Transcription profiling of Prss16 (Tssp) can be used to find additional peptidase genes that are candidates for self-peptide generation in the thymus

Positive selection (PS) in the thymus involves the presentation of self-peptides that are bound to MHC class II on the surface of cortical thymus epithelial cells (cTECs). Prss16 gene corresponds to one important element regulating the PS of CD4(+) T lymphocytes, which encodes Thymus-specific serine protease (Tssp), a cTEC serine-type peptidase involved in the proteolytic generation of self-peptides. Nevertheless, additional peptidase genes participating in the generation of self-peptides need to be found. Because of its role in the mechanism of PS and its expression in cTECs, the Prss16 gene might be used as a transcriptional marker to identify new genes that share the same expression profile and that encode peptidases in the thymus. To test this hypothesis, we compared the differential thymic expression of 4,500 mRNAs of wild-type (WT) C57BL/6 mice with their respective Prss16-knockout (KO) mutants by using microarrays. From these, 223 genes were differentially expressed, of which 115 had known molecular/biological functions. Four endopeptidase genes (Casp1, Casp2, Psmb3 and Tpp2) share the same expression profile as the Prss16 gene; i.e., induced in WT and repressed in KO while one endopeptidase gene, Capns1, features opposite expression profile. The Tpp2 gene is highlighted because it encodes a serine-type endopeptidase functionally similar to the Tssp enzyme. Profiling of the KO mice featured down-regulation of Prss16, as expected, along with the genes mentioned above. Considering that the Prss16-KO mice featured impaired PS, the shared regulation of the four endopeptidase genes suggested their participation in the mechanism of self-peptide generation and PS.

INVESTIGAÇÃO ELETROQUÍMICA E CALORIMÉTRICA DA INTERAÇÃO DE NOVOS AGENTES ANTITUMORAIS BISCATIÔNICOS COM DNA

ELECTROCHEMICAL AND CALORIMETRIC INVESTIGATION OF INTERACTION OF NOVEL BISCATIONIC ANTICANCER AGENTS WITH DNA. Biscationic amidines bind in the DNA minor groove and present biological activity against a range of infectious diseases. Two new biscationic compounds (bis-alpha,omega-S-thioureido, amino and sulfide analogues) were synthesized in good yields and fully characterized, and their interaction with DNA was also investigated. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic properties of binding interactions between DNA and these ligands. A double stranded calf thymus DNA immobilized on an electrode surface was used to study the possible DNA-interacting abilities of these compounds towards dsDNA in situ. A remarkable interaction of these compounds with DNA was demonstrated and their potential application as anticancer agents was furthered.

Thymopoiesis and regulatory T cells in healthy children and adolescents

OBJECTIVES: The purpose of this study was to investigate the association between T cell receptor excision circle levels in peripheral blood mononuclear cells and regulatory T cells that co-express CD25 and Foxp3 in healthy children and adolescents of different ages. MATERIALS AND METHODS: The quantification of signal-joint T-cell receptor excision circle levels in the genomic DNA of peripheral blood mononuclear cells was performed using real-time quantitative PCR. The analysis of CD4, CD8, CD25, and Foxp3 expression was performed using flow cytometry. RESULTS: Ninety-five healthy controls (46 females and 49 males) ranging in age from 1 to 18 years were analyzed. The mean T-cell receptor excision circle count in all individuals was 89.095 +/- 36.790 T-cell receptor excision circles per microgram of DNA. There was an inverse correlation between T-cell receptor excision circles counts and age (r = -0.846; p < 0.001) as well as between the proportion of CD4(+)CD25(+)Foxp3(+) T cells and age (r = -0.467; p = 0.04). In addition, we observed a positive correlation between the amount of CD4(+)CD25(+)Foxp3(+) T cells and the amount of T-cell receptor excision circles per microgram of DNA in individuals of all ages (r = -0.529; p = 0.02). CONCLUSIONS: In this study, we observed a decrease in the thymic function with age based on the fact that the level of T-cell receptor excision circles in the peripheral blood positively correlated with the proportion of regulatory T cells in healthy children and adolescents. These findings indicate that although T-cell receptor excision circles and regulatory T cells levels decrease with age, homeostasis of the immune system and relative regulatory T cells population levels are maintained in the peripheral blood.

