Enhanced Proinflammatory Cytokine Response to Bacterial Lipopolysaccharide in the Adult Male Rat after either Neonatal or Prepubertal Ablation of Biological Testosterone Activity.

A sex steroid-dependent modulation of the immune function in mammals is accepted, and evidence suggests that while estrogens enhance, androgens inhibit the immune response. The aim of this study was to explore in the adult male rat the effect of either neonatal flutamide (FTM) treatment or prepubertal orchidectomy (ODX) on endocrine markers in the basal condition and peripheral tumor necrosis factor alpha (TNFα) levels during inflammatory stress. For these purposes, (1) 5-day-old male rats were subcutaneously injected with either sterile vehicle alone or containing 1.75 mg FTM, and (2) 25-day-old male rats were sham operated or had ODX. Rats were sacrificed (at 100 days of age) in the basal condition for determination of peripheral metabolite levels. Additional rats were intravenously injected with bacterial lipopolysaccharide (LPS; 25 μg/kg body weight, i.v.) and bled for up to 4 h. Data indicate that (1) ODX increased peripheral glucocorticoid levels and reduced those of testosterone, whereas FTM-treated rats displayed low circulating leptin concentrations, and (2) LPS-induced TNFα secretion in plasma was significantly enhanced in the FTM and ODX groups. Our study supports that neonatal FTM treatment affected adiposity function, and adds data maintaining that androgens have a suppressive role in proinflammatory cytokine release in plasma during inflammation.

Effect of sequential heat and cold shocks on nuclear phenotypes of the blood-sucking insect, Panstrongylus megistus (Burmeister) (Hemiptera, Reduviidae)

Thermal shocks induce changes in the nuclear phenotypes that correspond to survival (heterochromatin decondensation, nuclear fusion) or death (apoptosis, necrosis) responses in the Malpighian tubules of Panstrongylus megistus. Since thermal tolerance increased survival and molting rate in this species following sequential shocks, we investigated whether changes in nuclear phenotypes accompanied the insect survival response to sequential thermal shocks. Fifth instar nymphs were subjected to a single heat (35 or 40°C, 1 h) or cold (5 or 0°C, 1 h) shock and then subjected to a second shock for 12 h at 40 or 0°C, respectively, after 8, 18, 24 and 72 h at 28°C (control temperature). As with specimen survival, sequential heat and cold shocks induced changes in frequency of the mentioned nuclear phenotypes although their patterns differed. The heat shock tolerance involved decrease in apoptosis simultaneous to increase in cell survival responses. Sequential cold shocks did not involve cell/nuclear fusion and even elicited increase in necrosis with advancing time after shocks. The temperatures of 40 and 0ºC were more effective than the temperatures of 35 and 5ºC in eliciting the heat and cold shock tolerances, respectively, as shown by cytological analysis of the nuclear phenotypes. It is concluded that different sequential thermal shocks can trigger different mechanisms of cellular protection against stress in P. megistus, favoring the insect to adapt to various ecotopes.

Five new CYLD mutations in skin appendage tumors and evidence that aspartic acid 681 in CYLD is essential for deubiquitinase activity.

Brooke-Spiegler syndrome, familial cylindromatosis, and familial trichoepithelioma are autosomal-dominant genetic predispositions for benign tumors of skin appendages caused by mutations in the CYLD gene localized on chromosome 16q12-q13. The encoded protein functions as ubiquitin-specific protease (UBP), which negatively regulates NF-kappaB and c-Jun N-terminal kinase (JNK) signaling. We investigated five families affected with these skin neoplasms and identified four premature stop codons and the novel missense mutation D681G in a family in which 11 of 12 investigated tumors were trichoepitheliomas. CYLD protein harboring this missense mutation had a significant reduced ability to inhibit TNF receptor-associated factor (TRAF)2- and TRAF6-mediated NF-kappaB activation, tumor necrosis factor-alpha (TNFalpha)-induced JNK signaling, and to deubiquitinate TRAF2. CYLD-D681G was coimmunoprecipitated by TRAF2, but was unable to cleave K63-linked polyubiquitin chains. Aspartic acid 681 is highly conserved in CYLD homologues and other members of the UBP family, but does not belong to the Cys and His boxes providing the CYLD catalytic triad (Cys601, His871, and Asp889). As reported previously, the homologous residue D295 of HAUSP/USP-7 forms a hydrogen bond with the C-terminal end of ubiquitin and is important for the enzymatic activity. These results underline that D681 in CYLD is required for cleavage of K63-linked polyubiquitin chains.

