpha), C-reactive protein, and procalcitonin (PCT) levels in 79 potential heart or lung donors. Measurements were performed before and after 4 hr of algorithm-based donor management to optimize cardiorespiratory function and +/-hormone treatment. Donors were assigned to receive T3, MP, both drugs, or placebo. RESULTS: Initial IL-1 was elevated in 16% donors, IL-6 in 100%, TNF-alpha in 28%, CRP in 98%, and PCT in 87%. Overall biomarker concentrations did not change between initial and later measurements and neither T3 nor MP effected any change. Both PCT (P =0.02) and TNF-alpha (P =0.044) levels were higher in donor hearts with marginal hemodynamics at initial assessment. Higher PCT levels were related to worse cardiac index and right and left ventricular ejection fractions and a PCT level more than 2 ng x mL(-1) may attenuate any improvement in cardiac index gained by donor management. No differences were observed between initially marginal and nonmarginal donor lungs. A PCT level less than or equal to 2 ng x mL(-1) but not other biomarkers predicted transplant suitability following management. CONCLUSIONS: There is high prevalence of a proinflammatory environment in the organ donor that is not affected by tri-iodothyronine or MP therapy. High PCT and TNF-alpha levels are associated with donor heart dysfunction. (C) 2009 Lippincott Williams & Wilkins, Inc.

A molecular analysis of the interactions of escherichia coli and macrophages

Escherichia coli (E.coli) is a diverse bacterial species that primarily forms a beneficial symbiotic relationship with the host in the human lower gastrointestinal track (GIT), however it can also be pathogenic in this environment. Furthermore, some strains can diverge from the GIT and occupy niches such as the urinary tract. In all these environments, E. coli interacts with the immune system and macrophages represent the front line of the innate immune system. In this study we characterise the immune response by macrophages to E. coli infection. It was shown that E. coli broadly provoke a similar cytokine response during macrophages infection and furthermore are degraded primarily by the phagocytosis pathway. Recently a new group of E. coli called Adherent Invasive Escherichia coli (AIEC) has been described. AIEC are present in the guts of Crohn’s disease (CD) patients at a higher frequency than in healthy patients. AIEC can replicate in macrophages but the mechanism for this is not fully understood. The processing of AIEC by macrophages was investigated and it was shown that AIEC only replicated in permissive macrophages. Furthermore, even in a permissive macrophages AIEC are trafficked through macrophages in a similar manner to commensal E. coli. This supports the hypothesis that AIEC are highly similar to commensal E. coli and only cause pathogenicity when present in the permissive environment of the gut of CD patients. Replication in macrophages requires functioning metabolic pathways and it was identified that glycolysis is important for AIEC survival in macrophages. AIEC mutants without a fully functioning glycolysis pathway induced less IL-1β cytokine release from macrophages than wild type strain suggesting that metabolism plays a role in inflammasome activation. Furthermore, AIEC mutants that could not produce the glycolytic end product acetate induced significantly reduced IL-1β release during infection. This suggest that the acetate molecule or a phenotypic effect of its production may be a driver of IL-1β release from AIEC infected macrophages. The interaction of uropathogenic E. coli (UPEC) with macrophages was also investigated. UPEC induced very high levels of cytotoxicity in human macrophages which was shown to be dependent on the production of the pore forming toxin α-hemolysin. However, UPEC did not induced high levels of cytotoxicity in murine macrophages suggesting there are species specific sensitivity to α-hemolysin that should be considered when studying UPEC pathogenicity in murine models.

La concentración de calcio iónico se asocia a la presencia de fibrilación auricular post operatoria en pacientes llevados a revascularización miocardica

