A new small molecule C5a receptor antagonist inhibits the reverse-passive arthus reaction and endotoxic shock in rats

C5a is implicated as a pathogenic factor in a wide range of immunoinflammatory diseases, including sepsis and immune complex disease, Agents that antagonize the effects of C5a could be useful in these diseases. We have developed some novel C5a antagonists and have determined the acute anti-inflammatory properties of a new small molecule C5a receptor antagonist against C5a- and LPS-induced neutrophil adhesion and cytokine expression, as well as against some hallmarks of the reverse Arthus reaction in rats. We found that a single i.v. dose (1 mg/kg) of this antagonist inhibited both C5a- and LPS-induced neutropenia and elevated levels of circulating TNF-alpha, as well as polymorphonuclear leukocyte migration, increased TNF-alpha levels and vascular leakage at the site of immune complex deposition. These results indicate potent anti-inflammatory activities of a new C5a receptor antagonist and provide more evidence for a key early role for C5a in sepsis and the reverse Arthus reaction. The results support a role for antagonists of C5a receptors in the therapeutic intervention of immunoinflammatory disease states such as sepsis and immune complex disease.

Increased circulating pro-inflammatory cytokines and imbalanced regulatory T-cell cytokines production in chronic idiopathic urticaria

The immunologic characterization of chronic idiopathic urticaria (CIU), mainly regarding cytokine profile needs more investigation. We examined circulating inflammatory cytokine levels, T-cell induced secretion, and cytokine mRNA expression in patients with CIU subjected to the intradermal autologous serum skin test (ASST). Increased levels of circulating pro-inflammatory cytokines, such as TNF-alpha, IL-1 beta, IL-12p70, and IL-6 have been observed in most of patients with CIU, together with an enhancement of IL-2 secretion following T-cell stimulation. Highlighting the inflammatory profile in CIU found in ASST positive, is the enhanced B-cell proliferative responsiveness and increased IL-17 secretion levels. ASST-positive patients also exhibited impaired IL-4 secretion associated with increased IL-10 production. Altered cytokine expression in patients with ASST-negative, was the down-modulation of spontaneous IL-10 mRNA expression levels in peripheral blood mononuclear cells. Our findings support the concept of immunologic dysregulation in CIU, revealing a systemic inflammatory profile associated with disturbed cytokine production by T cells, mainly related to IL-17 and IL-10 production. (c) 2008 Elsevier B.V. All rights reserved.

Interleukin-33 attenuates sepsis by enhancing neutrophil influx to the site of infection

Sepsis is a systemic inflammatory condition following bacterial infection with a high mortality rate and limited therapeutic options(1,2). Here we show that interleukin-33 (IL-33) reduces mortality in mice with experimental sepsis from cecal ligation and puncture (CLP). IL-33-treated mice developed increased neutrophil influx into the peritoneal cavity and more efficient bacterial clearance than untreated mice. IL-33 reduced the systemic but not the local proinflammatory response, and it did not induce a T helper type 1 (T(H)1) to T(H)2 shift. The chemokine receptor CXCR2 is crucial for recruitment of neutrophils from the circulation to the site of infection(3). Activation of Toll-like receptors (TLRs) in neutrophils downregulates CXCR2 expression and impairs neutrophil migration(4). We show here that IL-33 prevents the downregulation of CXCR2 and inhibition of chemotaxis induced by the activation of TLR4 in mouse and human neutrophils. Furthermore, we show that IL-33 reverses the TLR4-induced reduction of CXCR2 expression in neutrophils via the inhibition of expression of G protein coupled receptor kinase-2 (GRK2), a serine-threonine protein kinase that induces internalization of chemokine receptors(5,6). Finally, we find that individuals who did not recover from sepsis had significantly more soluble ST2 (sST2, the decoy receptor of IL-33) than those who did recover. Together, our results indicate a previously undescribed mechanism of action of IL-33 and suggest a therapeutic potential of IL-33 in sepsis.

Th1 cytokines in oral lichen planus

Background: Cell-mediated immune responses in oral lichen planus (OLP) may be regulated by cytokines and their receptors. Methods: In situ cytokine expression and in vitro cytokine secretion in OLP were determined by immunohistochemistry and ELISA. Resulults: The majority of subepithelial and intraepithelial mononuclear cells in OLP were CD8(+) . In some cases, intraepithelial CD8(+) cells were adjacent to degenerating keratinocytes. CD4(+) cells were observed mainly in the deep lamina propria with occasional CD4(+) cells close to basal keratinocytes. Mononuclear cells expressed IFN-gamma in the superficial lamina propria and TNF-alpha adjacent to basal keratinocytes. Basal keratinocytes expressed TNF-alpha as a continuous band. TNF R1 was expressed by mononuclear cells and basal and suprabasal keratinocytes. There was variable expression of TGF-beta1 in the subepithelial infiltrate while all intraepithelial mononuclear cells were TGF-beta1(-) . Keratinocytes in OLP stained weakly for TGF-beta1. Unstimulated OLP lesional T cells secreted IFN-gammain vitro . TNF-alpha stimulation down-regulated IFN-gamma secretion and up-regulated TNF-alpha secretion. IL-4, IL-10 and TGF-beta1 secretion were not detected. Conclusions: These data suggest the development of a T helper 1 immune response that may promote CD8(+) cytotoxic T-cell activity in OLP.