Histologia e ultraestrutura do rim e rim cefálico do pacu

O pacu, Piaractus mesopotamicus, é um teleósteo da Família Characidae, intensivamente cultivado no Brasil devido sua rusticidade, crescimento rápido e fácil adaptação. O conhecimento morfológico dos sistemas corpóreos, incluído órgãos linfóide, se faz necessário, para uma melhor produção no cultivo de peixes, fornecendo subsídios na manutenção dos estoques. O objetivo deste estudo foi descrever morfologicamente o rim e rim cefálico de Piaractus mesopotamicus, analisando os perfis celulares de cada órgão com o uso de microscopia de luz e microscopia eletrônica de transmissão. O resultado da análise macroscópica mostrou que a localização do rim e rim cefálico são as mesmas encontradas na maioria dos teleósteos. O rim apresentou uma forma em "H", onde a região média se expandia sobre a bexiga natatória. O rim cefálico se apresentou como uma dilatação na região cranial do rim, mostrando-se bem visível. Na microscopia eletrônica de transmissão também foram observadas similaridades ultraestruturais com outros teleósteos. Observando nossos resultados concluímos que histologicamente e ultraestruturalmente, os órgãos linfóides rim e rim cefálico de Piaractus mesopotamicus são similares aos de outros teleósteos.

Acute heat stress impairs performance parameters and induces mild intestinal enteritis in broiler chickens: Role of acute hypothalamic-pituitary-adrenal axis activation

Studies on the environmental consequences of stress are relevant for economic and animal welfare reasons. We recently reported that long-term heat stressors (31 +/- 1 degrees C and 36 +/- 1 degrees C for 10 h/d) applied to broiler chickens (Gallus gallus domesticus) from d 35 to 42 of life increased serum corticosterone concentrations, decreased performance variables and the macrophage oxidative burst, and produced mild, multifocal acute enteritis. Being cognizant of the relevance of acute heat stress on tropical and subtropical poultry production, we designed the current experiment to analyze, from a neuroimmune perspective, the effects of an acute heat stress (31 +/- 1 degrees C for 10 h on d 35 of life) on serum corticosterone, performance variables, intestinal histology, and peritoneal macrophage activity in chickens. We demonstrated that the acute heat stress increased serum corticosterone concentrations and mortality and decreased food intake, BW gain, and feed conversion (P < 0.05). We did not find changes in the relative weights of the spleen, thymus, and bursa of Fabricius (P > 0.05). Increases in the basal and the Staphylococcus aureus-induced macrophage oxidative bursts and a decrease in the percentage of macrophages performing phagocytosis were also observed. Finally, mild, multifocal acute enteritis, characterized by the increased presence of lymphocytes and plasmocytes within the lamina propria of the jejunum, was also observed. We found that the stress-induced hypothalamic-pituitary-adrenal axis activation was responsible for the negative effects observed on chicken performance and immune function as well as for the changes in the intestinal mucosa. The data presented here corroborate with those presented in other studies in the field of neuroimmunomodulation and open new avenues for the improvement of broiler chicken welfare and production performance.

Lower numbers of natural killer T cells in HIV-1 and Mycobacterium leprae co-infected patients

Natural killer T (NKT) cells are a heterogeneous population of lymphocytes that recognize antigens presented by CD1d and have attracted attention because of their potential role linking innate and adaptive immune responses. Peripheral NKT cells display a memory-activated phenotype and can rapidly secrete large amounts of pro-inflammatory cytokines upon antigenic activation. In this study, we evaluated NKT cells in the context of patients co-infected with HIV-1 and Mycobacterium leprae. The volunteers were enrolled into four groups: 22 healthy controls, 23 HIV-1-infected patients, 20 patients with leprosy and 17 patients with leprosy and HIV-1-infection. Flow cytometry and ELISPOT assays were performed on peripheral blood mononuclear cells. We demonstrated that patients co-infected with HIV-1 and M.leprae have significantly lower NKT cell frequencies [median 0.022%, interquartile range (IQR): 0.0070.051] in the peripheral blood when compared with healthy subjects (median 0.077%, IQR: 0.0320.405, P < 0.01) or HIV-1 mono-infected patients (median 0.072%, IQR: 0.0300.160, P < 0.05). Also, more NKT cells from co-infected patients secreted interferon-? after stimulation with DimerX, when compared with leprosy mono-infected patients (P = 0.05). These results suggest that NKT cells are decreased in frequency in HIV-1 and M.leprae co-infected patients compared with HIV-1 mono-infected patients alone, but are at a more activated state. Innate immunity in human subjects is strongly influenced by their spectrum of chronic infections, and in HIV-1-infected subjects, a concurrent mycobacterial infection probably hyper-activates and lowers circulating NKT cell numbers.