Molecular mechanisms for the transcriptional regulation of the Connexin36 gene expression in insulin-secreting cells

Résumé Régulation de l'expression de la Connexin36 dans les cellules sécrétrices d'insuline La communication intercellulaire est en partie assurée via des jonctions communicantes de type "gap". Dans la cellule ß pancréatique, plusieurs observations indiquent que le couplage assuré par des jonctions gap formées parla Connexine36 (Cx36) est impliqué dans le contrôle de la sécrétion de l'insuline. De plus, nous avons récemment démontré qu'un niveau précis d'expression de la Cx36 est nécessaire pour maintenir une bonne coordination de l'ensemble des cellules ß, et permettre ainsi une sécrétion synchrone et contrôlée d'insuline. Le développement du diabète et du syndrome métabolique est partiellement dû à une altération de la capacité des cellules ß à sécréter de l'insuline en réponse à une augmentation de la glycémie. Cette altération est en partie causée par l'augmentation prolongée des taux circulant de glucose, mais aussi de lipides, sous la forme d'acides gras libres, et de LDL (Low Density Lipoproteins), particules assurant le transport des acides gras et du cholestérol dans le sang. Nous avons étudié la régulation de l'expression de la Cx36 dans différentes conditions reflétant la physiopathologie du diabète de type 2 et du syndrome métabolique et démontré qu'une exposition prolongée à des concentrations élevées de glucose, de LDL, ainsi que de palmitate (acide gras saturé le plus abondant dans l'organisme), inhibent l'expression de la Cx36 dans les cellules ß. Cette inhibition implique l'activation de la PKA (Proteine Kinase A), qui stimule à son tour l'expression du facteur de transcription ICER-1 (Inductible cAMP Early Repressor-1). Ce puissant répresseur se fixe spécifiquement sur un motif CRE (cAMP Response Element), situé dans le promoteur du gène de la Cx36, inhibant ainsi son expression. Nous avons de plus démontré que des cytokines pro-inflammatoires, qui pourraient contribuer au développement du diabète, inhibent également l'expression de la Cx36. Cependant, les cytokines agissent indépendamment du répresseur ICER-1, mais selon un mécanisme requérant l'activation de l'AMPK (AMP dependant protein kinase). Sachant qu'un contrôle précis des niveaux d'expression de la Cx36 est un élément déterminant pour une sécrétion optimale de l'insuline, nos résultats suggèrent que la Cx36 pourrait être impliquée dans l'altération de la sécrétion de l'insuline contribuant à l'apparition du diabète de type 2. Summary A particular way by which cells communicate with each other is mediated by gap junctions, transmembrane structures providing a direct pathway for the diffusion of small molecules between adjacent cells. Gap junctional communication is required to maintain a proper functioning of insulin-secreting ß-cells. Moreover, the expression levels of connexin36 (Cx36), the sole gap junction protein expressed in ß-cells, are critical in maintaining glucose-stimulated insulin secretion. Chronic hyperglycemia and hyperlipidemia exert deleterious effects on insulin secretion and may contribute to the progressive ß-cell failure linked to the development of type 2 diabetes and metabolic syndrome. Since modulations of the Cx36 levels might impair ß-cell function, the general aim of this work was to elucidate wether elevated levels of glucose and lipids affect Cx36 expression. The first part of this work was dedicated to the study of the effect of high glucose concentrations on Cx36 expression. We demonstrated that glucose transcriptionally down-regulates the expression of Cx36 in insulin-secreting cells through activation of the protein kinase A (PKA), which in turn stimulates the expression of the inducible cAMP early repressor-1 (ICER-1). This repressor binds to a highly conserved cAMP response element (CRE) located in the Cx36 promoter, thereby inhibiting Cx36 expression. The second part of this thesis consisted in studying the effects of sustained exposure to free fatty acids (FFA) and human lipoproteins on Cx36 levels. The experiments revealed that the most abundant FFA, palmitate, as well as the atherogenic low density lipoproteins (LDL), also stimulate ICER-1 expression, resulting in Cx36 down-regulation. Finally, the third part of the work focused on the consequences of long-term exposure to proinflammatory cytokines on Cx36 content. Interleukin-1 ß (IL-1 ß) inhibits Cx36 expression and its effect is potentialized by tumor necrosis factor α (TNFα) and interferon γ (IFNγ). We further unveiled that the cytokines effect on Cx36 levels requires activation of the AMP dependent protein kinase (AMPK). Prolonged exposures to glucose, palmitate, LDL, and pro-inflammatory cytokines have all been proposed to contribute to the development of diabetes and metabolic syndrome. Since Cx36 expression levels are critical to maintain ß-cell function, Cx36 down-regulation by glucose, lipids, and cytokines might participate to the ß-cell failure associated with diabetes development.