A protective effect of early pregnancy factor on experimental autoimmune encephalomyelitis induced in Lewis rats by inoculation with myelin basic protein

Experimental antoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease characterised by inflammation and demyelination of the central nervous system and is the best available animal model of multiple sclerosis (MS). Since previous studies have shown that EAE is less severe or is delayed in onset during pregnancy and that administration of the pregnancy hormone early pregnancy factor (EPF) down-regulates EAE, experiments in the present study were designed to explore further the role of EPF in EAE. By using the rosette inhibition test, the standard bioassay for EPF and, by semi-quantitative RT-PCR techniques, we have now shown that inflammatory cells from the spinal cord of rats with EAE can produce and secrete EPF, with production being greatest during recovery from disease. Administration of EPF to rats with EAE resulted in a significant increase in the expression of IL-4 and IL-10 mRNA and a significant decrease in IFN-gamma mRNA expression in spinal cord inflammatory cells. Encephalitogenic MBP-specific T cell lines were prepared from popliteal lymph nodes of rats with EAE. Proliferation assays using these cells demonstrated the ability of exogenous EPF to down-regulate the responses of T lymphocytes to MBP. (C) 2003 Elsevier B.V. All rights reserved.

The role of complex chromosome translocations in leukemia

Resumo A tumorigénese é um processo de transformação celular que se desenrola tipicamente em várias etapas. Os diferentes níveis de evolução tumoral resultam da acumulação sucessiva de mutações genéticas numa célula normal que lhe conferem uma vantagem selectiva no respectivo meio tecidular. As mutações podem manifestar-se sob a forma de alterações nucleotídicas pontuais ao nível da sequência de DNA, levando a uma desregulação da função proteíca ou à formação de proteínas não-funcionais, ou através de alterações cromossómicas numéricas ou estruturais. Na leucemia, por exemplo, os genes híbridos que resultam de translocações cromossómicas desempenham um importante papel no processo tumorigénico. Estes genes são transcritos sob a forma de um RNA mensageiro de fusão, o qual é traduzido numa proteína híbrida com função oncogénica. Frequentemente, os subtipos de doença leucémica estão associados com translocações cromossómicas que envolvem 2 pontos de quebra recorrentes e específicos. É disto exemplo a leucemia mielóide crónica, em que uma translocação recíproca entre os cromossomas 9 e 22 conduz à formação de um gene de fusão BCR-ABL1. Em diferentes subtipos de doença, existe também uma pequena proporção de casos que apresenta translocações cromossómicas complexas, que envolvem um ou mais pontos de quebra adicionais em outras localizações genómicas além das que estão implicadas na formação dos genes de fusão. Por vezes, os pontos de quebra estão também associados a delecções extensas de material genético que se pensa terem uma função importante na tumorigénese. No entanto, o papel destas regiões genómicas no desenvolvimento tumoral não tem sido um motivo recorrente de estudo. Neste contexto, o objectivo desta dissertação foi o de determinar o potencial papel tumorigénico de alterações génicas adicionais ocorridas nos pontos de quebra de translocações cromossómicas complexas. Para a prossecução do objectivo proposto, foram estudados 5 rearranjos cromossómicos distintos associados com diferentes tipos de doença hematológica maligna, nomeadamente a leucemia linfoblástica aguda de células B (2 casos), leucemia mielóide aguda, neoplasma mieloproliferativo e síndrome mielodisplásico/neoplasma ieloproliferativo, não classificável. O mapeamento dos pontos de quebra foi efectuado utilizando a hibridação fluorescente in situ e diferentes metodologias de biologia molecular, tendo como base a informação inicial da análise citogenética. Em casos seleccionados, o papel dos novos genes candidatos foi avaliado in vitro utilizando modelos de linhas celulares, nomeadamente no que respeita às funções de controlo da proliferação celular e de regulação transcricional. De entre os 5 casos estudados, quatro deles evidenciaram translocações complexas envolvendo 3 cromossomas, nomeadamente t(12;21;5)(p13;q22;q13), t(12;6;15)(p13;p24~25;q22), t(9;11;19)(p22;q23;p13) e t(X;20;16)(p11;q13;q23). No caso remanescente, foi observada uma translocação dicêntrica dic(9;12)(p11;p11) acompanhada de delecções extensas em ambos os pontos de quebra. Nos casos com t(12;21;5) e t(9;11;19) as translocações estavam associadas com a presença de genes de fusão recorrentes, nomeadamente TV6(12p13)-RUNX1(21q22) e TLL(11q23)-MLLT3(9p22), indicando que se tratavam de rearranjos complexos das translocações t(12;21) e t(9;11) associadas com a leucemia linfoblástica aguda de células B e a leucemia mielóide aguda, respectivamente. O papel dos pontos de quebra adicionais foi estudado em detalhe no caso com t(9;11;19). Através da metodologia de long distance inverse-polymerase chain reaction, foram identificados os pontos de quebra na sequência de DNA dos 3 cromossomas envolvidos