Supernumerary parathyroid glands in hyperparathyroidism associated with multiple endocrine neoplasia type 1

Objective: To evaluate frequency, anatomic presentation, and quantities of supernumerary parathyroids glands in patients with primary hyperparathyroidism (HPT1) associated with multiple endocrine neoplasia type 1 (MEN1), as well as the importance of thymectomy, and the benefits of localizing examinations for those glands. Methods: Forty-one patients with hyperparathyroidism associated with MEN1 who underwent parathyroidectomy between 1997 and 2007 were retrospectively studied. The location and number of supernumerary parathyroids were reviewed, as well as whether cervical ultrasound and parathyroid SESTAMIBI scan (MIBI) were useful diagnostic tools. Results: In five patients (12.2%) a supernumerary gland was identified. In three of these cases (40%), the glands were near the thyroid gland and were found during the procedure. None of the imaging examinations were able to detect supernumerary parathyroids. In one case, only the pathologic examination could find a microscopic fifth gland in the thymus. In the last case, the supernumerary gland was resected through a sternotomy after a recurrence of hyperparathyroidism, ten years after the initial four-gland parathyroidectomy without thymectomy. MIBI was capable of detecting this gland, but only in the recurrent setting. Cervical ultrasound did not detect any supernumerary glands. Conclusion: The frequency of supernumerary parathyroid gland in the HPT1/MEN1 patients studied (12.2%) was significant. Surgeons should be aware of the need to search for supernumerary glands during neck exploration, besides the thymus. Imaging examinations were not useful in the pre-surgical location of these glands, and one case presented a recurrence of hyperparathyroidism.

Tuning the reaction products of ruthenium and ciprofloxacin for studies of DNA interactions

This work presents two potential metallo-drugs, the ionic (C17H19FN3O3)(3)[RuCl6]center dot 3H(2)O (1) and the coordination [Ru(C17H17FN3O3)(3)]center dot 4H(2)O (2) compounds, obtained by the combination of ruthenium(III) and ciprofloxacin in different synthetic conditions. The ESI MS spectrum of 1 displayed a main peak at m/z = 994.6, assigned to the gaseous phase adduct (ciprofloxacin)(3)center dot H+, while 2 featured peaks at m/z 1093.3 and 547.1 ascribed to [Ru(C17H17FN3O3)(3)center dot H+-4H(2)O](+) and [Ru(C17H17FN3O3)(3)center dot 2H(+)-4H(2)O](2+). Thermal analysis corroborated the proposed water content for both complexes. Absorption spectra of the compounds in aqueous medium are dominated by ciprofloxacin transitions in the UV region but displayed weak bands in the visible region, assigned to ligand field transitions. The cyclic voltammograms of 2 exhibited a quasi-reversible process ascribed to the Ru(II)/(III) redox pair at -0.25V (vs. SHE) while 1 displayed this process at -0.11 V, showing that the central ruthenium ion is stabilized in the (III) oxidation state by the coordination to the hard oxygen atoms of ciprofloxacin. The solubility of 1 is pH dependent (as well as free ciprofloxacin) while 2 is fully water soluble and stable under physiological pH for at least 48 h. The compounds are also stable under incubation conditions (stomach pH and 37 degrees C) without significant pH lowering. An interaction study of 2 with ct-DNA showed a value of K-b = 2.47 (+/- 0.89) x 10(4) mol(-1) L for the intrinsic binding constant.