The role of APRIL and BAFF in lymphocyte activation.

The TNF family ligands BAFF (also called BLyS) and APRIL regulate lymphocyte survival and activation. BAFF binds to three receptors, BAFF-R, TACI and BCMA, whereas APRIL interacts with TACI, BCMA and proteoglycans. The contribution of BAFF and APRIL to B-cell and plasma-cell survival, CD154 (CD40L)-independent antibody isotype switching, germinal center maintenance, T-dependent and T-independent antibody responses, and T cell co-stimulation are relatively well understood. Constitutive BAFF produced by stromal cells determines the size of the peripheral B cell pool, whereas inducible BAFF produced by myeloid and other cells supports local survival of B lymphocytes and can be associated with development of autoimmunity when deregulated.

Local moderate magnetically induced hyperthermia using an implant formed in situ in a mouse tumor model.

Purpose: We investigate a new heat delivery technique for the local treatment of solid tumors. The technique involves injecting a formulation that solidifies to form an implant in situ. This implant entraps superparamagnetic iron oxide nanoparticles (SPIONs) embedded in silica microbeads for magnetically induced moderate hyperthermia. Particle entrapment prevents phagocytosis and distant migration of SPIONs. The implant can be repeatedly heated by magnetic induction. Methods: We evaluated heating and treatment efficacies by means of thermometry and survival studies in nude mice carrying subcutaneous human colocarcinomas. At day 1, we injected the formulation into the tumor. At day 2, a single 20-min hyperthermia treatment was delivered by 141-kHz magnetic induction using field strengths of 9 to 12 mT under thermometry. Results: SPIONs embedded in silica microbeads were effectively confined within the implant at the injection site. Heat-induced necro-apoptosis was assessed by histology on day 3. On average, 12 mT resulted in tumor temperature of 47.8 degrees C, and over 70% tumor necrosis that correlated to the heat dose (AUC = 282 degrees C.min). In contrast, a 9-mT field strength induced tumoral temperature of 40 degrees C (AUC = 131 degrees C.min) without morphologically identifiable necrosis. Survival after treatment with 10.5 or 12 mT fields was significantly improved compared to non-implanted and implanted controls. Median survival times were 27 and 37 days versus 12 and 21 days respectively. Conclusion: Five of eleven mice (45%) of the 12 mT group survived one year without any tumor recurrence, holding promise for tumor therapy using magnetically induced moderate hyperthermia through injectable implants.

Nodular typhlitis associated with the nematodes Heterakis gallinarum and Heterakis isolonche in pheasants: frequency and pathology with evidence of neoplasia

An investigation related to the frequency and pathology of Heterakis gallinarum and pathology of Heterakis isolonche in pheasants from Rio de Janeiro, Brazil was conducted by means of clinical examinations, necropsies, and histopathological analysis in 50 ring-necked pheasants from backyard flocks of 11 localities; also, histological sections of caeca of golden pheasants deposited in the Helminthological Collection of the Oswaldo Cruz Institute (CHIOC) have been considered in the present study. During necropsies, only specimens of H. gallinarum were recovered with a prevalence of 90%, mean intensity of 81.9 and range of infection of 1-413. Gross lesions were characterized by congestion, thickening, petechial haemorrhages of the mucosa, intussusception, and nodules in the cecal wall. Under microscopy, chronic difuse typhlitis, haemosiderosis, granulomas with necrotic center in the submucosa and leiomyomas in the submucosa, muscular and serosa associated with immature H. gallinarum worms were observed. The examination of histological sections previously deposited in the CHIOC, revealed more severe alterations associated with concomitant infections with H. gallinarum and H. isolonche in golden pheasants, and were characterized by several necrotic areas with cholesterol clefts in the submucosa, giant cell granulomas in the submucosa, and serosa centralized by necrosis and worm sections and neoplastic nodules in the muscular and submucosa.