na translocação. Além dos pontos de quebra nos genes MLL e MLLT3, foi observado que o local de quebra no cromossoma 19 interrompeu a sequência de um novo gene, designado CCDC94,conduzindo à sua haplo-insuficiência nas células com t(9;11;19). Através de ensaios de reverse transcription-polymerase chain reaction verificámos que o gene CCDC94 é expresso ubiquitariamente em tecidos humanos normais. A análise informática da sequência prevista da proteína CCDC94 indicou uma elevada identidade de aminoácidos com a proteína cwf16, envolvida na regulação do ciclo celular da levedura Schizosaccharomyces pombe. Através da clonagem do DNA complementar de CCDC94 em vectores de expressão, e após a transfecção destes em culturas de linhas celulares in vitro, observámos que este gene codifica uma proteína de localização exclusivamente nuclear. A expressão ectópica da proteína CCDC94 diminuiu a progressão do ciclo celular e a proliferação das células em cultura. Inversamente, a supressão do transcrito do gene CCDC94 através de interferência de RNA conduziu a um aumento significativo da proliferação celular, confirmando que CCDC94 regula negativamente a proliferação e a progressão do ciclo celular. Estes resultados mostram que os pontos de quebra adicionais, presentes em translocações cromossómicas complexas em leucemia, podem resultar na haplo-insuficiência de genes controladores dos mecanismos proliferativos, cooperando desta forma com a acção das proteínas de fusão para proporcionar ao clone leucémico uma proliferação celular descontrolada. Nos restantes 3 casos estudados não foram identificados genes de fusão. Ao invés, todos aqueles apresentaram delecções de extensão variável associadas com os pontos de quebra cromossómicos. No caso com t(12;6;15), identificámos uma delecção de 1.2 megabases de DNA na banda 12p13 que resultou na eliminação de 9 genes incluindo ETV6 e CDKN1B. O gene ETV6 codifica um factor de transcrição que é essencial para a formação das diferentes linhagens hematopoiéticas na medula óssea, enquanto CDKN1B é traduzido numa proteína responsável por bloquear a entrada das células na fase G1 do ciclo celular e,consequentemente, por travar a proliferação celular. Neste contexto, os resultados obtidos indicam que a perda simultânea de ETV6 e de CDKN1B, através de uma translocação cromossómica complexa, constituiu uma acção cooperativa na leucemogénese. A mesma noção pode aplicar-se ao caso com dic(9;12), no qual pelo menos 2 genes que codificam para factores de transcrição importantes na linhagem hematopoiética, PAX5 no cromossoma 9 e ETV6 no cromossoma 12, estavam deleccionados como resultado do rearranjo cromossómico. Dado que o factor de transcrição PAX5 regula negativamente a expressão do gene FLT3, que desempenha uma função pró-proliferativa, é expectável que a haplo-insuficiência de PAX5 no caso com dic(9;12) terá tido como consequência uma elevação dos níveis de expressão de FLT3, contribuindo deste modo para uma proliferação celular aumentada. A t(X;20;16) foi identificada num doente com trombocitémia essencial (TE), uma doença que está intimamente relacionada com alterações de vias intracelulares reguladas por citocinas. Neste caso, através da utilização de um array genómico, identificámos a presença de pequenas delecções associadas com os pontos de quebra nos cromossomas 16 e 20. No cromossoma 16 apenas um gene, MAF, estava deleccionado, enquanto no cromossoma 20 a delecção tinha abrangido 3 genes. Dos genes deleccionados, dois deles, NFATC2 (20q13) e MAF (16q23), codificam proteínas que operam como reguladores transcricionais de citocinas hematopoiéticas. Dado que NFATC2 se localiza numa região que constitui um alvo frequente de delecções em neoplasmas ieloproliferativos, incluindo a trombocitémia essencial,efectuámos um estudo detalhado do papel deste gene na proliferação megacariocítica e na regulação da expressão de uma citocina hematopoiética (GM-CSF), implicada na maturação das diferentes linhagens mielóides. Utilizando um modelo de linha celular de trombocitémia essencial, verificámos que a supressão do transcrito do gene NFATC2 in vitro, por interferência de RNA, estava associada com um aumento da proliferação celular. Em concordância, o bloqueio da activação da proteína NFATC2 através de um inibidor específico da sua interacção com a calcineurina, conduziu a um aumento da proliferação celular in vitro. Utilizando a PCR quantitativa em tempo real, detectou-se um aumento da produção do RNA de GM-CSF em ambos os ensaios celulares, indicando que o factor de transcrição NFATC2 pode regular negativamente a expressão de GM-CSF em células de trombocitémia essencial. No geral, estes resultados mostram que a redução dos níveis fisiológicos do transcrito NFATC2, ou a redução da respectiva actividade proteica, estão relacionados com a proliferação de megacariocitos através do aumento da produção de GM-CSF. De acordo com estes resultados, verificámos que as células dos doentes com TE apresentam níveis mais baixos do transcrito NFATC2 do que a população normal. Dado que o factor de transcrição MAF desempenha igualmente um papel como regular transcricional de citocinas, é