Myasthenia gravis and neuromyelitis optica spectrum disorder A multicenter study of 16 patients

Objective: To describe 16 patients with a coincidence of 2 rare diseases: aquaporin-4 antibody (AQP4-Ab)-mediated neuromyelitis optica spectrum disorder (AQP4-NMOSD) and acetylcholine receptor antibody (AChR-Ab)-mediated myasthenia gravis (AChR-MG). Methods: The clinical details and antibody results of 16 patients with AChR-MG and AQP4-NMOSD were analyzed retrospectively. Results: All had early-onset AChR-MG, the majority with mild generalized disease, and a high proportion achieved remission. Fifteen were female; 11 were Caucasian. In 14/16, the MG preceded NMOSD (median interval: 16 years) and 11 of these had had a thymectomy although 1 only after NMOSD onset. In 4/5 patients tested, AQP4-Abs were detectable between 4 and 16 years prior to disease onset, including 2 patients with detectable AQP4-Abs prior to thymectomy. AChR-Abs decreased and the AQP4-Ab levels increased over time in concordance with the relevant disease. AChR-Abs were detectable at NMOSD onset in the one sample available from 1 of the 2 patients with NMOSD before MG. Conclusions: Although both conditions are rare, the association of MG and NMOSD occurs much more frequently than by chance and the MG appears to follow a benign course. AChR-Abs or AQP4-Abs may be present years before onset of the relevant disease and the antibody titers against AQP4 and AChR tend to change in opposite directions. Although most cases had MG prior to NMOSD onset, and had undergone thymectomy, NMOSD can occur first and in patients who have not had their thymus removed. Neurology (R) 2012;78:1601-1607

RNA interference-mediated knockdown of CD49e (α5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion

Abstract Background The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin α5β1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. Results Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. Conclusion Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation.

Dietary restriction abrogates antibody production induced by a DNA vaccine encoding the mycobacterial 65 kDa heat shock protein

Abstract Background Protein-calorie malnutrition (PCM) is the most common type of malnutrition. PCM leads to immunodeficiency and consequent increased susceptibility to infectious agents. In addition, responses to prophylactic vaccines depend on nutritional status. This study aims to evaluate the ability of undernourished mice to mount an immune response to a genetic vaccine (pVAXhsp65) against tuberculosis, containing the gene coding for the heat shock protein 65 from mycobacteria. Methods Young adult female BALB/c mice were fed ad libitum or with 80% of the amount of food consumed by a normal diet group. We initially characterized a mice model of dietary restriction by determining body and spleen weights, hematological parameters and histopathological changes in lymphoid organs. The ability of splenic cells to produce IFN-gamma and IL-4 upon in vitro stimulation with LPS or S. aureus and the serum titer of specific IgG1 and IgG2a anti-hsp65 antibodies after intramuscular immunization with pVAXhsp65 was then tested. Results Dietary restriction significantly decreased body and spleen weights and also the total lymphocyte count in blood. This restriction also determined a striking atrophy in lymphoid organs as spleen, thymus and lymphoid tissue associated with the small intestine. Specific antibodies were not detected in mice submitted to dietary restriction whereas the well nourished animals produced significant levels of both, IgG1 and IgG2a anti-hsp65. Conclusion 20% restriction in food intake deeply compromised humoral immunity induced by a genetic vaccine, alerting, therefore, for the relevance of the nutritional condition in vaccination programs based on these kinds of constructs.

Serial bone marrow transplantation reveals in vivo expression of the pCLPG retroviral vector

Abstract Background Gene therapy in the hematopoietic system remains promising, though certain aspects of vector design, such as transcriptional control elements, continue to be studied. Our group has developed a retroviral vector where transgene expression is controlled by p53 with the intention of harnessing the dynamic and inducible nature of this tumor suppressor and transcription factor. We present here a test of in vivo expression provided by the p53-responsive vector, pCLPG. For this, we used a model of serial transplantation of transduced bone marrow cells. Results We observed, by flow cytometry, that the eGFP transgene was expressed at higher levels when the pCLPG vector was used as compared to the parental pCL retrovirus, where expression is directed by the native MoMLV LTR. Expression from the pCLPG vector was longer lasting, but did decay along with each sequential transplant. The detection of eGFP-positive cells containing either vector was successful only in the bone marrow compartment and was not observed in peripheral blood, spleen or thymus. Conclusions These findings indicate that the p53-responsive pCLPG retrovirus did offer expression in vivo and at a level that surpassed the non-modified, parental pCL vector. Our results indicate that the pCLPG platform may provide some advantages when applied in the hematopoietic system.

