Control of pathogens by formation of abscesses and granulomas is a major strategy of the innate immune system, especially when effector mechanisms of adaptive immunity are insufficient. We show in human listeriosis that DCs expressing indoleamine 2,3-dioxygenase (IDO), together with macrophages, are major cellular components of suppurative granulomas in vivo. Induction of IDO by DCs is a cell-autonomous response to Listeria monocytogenes infection and was also observed in other granulomatous infections with intracellular bacteria, such as Bartonella henselae. Reporting on our use of the clinically applied anti-TNF-α antibody infliximab, we further demonstrate in vitro that IDO induction is TNF-α dependent. Repression of IDO therefore might result in exacerbation of granulomatous diseases observed during anti-TNF-α therapy. These findings place IDO + DCs not only at the intersection of innate and adaptive immunity but also at the forefront of bacterial containment in granulomatous infections.
Deficiencies of natural anticoagulant proteins including antithrombin (AT), protein C (PC) and protein S (PS) are important causes of inherited thrombophilia. This study aimed to report on the practical experience gained in performing genetic analyses of a large cohort of patients with AT, PC and PS deficiencies and to relate this knowledge to clinical application. We genotyped a large cohort of 709 unrelated patients with AT (231), PC (234) and PS (244) deficiencies referred to us by physicians throughout Germany. Mutations were detected by direct sequencing and multiplex ligation-dependent probe amplification (MLPA). The highest mutation detection rate (MDR) was found for the SERPINC1 gene (83.5%), followed by the PROC (69%) and PROS1 (43%) genes. Even at AT activities close to the normal range (75%), the MDR was 70%. Contrastingly, for PC and PS deficiencies, the MDR dropped significantly and mildly lowered to subnormal values. At PS activities >55% for PS no mutations were detected. Mutation profiles of all three genes were similar with the highest prevalence for missense mutations (63-78%), followed by nonsense (7-11%), splice-site mutations (7-13%), small deletions (1-8%), small insertions/duplications (1-4%) and large deletions (3-6%). In conclusion, genetic testing is a useful diagnostic tool for diagnosing thrombophilia. Based on our data, genetic analysis for patients with AT deficiency is indicated for all subnormal activities. In contrast, genotyping is not advisable for PC activities >70% and for PS activities >55%.
Von Willebrand disease (VWD) is the most common inherited bleeding disorder caused by quantitative or qualitative defects of the von Willebrand factor (VWF). VWD is classified into three types--type 1 (partial quantitative deficiencies), type 2 (qualitative defects) and type 3 (complete deficiency of VWF). In this study we explored genotype and phenotype characteristics of patients with VWD with the aim of dissecting the distribution of mutations in different types of VWD. One hundred fourteen patients belonging to 78 families diagnosed to have VWD were studied. Mutation analysis was performed by direct sequencing of the VWF . Large deletions were investigated by multiplex ligation-dependent probe amplification (MLPA) analysis. The impact of novel candidate missense mutations and potential splice site mutations was predicted by in silico assessments. We identified mutations in 66 index patients (IPs) (84.6%). Mutation detection rate was 68%, 94% and 94% for VWD type 1, 2 and 3, respectively. In total, 68 different putative mutations were detected comprising 37 missense mutations (54.4%), 10 small deletions (14.7%), two small insertions (2.9%), seven nonsense mutations (10.3%), five splice-site mutations (7.4%), six large deletions (8.8%) and one silent mutation (1.5%). Twenty-six of these mutations were novel. Furthermore, in type 1 and type 2 VWD, the majority of identified mutations (74% vs. 88.1%) were missense substitutions while mutations in type 3 VWD mostly caused null alleles (82%). Genotyping in VWD is a helpful tool to further elucidate the pathogenesis of VWD and to establish the relationship between genotype and phenotype.
Many tumors, including Hodgkin's lymphoma, are associated with decreased cellular immunity and elevated levels of prostaglandin E 2 (PGE 2 ), a known inhibitor of CD4 + T cell activation, suggested to be involved in immune deviation in cancer. To address the molecular mechanisms tumor-derived PGE 2 might have on primary human CD4 + T cells, we used a whole genome-based transcriptional approach and show that PGE 2 severely limited changes of gene expression induced by signaling through the T cell receptor and CD28. This data suggests an interference of PGE 2 at an early step of T cell receptor signaling: indeed, PGE 2 stimulation of T cells leads to inactivation of lck and reduced phosphorylation of ZAP70. Antiapoptotic genes escaped PGE 2 -induced inhibition resulting in partial protection from apoptosis in response to irradiation or Fas-mediated signaling. As a functional consequence, PGE 2 -treated CD4 + T cells are arrested in the cell cycle associated with up-regulation of the cyclin/cyclin-dependent kinase inhibitor p27 kip1 . Most importantly, CD4 + T cells in Hodgkin's lymphoma show similar regulation of genes that were altered in vitro by PGE 2 in T cells from healthy individuals. These data strongly suggest that PGE 2 is an important factor leading to CD4 + T cell impairment observed in Hodgkin's lymphoma. (Cancer Res 2006; 66(2): 1114-22)