plausível que a haplo-insuficiência dos genes NFATC2 e MAF, resultante do rearranjo cromossómico complexo t(X;20;16), teve um efeito cooperativo importante na patogénese da trombocitémia essencial através da alteração do padrão normal de expressão das citocinas hematopoiéticas. Em síntese, efectuámos nesta dissertação um estudo citogenético de 4 translocações cromossómicas complexas incluindo t(12;21;5), t(12;6;15), t(9;11;19) e t(X;20;16), e de uma translocação dicêntrica dic(9;12), associadas com diferentes neoplasmas hematológicos. Em casos seleccionados efectuámos também um estudo molecular detalhado das regiões dos pontos de quebra. Esta análise permitiu-nos identificar 2 genes, CCDC94 no cromossoma 19 e NFATC2 no cromossoma 20, cuja haplo-insuficiência pode promover o aumento da proliferação celular das células leucémicas. A partir destes estudos podem ser retiradas 2 noções principais: (i) Os pontos de quebra adicionais, que ocorrem em translocações complexas associadas com a formação de genes de fusão, podem ter como consequência a desregulação de genes controladores da proliferação celular (e.g., CCDC94); (ii) As translocações complexas caracterizadas pela ausência de genes de fusão recorrentes poderão estar preferencialmente associadas com a presença de delecções, envolvendo um ou mais genes, nos pontos de quebra; nestas situações, serão necessários pelo menos 2 genes com funções celulares semelhantes (e.g., NFATC2 e MAF) ou complementares (e.g., ETV6 e CDKN1B) para, quando deleccionados, promoverem de forma cooperativa a leucemogénese. Nestes termos, o modelo de alterações genéticas sequenciais que caracteriza o desenvolvimento do cancro pode ser substituído por um modelo em que vários genes-alvo são simultaneamente desregulados pela formação de uma translocação cromossómica complexa, evitando deste modo a necessidade de ocorrência de alterações genéticas subsequentes.----------------------ABSTRACT: Tumourigenesis is a multistep process which results from the accumulation of successive genetic mutations in a normal cell. In leukemia for instance, recurrent translocations play a part in this process by generating fusion genes which lead to the production of hybrid proteins with an oncogenic role. However, a minor subset of chromosomal translocations referred to as complex or variant involves extra breakpoints at variable genome locations in addition to those implicated in the formation of fusion genes. We aimed to describe in this work the role, if any, of genes located at extra breakpoint locations or which are affected by breakpoint-adjacent deletions through the study of 5 leukemia patients.Two of the patients presented with TV6(12p13)-RUNX1(21q22) and MLL(11q23)- MLLT3(9p22) fusion genes as a result of a t(12;21;5) and a t(9;11;19), respectively. Detailed molecular characterization of the extra breakpoint at chromosome 19 in the latter case revealed that a novel ubiquitously expressed gene, CCDC94, with a potential role in cell cycle regulation, was disrupted by the breakpoint. We demonstrated using in vitro cellular assays that this gene codifies for a nuclear protein which negatively regulates cell cycle progression. These data shows that extra breakpoint locations of complex translocations may result in haplo-insufficiency of critical proliferation genes, thereby cooperating with the generation of hybrid proteins to provide unrestrained cell proliferation. In the other 3 patients there were reakpoint-associated deletions which precluded the formation of putative fusion genes. In a case with a t(12;6;15) we characterized a deletion at 12p13 which eliminated ETV6 and 8 other genes including CDKN1B. These findings indicate that concomitant loss of ETV6 and CDKN1B, which encodes a cyclin-dependent kinase inhibitor responsible for blocking entry of cells into the G1 phase of the cell cycle, acted cooperatively to promote leukemogenic proliferation. The same notion applied to a case with a dic(9;12) in which 2 genes encoding hematopoietic transcription factors - ETV6 and PAX5 (9p13)- were deleted as a result of breakpoint-adjacent deletions. Similarly, we found that 2 transcription factor genes involved in the regulation of cytokine expression, NFATC2 (20q13) and MAF (16q23), were involved in deletions contiguous to the breakpoints in a patient with a t(X;20;16). In vitro suppression of NFATC2 mRNA or inhibiton of NFATC2 protein activity enhanced cell proliferation as a result of an increase in the production of a myeloid-lineage stimulating hematopoietic cytokine, GM-CSF. These results suggest that haplo-insufficiency of NFATC2 and MAF genes had a cooperative effect in inducing cell proliferation as a result of a disregulation of cytokine production. Two main conclusions may be drawn from our studies: (i) In complex translocations associated with the production of fusion genes, additional breakpoints may cooperate in tumourigenesis by targeting genes that control cell proliferation; (ii) In complex translocations associated with small breakpoint-adjacent deletions, at least 2 genes with similar or complementary functions need to be deregulated to promote tumourigenesis.