Investigação eletroquímica e calorimétrica da interação de novos agentes antitumorais biscatiônicos com DNA

Biscationic amidines bind in the DNA minor groove and present biological activity against a range of infectious diseases. Two new biscationic compounds (bis-α,ω-S-thioureido, amino and sulfide analogues) were synthesized in good yields and fully characterized, and their interaction with DNA was also investigated. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic properties of binding interactions between DNA and these ligands. A double stranded calf thymus DNA immobilized on an electrode surface was used to study the possible DNA-interacting abilities of these compounds towards dsDNA in situ. A remarkable interaction of these compounds with DNA was demonstrated and their potential application as anticancer agents was furthered.

Aspectos morfométricos do timo em gatos domésticos (Felis domesticus)

O timo é um órgão linfático primário que desenvolve sua atividade em organismos jovens. Apesar de sua função ser responsável por mecanismos fundamentais na aquisição das defesas e conseqüentes respostas orgânicas, ela ainda não está totalmente esclarecida, nem tampouco as bases morfológicas que respondem por tais funções, como o processo de desenvolvimento e involução do órgão. Objetivou-se analisar e caracterizar os aspectos morfológicos do timo, tais como seu tamanho e volume, e aspectos histológicos do timo em gatos, correlacionando o sexo e o desenvolvimento etário. Doze timos provenientes de fetos de gatos domésticos (Felis domesticus) sem raça definida (SRD), machos e fêmeas, separados em três grupos etários. O timo apresentou-se com uma coloração rosa-pálida e com duas porções, a torácica e a cervical, sendo que cada uma delas possuía um lobo direito e um lobo esquerdo em sua maioria. A porção torácica localizava-se em região de mediastino cranial, entre os pulmões e à base do coração. E a porção cervical estendia-se além das costelas em sentido cranial, estando localizada ventralmente à traqueia. A estrutura celular do timo demonstrou-se organizada com a presença de agregados concêntricos, os chamados corpúsculos tímicos, formados por células epiteliais, sustentada por uma cápsula de tecido conjuntivo de onde partiam septos que ao penetrar no órgão dividia-o em lóbulos. Ocorreram variações significativas quanto à lobação e as dimensões do timo entre indivíduos da mesma faixa etária, e entre sexos diferentes. Os valores relativos ao comprimento, espessura e largura, de maneira geral, apresentaram aumento, em conformidade ao desenvolvimento dos animais, mas com diferenças entre os sexos.

Ovariectomized OVA-sensitized mice display increased frequency of CD4+Foxp3+ T regulatory cells in the periphery

It is well established that female sex hormones have a pivotal role in inflammation. For instance, our group has previously reported that estradiol has proinflammatory actions during allergic lung response in animal models. Based on these findings, we have decided to further investigate whether T regulatory cells are affected by female sex hormones absence after ovariectomy. We evaluated by flow cytometry the frequencies of CD4+Foxp3+ T regulatory cells (Tregs) in central and peripheral lymphoid organs, such as the thymus, spleen and lymph nodes. Moreover, we have also used the murine model of allergic lung inflammation a to evaluate how female sex hormones would affect the immune response in vivo. To address that, ovariectomized or sham operated female Balb/c mice were sensitized or not with ovalbumin 7 and 14 days later and subsequently challenged twice by aerosolized ovalbumin on day 21. Besides the frequency of CD4+Foxp3+ T regulatory cells, we also measured the cytokines IL-4, IL-5, IL-10, IL-13 and IL-17 in the bronchoalveolar lavage from lungs of ovalbumine challenged groups. Our results demonstrate that the absence of female sex hormones after ovariectomy is able to increase the frequency of Tregs in the periphery. As we did not observe differences in the thymus-derived natural occurring Tregs, our data may indicate expansion or conversion of peripheral adaptive Tregs. In accordance with Treg suppressive activity, ovariectomized and ovalbumine-sensitized and challenged animals had significantly reduced lung inflammation. This was observed after cytokine analysis of lung explants showing significant reduction of pro-inflammatory cytokines, such as IL-4, IL-5, IL-13 and IL-17, associated to increased amount of IL-10. In summary, our data clearly demonstrates that OVA sensitization 7 days after ovariectomy culminates in reduced lung inflammation, which may be directly correlated with the expansion of Tregs in the periphery and further higher IL-10 secretion in the lungs.