RESUMO
Inflammatory bowel disease (Crohn's disease (CD) and ulcerative colitis (UC)) is a multifactorial disease resulting from immune dysregulation in the gut. The underlying colitis is characterized by high levels of inflammatory cytokines, including TNFα. Biological intervention for IBD patients using anti-TNFα antibodies is often an effective therapeutic solution. However, TNFα neutralization fails to induce remission in a subgroup of IBD patients, primarily in UC patients. There is a dearth of suitable animal models representing TNFα non-responders. Here we have combined one of the best UC models currently available, namely Winnie and the TNFαKO mouse to generate a TNFα-deficient Winnie to study early onset colitis. The induced TNFα deficiency with underlying colitis does not influence general health (viability and body weight) or clinical parameters (colon weight, colon length and histological colitis) when compared with the Winnie genotype alone. The molecular characterization resulted in identification of Il1ß as the major elevated cytokine during early phases of colitis. Further, in vitro functional assay using bone marrow-derived dendritic cells confirmed IL-1ß as the major cytokine released in the absence of TNFα. This study has generated a successful model of colitis that remains TNFα non-responsive and has demonstrated that IL-1ß expression is a major pathway for the progression of colitis in this system. These data also suggest that IL-1ß can be a potential target for clinical intervention of UC patients who fail to respond to TNFα neutralization.
Assuntos
Colite/metabolismo , Interleucina-1beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Colite/genética , Colite/patologia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Feminino , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Necrose Tumoral alfa/deficiênciaRESUMO
Fifty-seven genital tracts of regularly slaughtered culled Piedmontese cows, aged 7.4 ± 4.3 years (mean ± SD), range: 2.6-15.6 years, were grossly and microscopically examined. DNA extracted from oviducts was subjected to PCR to evaluate the presence of Chlamydia spp. The 15 PCR-positive oviducts were subjected to Sanger sequencing and showed the presence of Chamydia abortus, with an identity range between 99 and 100%. Nine of the PCR-positive samples belonged to the 24 animals with a normal macroscopic appearance of the whole genital tract (percentage of positive oviducts in normal genital tracts 9/24 = 37.5%), while six belonged to the 33 genital tracts with lesions in one or more organs (percentage of positive oviducts in pathological genital tracts 6/33 = 18.1%); of these, a single animal had salpingitis. The detection of C. abortus in bovine oviducts is of particular interest because it has never been previously investigated or reported.
Assuntos
Doenças dos Bovinos/microbiologia , Infecções por Chlamydia/veterinária , Chlamydia/isolamento & purificação , Oviductos/microbiologia , Animais , Bovinos , Chlamydia/classificação , Infecções por Chlamydia/diagnóstico , FemininoRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that occurs in two clinically indistinguishable forms: sporadic (SALS) and familial (FALS), the latter linked to several gene mutations, mostly inheritable in a dominant manner. Nearly 20% of FALS forms are linked to mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Research on ALS relies on transgenic models and particularly on mice carrying a glycine-to-alanine conversion at the 93rd codon (G93A) of the hSOD1 gene. Although G93A transgenic mice have been widely employed in clinical trials and basic research, doubts have been recently raised from numerous reliable sources about their suitability to faithfully reproduce human disease. Besides, the scientific community has already foreseen swine as an attractive and alternative model to nonhuman primates for modeling human diseases due to closer anatomical, physiological and biochemical features of swine rather than rodents to humans. On this basis, we have produced the first swine ALS model by in vitro transfection of cultured somatic cells combined with somatic cell nuclear transfer (SCNT). To achieve this goal we developed a SOD1(G93A) (superoxide dismutase 1 mutated in Gly93-Ala) vector, capable of promoting a high and stable transgene expression in primary porcine adult male fibroblasts (PAF). After transfection, clonal selection and transgene expression level assessment, selected SOD1(G93A) PAF colonies were used as nuclei donors in SCNT procedures. SOD1(G93A) embryos were transferred in recipient sows, and pregnancies developed to term. A total of 5 piglets survived artificial hand raising and weaning and developed normally, reaching adulthood. Preliminary analysis revealed transgene integration and hSOD1(G93A) expression in swine tissues and 360° phenotypical characterization is ongoing. We believe that our SOD1(G93A) swine would provide an essential bridge between the fundamental work done in rodent models and the reality of treating ALS.