Understanding how dentritic cell glycans affect antitumor immune responses

RESUMO: As células dendríticas (DCs) têm a capacidade única de induzir respostas imunitárias contra as células tumorais, fagocitando antigénios tumorais e apresentando-os às células T, provocando respostas imunitárias específicas que conduzem à eliminação de células de tumorais. Por induzirem memória imunológica de longa duração, as DCs são uma estratégia atrativa para o tratamento e/ou prevenção do cancro. No entanto, os resultados terapêuticos obtidos em ensaios clínicos com DCs são escassos e pouco eficientes. O nosso grupo demonstrou que ácidos siálicos que contêm glicanos desempenham um papel funcional importante em DCs geradas ex vivo. Com o objetivo de estabelecer um modelo in vitro para avaliar a resposta anti-tumoral específica realizou-se um tratamento enzimático a DCs derivadas de monócitos (moDCs) com sialidase, enzima que cliva ácidos siálicos na superfície celular. O perfil de maturação de moDCs foi caracterizado por citometria de fluxo e expressão de citocinas. Os resultados mostram que a sialidase pode regular positivamente a expressão de moléculas co-estimuladoras na superfície de moDCs estimuladas com agonistas de Toll like receptors (TLRs). Para percebermos se o tratamento com sialidase afeta a sinalização dos TLRs foram usadas células HEK transfectadas de forma estável com TLRs 2, 4 and 7/8. Os dados mostraram que a desialilação não afeta a sinalização através estes recetores. Para investigar o impacto funcional da sialidase na capacidade de moDCs em apresentar um antigénio e ativar células T, moDCs foram tratadas, ou não, com sialidase e cultivadas com clones de células T CD8+ específicas para os péptidos derivados do antigénio tumoral gp100. Os resultados mostram que DCs HLA*02:01+ desialiladas exibem maior cross-presentation do péptido gp100280-288 às células T CD8+ específicas. Além disso o tratamento com sialidase também aumenta a capacidade de DCs de induzir a proliferação de células T CD4+. Em conjunto, os resultados indicam que moDCs com menos ácidos siálicos na superfície, têm melhor potencial imuno-estimulador, com maior capacidade de induzir respostas imunes anti-tumorais.--------------------- ABSTRACT: Dendritic cells (DCs) have a unique capacity to induce immune responses against tumor cells. They can phagocyte tumor antigens, maturate and present them to T cells, triggering antigen-specific immune responses that may lead to the elimination of tumor cells. Since they induce long-lasting immunological memory, DCs become an attractive strategy as cellular targets for vaccines in the treatment and/or prevention of cancer. However, the therapeutic results obtained in clinical trials with DCs are scarce and only few patients effectively respond to the DC vaccines. Our group has shown that sialic acid containing glycans play an important functional role in ex vivo generated DC. Here we aimed to establish an in vitro model to assess specific antitumor responses. To achieve this, an enzymatic treatment of monocyte-derived DCs (moDCs) was performed using sialidase to cleave surface sialic acids. The maturation profile of the moDCs was characterized by flow cytometry and cytokine expression. The results show that sialidase treatment can upregulate co-stimulatory molecules on surface of moDCs stimulated with Toll like receptor (TLR) agonists. To understand whether sialidase treatment affected the TLR signaling, we have used HEK cells stably transfected with TLRs 2, 4 and 7/8. The data showed that desialylation of moDCs does not affect the signaling via these receptors. To investigate the functional impact of sialidase treatment in the capacity of moDCs to present antigen and to activate antigen specific T cells, sialidase treated and untreated moDCs were co-cultured with CD8+ T cell clones specific for peptides derived from the gp100 tumor antigen. Our results show that desialylated HLA02:01+ DCs are superior in cross-presentation of the peptide to gp100280–288 specific CD8+ T cells. In addition, sialidase treatment also increased the DC capacity to induce CD4+ T cells proliferation. Together, these data indicate that moDCs with altered cell surface sialic acids, through a sialidase treatment, have a better immunostimulatory potential which could improve anti-tumor immune responses.

Avaliação da expressão de mediadores imunitários em amostras de cetáceos capturados acidentalmente em Portugal Continental