IntroductionTumors frequently develop evasion strategies that may influence every stage of the tumor-specific immune response, from the activation of antigen-presenting cells to T-cell recruitment, activation, and effector function. [1][2][3] Hodgkin lymphoma (HL) seems to be a role model for an ineffective tumor immune response, because the minority of tumor cells, so called Reed-Sternberg (RS) cells, are surrounded mainly by a mixture of reactive cells including lymphocytes, plasma cells, eosinophils, and histiocytes. 4,5 The majority of lymphocytes in close proximity to the tumor cells are CD4 ϩ T cells with anergic phenotype, indicating poor immune surveillance of the tumor. 6 In fact, several immune evasion strategies are described in HL, including down-regulation of immunodominant Epstein-Barr Virus (EBV) antigens, and secretion of soluble factors such as transforming growth factor beta (TGF), interleukin-10 (IL10), or prostaglandin E 2 (PGE 2 ), that have been shown to inhibit the activation of specific cytotoxic T lymphocytes (CTLs) and professional antigen-presenting cells in vitro and in murine models. [7][8][9][10] Also cell-to-cell interactions via inhibitory receptors might lead to immune inhibition. For example, neoplastic CD20 ϩ B cells in nodular lymphocyte-predominant HL express PDL-1, and the respective inhibitory receptor PD-1 is expressed by T cells in close proximity to the tumor cells. 11 So far, a general limitation studying immune inhibition within the tumor microenvironment, particularly in humans, is the fact that we rely solely on indirect evidence. This is best exemplified for soluble factors such as TGF, which is found to be elevated within the tumor microenvironment of many human tumors including lymphomas. 12,13 It is also well established that T cells isolated from tumor tissue exhibit an anergic phenotype [14][15][16][17] and that such a phenotype can be induced in vitro in normal T cells exposed to inhibitory factors. 14,17,18 However, no direct evidence exists that the anergic phenotype observed in tumor infiltrating T cells is indeed due to exposure to inhibitory cytokines such as TGF, IL10, or PGE 2 . Moreover, there is not even direct evidence that these factors lead to signaling events within tumor-infiltrating T cells in vivo.HL is a very good example for the existing circumstantial evidence. Activated lymphocytes, binucleate RS cells, and mononuclear Hodgkin cells have been described to express activated TGF. 7,13 The expression of TGF as well as the functional phenotype of T cells in HL suggested that this cytokine might be involved in immunosuppression in HL, however, direct evidence is lacking. Other possible mechanisms of immune escape in HL might involve ligands for inhibitory receptors such as PDL-1 on the surface of the neoplastic cells. 11 PD-1 is a receptor of the Ig superfamily that negatively regulates T-cell antigen receptor signaling by interacting with the specific ligands (PDLs) and is suggested to play a role in the maintenance of self-tolerance. [19]...
Key Points• This study demonstrates allosteric RNA structure alteration resulting from an exonic variation, thereby interfering with splicing.• This study details a novel mechanism by which silent mutation distant to the 59 splice site could still result in intron retention.Disease-associated silent mutations are considered to affect the accurate premessenger RNA (mRNA) splicing either by influencing regulatory elements, leading to exon skipping, or by creating a new cryptic splice site. This study describes a new molecular pathological mechanism by which a silent mutation inhibits splicing and leads to intron retention. We identified a heterozygous silent mutation, c.7464C>T, in exon 44 of the von Willebrand factor (VWF) gene in a family with type 1 von Willebrand disease. In vivo and ex vivo transcript analysis revealed an aberrantly spliced transcript, with intron 44 retained in the mRNA, implying disruption of the first catalytic step of splicing at the 59 splice site (59ss). The abnormal transcript with the retained intronic region coded a truncated protein that lacked the carboxy-terminal end of the VWF protein. Confocal immunofluorescence characterizations of blood outgrowth endothelial cells derived from the patient confirmed the presence of the truncated protein by demonstrating accumulation of VWF in the endoplasmic reticulum. In silico pre-mRNA secondary and tertiary structure analysis revealed that this substitution, despite its distal position from the 59ss (85 bp downstream), induces cis alterations in pre-mRNA structure that result in the formation of a stable hairpin at the 59ss. This hairpin sequesters the 59ss residues involved in U1 small nuclear RNA interactions, thereby inhibiting excision of the pre-mRNA intronic region. This study is the first to show the allosteric-like/far-reaching effect of an exonic variation on pre-mRNA splicing that is mediated by structural changes in the pre-mRNA. (Blood. 2016;128(17):2144-2152
Myeloid dendritic cells (DC) and macrophages play an important role in pathogen sensing and antimicrobial defense. In this study we provide evidence that myeloid DC respond to infection with Listeria monocytogenes with simultaneous induction of multiple stimulatory and inhibitory molecules. However, the overall impact of infected DC during T cell encounter results in suppression of T cell activation, indicating that inhibitory pathways functionally predominate. Inhibitory activity of infected DC is effected mainly by IL-10 and cyclooxygenase 2-mediated mechanisms, with soluble CD25 acting as an IL-2 scavenger as well as by the products of tryptophan catabolism. These inhibitory pathways are strictly TNF-dependent. In addition to direct infection, DC bearing this regulatory phenotype can be induced in vitro by a combination of signals including TNF, TLR2, and prostaglandin receptor ligation and by supernatants derived from the infected cells. Both infection-associated DC and other in vitro-induced regulatory DC are characterized by increased resistance to infection and enhanced bactericidal activity. Furthermore, myeloid DC expressing multiple regulatory molecules are identified in vivo in granuloma during listeriosis and tuberculosis. Based on the in vivo findings and the study of in vitro models, we propose that in granulomatous infections regulatory DC may possess dual function evolved to protect the host from disseminating infection via inhibition of granuloma destruction by T cells and control of pathogen spreading.
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