Objetivo. Determinar en un grupo de pacientes llevados a revascularización miocárdica si existió asociación entre la presencia de niveles de calcio iónico inferiores a 1,1 en las 24 horas del post operatorio y la ocurrencia de fibrilación auricular post operatoria. Metodología. Estudio observacional, analítico de casos y controles, en donde de manera consecutiva se incluyeron 110 sujetos (57 en el grupo de casos con presencia de fibrilación auricular post operatoria y 54 en el grupo de controles sin evidencia de fibrilación auricular) estos sujetos fueron llevados a revascularización miocárdica en la Fundación Cardioinfantil en los años 2010 a 2015. Resultados. Hubo 13 casos de fibrilación auricular post operatoria en pacientes con niveles de calcio iónico inferiores a 1,1 mmol/l en las primeras 24 horas del post operatorio OR: 0,5, IC (0,2-1,2) p: 0,1. Sin determinarse asociación por limitaciones del estudio, sin embargo un 29% de los pacientes con fibrilación auricular tuvieron niveles de calcio inferiores a 1,1 mmol/l en las primeras 24 horas del post operatorio, este valor aumenta a 31% cuando se analizan por separado los valores de calcio obtenidos a las 12 horas. Conclusiones. Aunque no se logró determinar asociación entre la fibrilación auricular post operatoria y las concentraciones de calcio iónico, de manera exploratoria se pudo establecer que un 29% de los pacientes con fibrilación auricular tuvieron concentraciones de calcio iónico inferiores a 1,1 mmol/l, este valor aumenta a 31% cuando se analizan los niveles de calcio iónico por separado.

Immunotoxicity of aflatoxin B1: Impairment of the cell-mediated response to vaccine antigen and modulation of cytokine expression

Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus or A. parasiticus, is a frequent contaminant of food and feed. This toxin is hepatotoxic and immunotoxic. The present study analyzed in pigs the influence of AFB1 on humoral and cellular responses, and investigated whether the immunomodulation observed is produced through interference with cytokine expression. For 28 days, pigs were fed a control diet or a diet contaminated with 385, 867 or 1807 mu g pure AFB1/kg feed. At days 4 and 15, pigs were vaccinated with ovalbumin. AFB1 exposure, confirmed by an observed dose-response in blood aflatoxin-albumin adduct, had no major effect on humoral immunity as measured by plasma concentrations of total IgA, IgG and IgM and of anti-ovalbumin IgG. Toxin exposure did not impair the mitogenic response of lymphocytes but delayed and decreased their specific proliferation in response to the vaccine antigen, suggesting impaired lymphocyte activation in pigs exposed to AFB1. The expression level of pro-inflammatory (TNF-alpha, IL-1 beta, IL-6, IFN-gamma) and regulatory (IL-10) cytokines was assessed by real-time PCR in spleen. A significant up-regulation of all 5 cytokines was observed in spleen from pigs exposed to the highest dose of AFB1. In pigs exposed to the medium dose, IL-6 expression was increased and a trend towards increased IFN-gamma and IL-10 was observed. In addition we demonstrate that IL-6 impaired in vitro the antigenic- but not the mitogenic-induced proliferation of lymphocytes from control pigs vaccinated with ovalbumin. These results indicate that AFB1 dietary exposure decreases cell-mediated immunity while inducing an inflammatory response. These impairments in the immune response could participate in failure of vaccination protocols and increased susceptibility to infections described in pigs exposed to AFB1. (C) 2008 Elsevier Inc. All rights reserved.

Association of Human T Lymphotropic Virus 1 Amplification of Periodontitis Severity with Altered Cytokine Expression in Response to a Standard Periodontopathogen Infection

Background. Periodontal diseases (PDs) are infectious diseases in which periodontopathogens trigger chronic inflammatory and immune responses that lead to tissue destruction. Recently, viruses have been implicated in the pathogenesis of PDs. Individuals infected with human T lymphotropic virus 1 (HTLV-1) present with abnormal oral health and a marked increased prevalence of periodontal disease. Methods. In this study, we investigated the patterns of periodontopathogen infection and local inflammatory immune markers in HTLV-1-seropositive individuals with chronic periodontitis (CP/HTLV-1 group) compared with HTLV-1 -seronegative individuals with chronic periodontitis (CP group) and periodontally healthy, HTLV-1 -seronegative individuals (control group). Results. Patients in the CP/HTLV-1 group had significantly higher values of bleeding on probing, mean probing depth, and attachment loss than patients in the CP group. The expression of tumor necrosis factor a and interleukin (IL) 4 was found to be similar in the CP and CP/HTLV-1 groups, whereas IL-12 and IL-17 levels trended toward a higher expression in the CP/HTLV-1 group. A significant increase was seen in the levels of IL-1 beta and interferon gamma in the CP/HTLV-1 group compared with the CP group, whereas expression of the regulatory T cell marker FOXp3 and IL-10 was significantly decreased in the lesions from the CP/HTLV-1 group. Interestingly, similar frequency and/or load of periodontopathogens (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacter actinomycetemcomitans) and frequency of viruses (herpes simplex virus 1, human cytomegalovirus, and Epstein-Barr virus) characteristically associated with PDs were found in the CP/HTLV and CP groups. Conclusions. HTLV-1 may play a critical role in the pathogenesis of periodontal disease through the deregulation of the local cytokine network, resulting in an exacerbated response against a standard periodontopathogen infection.