Dissertação de Mestrado Integrado em Medicina Veterinária

Paper de la PAP en la resposta inflamatoria durant la pancreatitis aguda

Estudi elaborat a partir d’una estada al Institut national de la santé et de la recherche médicale, França, entre setembre i octubre del 2006. La PAP va ser identificada inicialment com una proteïna de secreció, que apareixia al suc pancreàtic després de la inducció d’una pancreatitis aguda experimental. Per la PAP s’ha suggerit diferents funcions, algunes no relacionades en aparença, però les més interessants en el camp de la pancreatitis són les activitats antiapoptótiques, mitogéniques i, especialment, antiinflamatóries. Per aprofundir en aquests aspectes de la PAP, s’ha emprat un model de ratolí PAP-/- per observar els efectes de la deleció del gen de la PAP durant la pancreatitis aguda.Per induir la pancreatitis es va fer servir el model de administració de ceruleina a dosi supra-màximes. En aquestes condicions es va observar que en els animals PAP-/-, la severitat del procés era menor. Els marcadors de necrosi pancreàtica, lipasa i amilasa, van presentar nivells menors en els animals PAP-/- que en els corresponents wild type. Per contra, el nombre de PMN infiltrats i la producció de citoquines pro-inflamatories va ser major en el pàncreas dels animals PAP-/-. La intensitat de la resposta inflamatòria observada suggereix que en condicions fisiològiques, el paper anti-inflamatori de la PAP es prou rellevant. Això ja s’havia suggerit en estudis in vitro, en els que es va demostrar que l’activitat antiinflamatòria de la PAP depenia de la via de transducció de senyal Jak/STAT/SOCS3. Aquesta hipòtesi s’ha comprovat in vivo, monitoritzant el nivell d’activació de STAT3 en els pàncreas dels animals després de la inducció de la pancreatitis.Tot plegat confirma que les funcions antiinflamatòries descrites in vitro per la PAP també es poden observar in vivo, de manera que la PAP sembla ser un agent important en la resposta de les cèl•lules pancreàtiques durant la pancreatitis aguda.

Waddlia chondrophila Infects and Multiplies in Ovine Trophoblast Cells Stimulating an Inflammatory Immune Response.

BACKGROUND: Waddlia chondrophila (W. chondrophila) is an emerging abortifacient organism which has been identified in the placentae of humans and cattle. The organism is a member of the order Chlamydiales, and shares many similarities at the genome level and in growth studies with other well-characterised zoonotic chlamydial abortifacients, such as Chlamydia abortus (C. abortus). This study investigates the growth of the organism and its effects upon pro-inflammatory cytokine expression in a ruminant placental cell line which we have previously utilised in a model of C. abortus pathogenicity. METHODOLOGY/PRINCIPAL FINDINGS: Using qPCR, fluorescent immunocytochemistry and electron microscopy, we characterised the infection and growth of W. chondrophila within the ovine trophoblast AH-1 cell line. Inclusions were visible from 6 h post-infection (p.i.) and exponential growth of the organism could be observed over a 60 h time-course, with significant levels of host cell lysis being observed only after 36 h p.i. Expression of CXCL8, TNF-α, IL-1α and IL-1β were determined 24 h p.i. A statistically significant response in the expression of CXCL8, TNF-α and IL-1β could be observed following active infection with W. chondrophila. However a significant increase in IL-1β expression was also observed following the exposure of cells to UV-killed organisms, indicating the stimulation of multiple innate recognition pathways. CONCLUSIONS/SIGNIFICANCE: W. chondrophila infects and grows in the ruminant trophoblast AH-1 cell line exhibiting a complete chlamydial replicative cycle. Infection of the trophoblasts resulted in the expression of pro-inflammatory cytokines in a dose-dependent manner similar to that observed with C. abortus in previous studies, suggesting similarities in the pathogenesis of infection between the two organisms.

Histone deacetylase inhibitors impair innate immune responses

SUMMARYThe innate immune system plays a central role in host defenses against invading pathogens. Innate immune cells sense the presence of pathogens through pattern recognition receptors that trigger intracellular signaling, leading to the production of pro-inflammatory mediators like cytokines, which shape innate and adaptive immune responses. Both by excess and by default inflammation may be detrimental to the host. Indeed, severe sepsis and septic shock are lethal complications of infections characterized by a dysregulated inflammatory response.In recent years, members of the superfamily of histone deacetylases have been the focus of great interest. In mammals, histone deacetylases are broadly classified into two main subfamilies comprising histone deacetylases 1-11 (HDAC1-11) and sirtuins 1-7 (SIRT1-7). These enzymes influence gene expression by deacetylating histones and numerous non-histone proteins. Histone deacetylases have been involved in the development of oncologic, metabolic, cardiovascular, neurodegenerative and autoimmune diseases. Pharmacological modulators of histone deacetylase activity, principally inhibitors, have been developed for the treatment of cancer and metabolic diseases. When we initiated this project, several studies suggested that inhibitors of HDAC 1-11 have anti-inflammatory activity. Yet, their influence on innate immune responses was largely uncharacterized. The present study was initiated to fill in this gap.In the first part of this work, we report the first comprehensive study of the effects of HDAC 1- 11 inhibitors on innate immune responses in vitro and in vivo. Strikingly, expression studies revealed that HDAC1-11 inhibitors act essentially as negative regulators of basal and microbial product- induced expression of critical immune receptors and antimicrobial products by mouse and human innate immune cells like macrophages and dendritic cells. Furthermore, we describe a new molecular mechanism whereby HDAC1-11 inhibitors repress pro-inflammatory cytokine expression through the induction of the expression and the activity of the transcriptional repressor Μί-2β. HDAC1-11 inhibitors also impair the potential of macrophages to engulf and kill bacteria. Finally, mice treated with an HDAC inhibitor are more susceptible to non-severe bacterial and fungal infection, but are protected against toxic and septic shock. Altogether these data support the concept that HDAC 1-11 inhibitors have potent anti-inflammatory and immunomodulatory activities in vitro and in vivo.Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that plays a central role in innate immune responses, cell proliferation and oncogenesis. In the second part of this manuscript, we demonstrate that HDAC1-11 inhibitors inhibit MIF expression in vitro and in vivo and describe a novel molecular mechanism accounting for these effects. We propose that inhibition of MIF expression by HDAC 1-11 inhibitors may contribute to the antitumorigenic and anti-inflammatory effects of these drugs.NAD+ is an essential cofactor of sirtuins activity and one of the major sources of energy within the cells. Therefore, sirtuins link deacetylation to NAD+ metabolism and energy status. In the last part of this thesis, we report preliminary results indicating that a pharmacological inhibitor of SIRT1-2 drastically decreases pro-inflammatory cytokine production (RNA and protein) and interferes with MAP kinase intracellular signal transduction pathway in macrophages. Moreover, administration of the SIRT1-2 inhibitor protects mice from lethal endotoxic shock and septic shock.Overall, our studies demonstrate that inhibitors of HDAC1-11 and sirtuins are powerful anti-inflammatory molecules. Given their profound negative impact on the host antimicrobial defence response, these inhibitors might increase the susceptibility to opportunistic infections, especially in immunocompromised cancer patients. Yet, these inhibitors might be useful to control the inflammatory response in severely ill septic patients or in patients suffering from chronic inflammatory diseases.