Assuntos
Esclerose Lateral Amiotrófica/genética , Animais Geneticamente Modificados , Modelos Animais de Doenças , Superóxido Dismutase/genética , Suínos/genética , Animais , Humanos , Masculino , Superóxido Dismutase-1RESUMO
Tumor-Associated Macrophages (TAMs) are abundantly present in the stroma of solid tumors and modulate several important biological processes, such as neoangiogenesis, cancer cell proliferation and invasion, and suppression of adaptive immune responses. Myeloid C-type lectin receptors (CLRs) constitute a large family of transmembrane carbohydrate-binding receptors that recognize pathogens as well as endogenous glycoproteins. Several lines of evidence demonstrate that some CLRs can inhibit the immune response. In this study we investigated TAM-associated molecules potentially involved in their immune suppressive activity. We found that TAMs isolated from human ovarian carcinoma samples predominantly express the CLRs Dectin-1, MDL-1, MGL, DCIR, and most abundantly the Mannose Receptor (MR). Components of carcinomatous ascites and purified tumoral mucins (CA125 and TAG-72) bound the MR and induced its internalization. MR engagement by tumoral mucins and by an agonist anti-MR antibody modulated cytokine production by TAM toward an immune-suppressive profile: increase of IL-10, absence of IL-12, and decrease of the Th1-attracting chemokine CCL3. This study highlights that tumoral mucin-mediated ligation of the MR on infiltrating TAM may contribute to their immune suppressive phenotype.
Assuntos
Lectinas Tipo C/imunologia , Macrófagos/imunologia , Lectinas de Ligação a Manose/imunologia , Mucinas/imunologia , Neoplasias Ovarianas/imunologia , Receptores de Superfície Celular/imunologia , Imunidade Adaptativa/efeitos dos fármacos , Imunidade Adaptativa/imunologia , Anticorpos/imunologia , Anticorpos/farmacologia , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Antígeno Ca-125/genética , Antígeno Ca-125/imunologia , Antígeno Ca-125/metabolismo , Quimiocina CCL3/imunologia , Quimiocina CCL3/metabolismo , Endocitose/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Humanos , Imuno-Histoquímica , Interferon gama/farmacologia , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-10/farmacologia , Interleucina-12/imunologia , Interleucina-12/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Receptor de Manose , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Mucinas/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
Intestinal dendritic cells (DCs) have been shown to display specialized functions, including the ability to promote gut tropism to lymphocytes, to polarize noninflammatory responses, and to drive the differentiation of adaptive Foxp3(+) regulatory T (T(reg)) cells. However, very little is known about what drives the mucosal phenotype of DCs. Here, we present evidence that the local microenvironment, and in particular intestinal epithelial cells (ECs), drive the differentiation of T(reg)-cell-promoting DCs, which counteracts Th1 and Th17 development. EC-derived transforming growth factor-beta (TGF-beta) and retinoic acid (RA), but not thymic stromal lymphopoietin (TSLP), were found to be required for DC conversion. After EC contact, DCs upregulated CD103 and acquired a tolerogenic phenotype. EC-conditioned DCs were capable of inducing de novo T(reg) cells with gut-homing properties that when adoptively transferred, protected mice from experimental colitis. Thus, we have uncovered an essential mechanism in which EC control of DC function is required for tolerance induction.
Assuntos
Colite/imunologia , Células Dendríticas/imunologia , Mucosa Intestinal/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Comunicação Celular/imunologia , Diferenciação Celular/imunologia , Colite/patologia , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Feminino , Tolerância Imunológica/imunologia , Interleucina-17/imunologia , Interleucina-17/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismo , Tretinoína/imunologia , Tretinoína/metabolismo , Linfopoietina do Estroma do TimoRESUMO
Fractalkine (FKN, CX3CL1) is a membrane-bound CX3C chemokine induced by primary proinflammatory signals in vascular endothelial cells (ECs). Here we examined the role of FKN in polarized Th1 or Th2 responses. Proinflammatory signals, including LPS, IL-1, TNF, and CD40 ligand, induced FKN, as did IFN-gamma, which had synergistic activity with TNF. IL-4 and IL-13 did not stimulate the expression of FKN and markedly reduced induction by TNF and IFN-gamma. TNF alone or combined with IFN-gamma also induced release of soluble FKN, which was inhibited by IL-4 and IL-13. In light of this differential regulation of FKN by the master cytokines that control polarized responses, we analyzed the interaction of FKN with natural killer (NK) cells and polarized T-cell populations. NK cells expressed high levels of the FKN receptor CX3CR1 and responded to FKN. CX3CR1 was preferentially expressed in Th1 compared with Th2 cells. Th1 but not Th2 cells responded to FKN. By immunohistochemistry, FKN was expressed on ECs in psoriasis, a Th1-dominated skin disorder, but not in Th2-driven atopic dermatitis. Similarly, ECs in Mycobacterium tuberculosis granulomatous lymphadenitis, but not those in reactive lymph node hyperplasia or in Castelman's disease, showed immunoreactive FKN. These results indicate that regulated expression of FKN in ECs participates in an amplification circuit of polarized type I responses.