Cytokine production in experimental paracoccidioidomycosis of murine selected lineages for acute inflammatory response (air)

Paracoccidioidomycosis is a systemic human mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis), an imperfect dimorphic fungus whose conidia are its infective form. Mice genetically selected for maximum (AIRmax) and minimum (AIRmin) acute inflammatory response were used as experimental paracoccidioidomycosis models. The animals were intraperitoneally inoculated with P. brasiliensis (strain 18) and killed 6, 12 and 24 hours or 3, 7 and 14 days after infection. In these periods, fragments from their spleen, liver and lung were removed for evaluation of the infection level by fungal cells, assessment of macrophagic activity by peritoneal and splenic macrophages - through the determination of nitric oxide (NO) concentrations and production of pro- and anti-inflammatory cytokines of lung and spleen homogenate supernatants. In the present study, it was observed that AIRmax lineages presented greater control of the infectious process than the AIRmin ones. Regarding NO production, AIRmax animals produced more metabolites in late periods, what may help control the infectious process. Concerning cytokine production, it was observed that the production of INF-gamma, TNF-alpha, IL-1, IL-6, IL-8 and IL-12 were increased in AIRmax lineages in most analyzed organs and periods, thus contributing to the greater resistance exhibited by such lineages against infection, except for IL-4 and IL-10 that showed decreased production in AIRmax lineage, reproducing its suppressive biological effect. From these results, it was observed that the AIRmax lineage was more effective in controlling the infectious process, with an important involvement of the analyzed cytokines. These findings are probably related to the genetically selected factors involved in the acute inflammatory response.

Cytokine gene expression in response to Haemonchus placei infections in Nelore cattle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Nerve growth factor inhibits sympathetic neurons’ response to an injury cytokine

Axonal damage to adult peripheral neurons causes changes in neuronal gene expression. For example, axotomized sympathetic, sensory, and motor neurons begin to express galanin mRNA and protein, and recent evidence suggests that galanin plays a role in peripheral nerve regeneration. Previous studies in sympathetic and sensory neurons have established that galanin expression is triggered by two consequences of nerve transection: the induction of leukemia inhibitory factor (LIF) and the reduction in the availability of the target-derived factor, nerve growth factor. It is shown in the present study that no stimulation of galanin expression occurs following direct application of LIF to intact neurons in the superior cervical sympathetic ganglion. Injection of animals with an antiserum to nerve growth factor concomitant with the application of LIF, on the other hand, does stimulate galanin expression. The data suggest that the response of neurons to an injury factor, LIF, is affected by whether the neurons still receive trophic signals from their targets.

Interferon β transduction of peripheral blood lymphocytes from HIV-infected donors increases Th1-type cytokine production and improves the proliferative response to recall antigens

We are developing a gene therapy method of HIV infection based on the constitutive low production of interferon (IFN) β. Peripheral blood lymphocytes (PBL) from HIV-infected patients at different clinical stages of infection were efficiently transduced with the HMB-HbHuIFNβ retroviral vector. The constitutive low production of IFN-β in cultured PBL from HIV-infected patients resulted in a decreased viral production and an enhanced survival of CD4+ cells, and this protective effect was observed only in the PBL derived from donors having a CD4+ cell count above 200 per mm3. In IFN-β-transduced PBL from healthy and from HIV-infected donors, the production of the Th1-type cytokines IFN-γ and interleukin (IL)-12 was enhanced. In IFN-β-transduced PBL from HIV-infected donors, the production of IL-4, IL-6, IL-10, and tumor necrosis factor α was maintained at normal levels, contrary to the increased levels produced by the untransduced PBL. The proliferative response to recall antigens was partially restored in IFN-β-transduced PBL from donors with an impaired antigen response. Thus, in addition to inhibiting HIV replication, IFN-β transduction of PBL from HIV-infected donors improves several parameters of immune function.