Low dose endoluminal photodynamic therapy improves murine T cell-mediated colitis.

BACKGROUND AND STUDY AIMS: Low dose photodynamic therapy (LDPDT) may modify the mucosal immune response and may thus provide a therapy for Crohn's disease. We evaluated the efficacy and safety of this technique in a murine T cell-mediated colitis model. METHODS: The safety of LDPDT was first tested in BALB/c mice. Naïve T cells were used to induce colitis in mice with severe combined immunodeficiency, which were followed up endoscopically, and a murine endoscopic index of colitis (MEIC) was developed. The efficacy of LDPDT (10 J/cm (2); delta-aminolevulinic acid, 15 mg/kg bodyweight) was then tested on mice with moderate colitis, while a disease control group received no treatment. The MEIC, weight, length, and histology of the colon, cytokine expression indices, number of mucosal CD4 (+) T cells, percentage of apoptotic CD4 (+) T cells, body weight, and systemic side effects were evaluated. RESULTS: LDPDT improved the MEIC ( P = 0.011) and the histological score ( P = 0.025), diminished the expression indices of the proinflammatory cytokines, interleukin-6 ( P = 0.042), interleukin-17 ( P = 0.029), and interferon-gamma ( P = 0.014), decreased the number of mucosal CD4 (+) T cells, and increased the percentage of apoptotic CD4 (+) T cells compared with the disease control group. No local or systemic side effects occurred. CONCLUSION: LDPDT improves murine T cell-mediated colitis, decreases the proinflammatory cytokines interleukin-6, interleukin-17, and interferon-gamma, and decreases the number of CD4 (+) T cells. No adverse events were observed. Therefore, this technique is now being evaluated in patients with inflammatory bowel disease.

Activin a inhibits antigen-induced allergy in murine epicutaneous sensitization.

Activin A, a member of the TGFβ superfamily, is involved in physiological processes such as cell differentiation, tissue homeostasis, wound healing, reproduction, and in pathological conditions, such as fibrosis, cancer, and asthma. Activin enhances mast cell maturation, as well as regulatory T-cell and Langerhans cell differentiation. In this study we investigated the potential role of activin in epicutaneous sensitization with ovalbumin (OVA), notably with respect to its effect on known Th2-polarization. For this purpose, transgenic mice overexpressing activin in keratinocytes and their wild-type (WT) controls were sensitized epicutaneously with OVA. Skin biopsies were analyzed with regard to histopathological features and mRNA expression of pro-inflammatory and Th1/Th2 cytokines, and Ig levels were measured in the serum. Unexpectedly, activin overexpressing animals were protected from Th2-cytokine expression and induction of OVA-specific IgE levels compared to WT animals. On the other hand, transgenic mice were more susceptible to inflammation compared to WT littermates after tape-stripping and saline (vehicle) or OVA application, as shown by increased pro-inflammatory cytokine mRNA levels and neutrophil accumulation at the site of the treatment. We conclude that activin protects from antigen-induced cutaneous Th2-polarization through modulation of the immune response. These findings highlight the role of activin in cutaneous sensitization, allergy, and in skin homeostasis.

Influence of age on retinochoroidal healing processes after argon photocoagulation in C57bl/6j mice.