Cellular immune responses and disease control in acute AIDS-associated Kaposi's sarcoma.

Kaposi's sarcoma-associated herpesvirus (KSHV) specific T cell responses and KSHV viremia were analyzed in seven HIV-infected patients with active Kaposi's sarcoma lesions who initiated highly active antiretroviral therapy, and were compared between patients with improved Kaposi's sarcoma and those with progressive Kaposi's sarcoma requiring further systemic chemotherapy. Patients with controlled Kaposi's sarcoma disease demonstrated undetectable Kaposi's sarcoma viremia together with KSHV-specific CD8 T cells secreting interferon-gamma and tumor necrosis factor-alpha, whereas progressors showed increasing viremia with weak or no T-cell responses. These data point toward a potential role of KSHV-specific immunity in the control of AIDS-associated Kaposi's sarcoma.

Maladies inflammatoires de l'intestin [Main therapeutic advances in IBD in 2008]

A coimmunosuppression with azathioprine or methotrexate in addition to infliximab does not improve the therapeutic efficacy in Crohn's disease but increase the risks of infectious complications and neoplasia.

Pulmonary tuberculosis: evaluation of interferon-gamma levels as an immunological healing marker based on the response to the Bacillus Calmette-Guerin

Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis whose interaction with the host may lead to a cell-mediated protective immune response. The presence of interferon-g (IFN-gamma) is related to this response. With the purpose of understanding the immunological mechanisms involved in this protection, the lymphoproliferative response, IFN-g and other cytokines like interleukin (IL-5, IL-10), and tumor necrosis factor alpha (TNF-a) were evaluated before and after the use of anti-TB drugs on 30 patients with active TB disease, 24 healthy household contacts of active TB patients, with positive purified protein derivative (PPD) skin tests (induration > 10 mm), and 34 asymptomatic individuals with negative PPD skin test results (induration < 5 mm). The positive lymphoproliferative response among peripheral blood mononuclear cells of patients showed high levels of IFN-g, TNF-a, and IL-10. No significant levels of IL-5 were detected. After treatment with rifampicina, isoniazida, and pirazinamida, only the levels of IFN-g increased significantly (p < 0.01). These results highlight the need for further evaluation of IFN-g production as a healing prognostic of patients treated.

The in vitro cytopathology of a porcine and the simian (SA-11) strains of rotavirus

Rotaviruses have been implicated as the major causal agents of acute diarrhoea in mammals and fowls. Experimental rotavirus infection have been associated to a series of sub-cellular pathologic alterations leading to cell lysis which may represent key functions in the pathogenesis of the diarrhoeic disease. The current work describes the cytopathic changes in cultured MA-104 cells infected by a simian (SA-11) and a porcine (1154) rotavirus strains. Trypan blue exclusion staining showed increased cell permeability after infection by both strains, as demonstrated by cell viability. This effect was confirmed by the leakage of infected cells evaluated by chromium release. Nuclear fragmentation was observed by acridine orange and Wright staining but specific DNA cleavage was not detected. Ultrastructural changes, such as chromatin condensation, cytoplasm vacuolisation, and loss of intercellular contact were shown in infected cells for both strains. In situ terminal deoxynucleotidyl transferase (Tunel) assay did not show positive result. In conclusion, we demonstrated that both strains of rotavirus induced necrosis as the major degenerative effect.

Involvement of PPAR nuclear receptors in tissue injury and wound repair.

Tissue damage resulting from chemical, mechanical, and biological injury, or from interrupted blood flow and reperfusion, is often life threatening. The subsequent tissue response involves an intricate series of events including inflammation, oxidative stress, immune cell recruitment, and cell survival, proliferation, migration, and differentiation. In addition, fibrotic repair characterized by myofibroblast transdifferentiation and the deposition of ECM proteins is activated. Failure to initiate, maintain, or stop this repair program has dramatic consequences, such as cell death and associated tissue necrosis or carcinogenesis. In this sense, inflammation and oxidative stress, which are beneficial defense processes, can become harmful if they do not resolve in time. This repair program is largely based on rapid and specific changes in gene expression controlled by transcription factors that sense injury. PPARs are such factors and are activated by lipid mediators produced after wounding. Here we highlight advances in our understanding of PPAR action during tissue repair and discuss the potential for these nuclear receptors as therapeutic targets for tissue injury.

Identification of proteoglycans as the APRIL-specific binding partners.

B cell activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferation-inducing ligand (APRIL) are closely related ligands within the TNF superfamily that play important roles in B lymphocyte biology. Both ligands share two receptors--transmembrane activator and calcium signal--modulating cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA)--that are predominantly expressed on B cells. In addition, BAFF specifically binds BAFF receptor, whereas the nature of a postulated APRIL-specific receptor remains elusive. We show that the TNF homology domain of APRIL binds BCMA and TACI, whereas a basic amino acid sequence (QKQKKQ) close to the NH2 terminus of the mature protein is required for binding to the APRIL-specific "receptor." This interactor was identified as negatively charged sulfated glycosaminoglycan side chains of proteoglycans. Although T cell lines bound little APRIL, the ectopic expression of glycosaminoglycan-rich syndecans or glypicans conferred on these cells a high binding capacity that was completely dependent on APRIL's basic sequence. Moreover, syndecan-1-positive plasma cells and proteoglycan-rich nonhematopoietic cells displayed high specific, heparin-sensitive binding to APRIL. Inhibition of BAFF and APRIL, but not BAFF alone, prevented the survival and/or the migration of newly formed plasma cells to the bone marrow. In addition, costimulation of B cell proliferation by APRIL was only effective upon APRIL oligomerization. Therefore, we propose a model whereby APRIL binding to the extracellular matrix or to proteoglycan-positive cells induces APRIL oligomerization, which is the prerequisite for the triggering of TACI- and/or BCMA-mediated activation, migration, or survival signals.

Cytokine profile associated with human chronic schistosomiasis mansoni

This study objective was to evaluate the cytokines associated with early events of hepatic fibrosis in schistosomiasis mansoni. Hepatic fibrosis was classified by ultrasonography in 94 patients. Immunological evaluation was performed by measurement of secreted cytokines (interleukin IL-5, IL-10, IL-13, interferon-gamma, tumor necrosis factor-alpha and transforming growth factors-beta) in peripherl blood mononuclear cells stimulated by Schistosoma mansoni antigens. Significantly, higher levels of IL-5, IL-10 and IL-13 were found in supernatants of SEA-stimulated PBMC from subjects with degree III hepatic fibrosis as compared to patients with degree I or II fibrosis, Significant increases in IL-5 and IL-13 levels were also observed in some of the subjects who remained untreated for one year following initial assessment and developed more serious fibrosis during this period. The data suggests a role for type 2 cytokines in early stages of hepatic fibrosis in human schistosomiasis mansoni.

Schistosomal hepatopathy

Gross anatomical features and a complex set of vascular changes characterize schistosomal hepatopathy as a peculiar form of chronic liver disease, clinically known as "hepatosplenic schistosomiasis". It differs from hepatic cirrhosis, although clinical and pathological aspects may sometimes induce confusion between these two conditions. Intrahepatic portal vein obstruction and compensatory arterial hypertrophy render the hepatic parenchyma vulnerable to ischemic insult. This may lead to focal necrosis, which may give place to focal post-necrotic scars. These events are of paramount importance for the clinico-pathological evolution of schistosomal hepatopathy. Although portal fibrosis due to schistosomiasis sometimes reveals numerous myofibroblasts, it does not mean that such fibrosis belongs to a peculiar type. Damage to the muscular walls of the portal vein may be followed by dissociation of smooth muscle cells and their transition toward myofibroblasts, which appear only as transient cells in schistosomal portal fibrosis. Studies made with plastic vascular casts, especially those with the murine model of "pipestem" fibrosis have helped to reveal the mechanisms involved in systematized portal fibrosis formation. However, the factors involved in the pathogenesis of hepatosplenic disease remain poorly understood. A process of chronic hepatitis is a common accompaniment of portal fibrosis in schistosomiasis. Most of the times it is caused by concomitant viral infection. However, no especial interaction seems to exist between schistosomal hepatopathy and viral hepatitis.