PURPOSE: To analyze the influence of age on retinochoroidal wound healing processes and on glial growth factor and cytokine mRNA expression profiles observed after argon laser photocoagulation. METHODS: A cellular and morphometric study was performed that used 44 C57Bl/6J mice: 4-week-old mice (group I, n=8), 6-week-old mice (group II, n=8), 10-12-week-old mice (group III, n=14), and 1-year-old mice (group IV, n=14). All mice in these groups underwent a standard argon laser photocoagulation (50 microm, 400 mW, 0.05 s). Two separated lesions were created in each retina using a slit lamp delivery system. At 1, 3, 7, 14, 60 days, and 4 months after photocoagulation, mice from each of the four groups were sacrificed by carbon dioxide inhalation. Groups III and IV were also studied at 6, 7, and 8 months after photocoagulation. At each time point the enucleated eyes were either mounted in Tissue Tek (OCT), snap frozen and processed for immunohistochemistry or either flat mounted (left eyes of groups III and IV). To determine, by RT-PCR, the time course of glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and monocyte chemotactic protein-1 (MCP-1) gene expression, we delivered ten laser burns (50 microm, 400 mW, 0.05 s) to each retina in 10-12-week-old mice (group III', n=10) and 1-year-old mice (group IV', n=10). Animals from Groups III' and IV' had the same age than those from Groups III and IV, but they received ten laser impacts in each eye and served for the molecular analysis. Mice from Groups III and IV received only two laser impacts per eye and served for the cellular and morphologic study. Retinal and choroidal tissues from these treated mice were collected at 16 h, and 1, 2, 3, and 7 days after photocoagulation. Two mice of each group did not receive photocoagulation and were used as controls. RESULTS: In the cellular and morphologic study, the resultant retinal pigment epithelium interruption expanse was significantly different between the four groups. It was more concise and smaller in the oldest group IV (112.1 microm+/-11.4 versus 219.1 microm+/-12.2 in group III) p<0.0001 between groups III and IV. By contrast, while choroidal neovascularization (CNV) was mild and not readily identifiable in group I, at all time points studied, CNV was more prominent in the (1-year-old mice) Group IV than in the other groups. For instance, up to 14 days after photocoagulation, CNV reaction was statistically larger in group IV than in group III ((p=0.0049 between groups III and IV on slide sections and p<0.0001 between the same groups on flat mounts). Moreover, four months after photocoagulation, the CNV area (on slide sections) was 1,282 microm(2)+/-90 for group III and 2,999 microm(2)+/-115 for group IV (p<0.0001 between groups III and IV). Accordingly, GFAP, VEGF, and MCP-1 mRNA expression profiles, determined by RT-PCR at 16 h, 1, 2, 3, and 7 days postphotocoagulation, were modified with aging. In 1-year-old mice (group IV), GFAP mRNA expression was already significantly higher than in the younger (10-12 week) group III before photocoagulation. After laser burns, GFAP mRNA expression peaked at 16-24 h and on day 7, decreasing thereafter. VEGF mRNA expression was markedly increased after photocoagulation in old mice eyes, reaching 2.7 times its basal level at day 3, while it was only slightly increased in young mice (1.3 times its level in untreated young mice 3 days postphotocoagulation). At all time points after photocoagulation, MCP-1 mRNA expression was elevated in old mice, reaching high levels of expression at 16 h and day 3 respectively. CONCLUSIONS: Our results were based on the study of four different age groups and included not only data from morphological observations but also from a molecular analysis of the various alterations of cytokine signaling and expression. One-year-old mice demonstrated more extensive CNV formation and a slower pace of regression after laser photocoagulation than younger mice. These were accompanied by differences in growth factors and cytokine expression profiles indicate that aging is a factor that aggravates CNV. The above results may provide some insight into possible therapeutic strategies in the future.

Toll-Interacting Protein Deficiency Leads to Increased Susceptibility to Acute and Chronic Colitis.

BACKGROUND: Sensing of bacterial products via Toll-like receptors is critical to maintain gut immune homeostasis. The Toll-Interacting Protein (Tollip) inhibits downstream signaling through the IL-1 receptor, TLR-2 and TLR-4. Here,we aimed to address the role of Tollip in acute and chronic inflammatory responses in the gut. MATERIAL AND METHODS: WT or Tollip-deficient mice were exposed to dextran sulfate sodium (DSS) 1.5% in the drinking water during 7 days. To generate bone-marrow chimeras, WT or Tollip deficient mice were 900-rads irradiated, transplanted with WT or Tollip deficient bone-marrow cells and challenged with DSS 2-3 months after transplantation. IL-10 deficient mice were bred with Tollip deficient mice and colitis was compared at various time points. RESULTS: Upon DSS exposure, Tollip-deficient mice had increased body weight loss and increased pro-inflammatory cytokine expression compared to WT controls. Challenge of bone-marrow chimeras showed that colitis susceptibility was also increased when Tollip deficiency was restricted to non-hematopoietic cells. DSS-exposure lead to a disorganized distribution of zona-occludens-1, a tight junction marker and increased number of apoptotic, cleaved caspase 3 positive, epithelial cells in Tollip-deficient compared to WT mice. Chronic colitis was also affected by Tollip deficiency as Tollip/IL-10 deficient mice had more severe histological stigmata of colitis and higher IL-17 expression than IL-10 deficient controls. CONCLUSION: Tollip in non-hematopoietic cells is critical for adequate response to a chemical-induced stress in the gut and to hamper chronic bacteria-driven colitis. Modulation of epithelial cell integrity via Tollip likely contributes to the observed defects.