Cytokines and acute-phase serum proteins as markers of inflammatory regression during pulmonary tuberculosis treatment

Tuberculosis is still increasing and was declared a worldwide sanitary emergency by the World Health Organization (WHO) in 1995. Its control is difficult due to long treatment duration and lack of markers of treatment success or failure. Cytokines such as IFN-gamma and TNF-alpha, a central factor in immune response against Mycobacterium tuberculosis, are responsible for the interaction between T lymphocytes and the infected macrophage and are also produced during this interaction. As proinflammatory cytokines have a close relationship with mycobacteria clearance, in fact even preceding it, they could be used as markers for inflammatory activity and response to treatment. Proinflammatory cytokines act in the liver and stimulate a strong local and systemic acute-phase response as a result of homeostatic and physiological responses also induced by them. Acute-phase proteins produced by cytokine activity are useful diagnostic markers that could also be used to monitor treatment response as they can be serially quantified. The objective of this study was to evaluate IFN-gamma, TNF-alpha, IL-10 and TGF-beta production in supernatant of peripheral blood mononuclear cell (PBMC) and monocyte (MO) cultures, as well as serum acute-phase response through total protein, albumin, globulin, C-reactive protein (CRP), alpha-1-acid glycoprotein (AGP), and erythrocyte sedimentation rate (ESR) as regression markers of inflammatory response during pulmonary tuberculosis treatment. Twenty blood donors (G1) from the Blood Bank at Botucatu School of Medicine's University Hospital (BSM-UH) were evaluated once and 28 pulmonary tuberculosis patients (G2): 13 from BSM-UH and 15 from the Bauru State Health Secretariat. Patients were evaluated at three moments of treatment: before (M1), at three months (M2), and at the end (M3). Cytokines were determined in 20ml of peripheral blood (ELISA), with or without activation: lipopolysaccharide (LPS) for MO culture and phytohemagglutinin (PHA) for PBMC culture. Acute-phase protein behavior in G2 throughout treatment was: Globulins: M1> M2, M1> M3 (rho < 0.001); CRP: M1> M2> M3 (.< 0.001); AGP for men: M1> M2, M1> M3 (rho < 0.001); ESR for men: M1> M2, M1> M3 (rho < 0.0016) and for women: M1> M2 (.< 0.025). Comparison between cytokine levels found in supernatant of MO and PBMC cultures, with and without stimulus, in G1 and G2 during treatment showed: TNF-alpha (with/ without LPS) at M1: G2> G1; at M2: G2> G1 (rho < 0.001); (without LPS) at M3: G2> G1 (rho < 0.001), (with LPS) at M3: G2> G1 (rho < 0.028); IFN-. (with and without PHA) at M1: G2> G1; at M2: G2> G1 (rho < 0.001); IL-10 (with and without LPS) at M1: G2> G1; at M2: G2> G1; at M3: G2> G1 (rho < 0.001); TGF-beta (with and without LPS) at M1: G2> G1; at M2: G2> G1 (rho < 0.001), (without LPS) at M3: G2> G1 (rho < 0.001). In G2, all cytokines in supernatant of MO and PBMC cultures, with and without stimulus, showed: M1> M2> M3 (rho < 0.01). Levels of globulins, CRP, AGP, and ESR in patients with pulmonary tuberculosis before treatment (M1) were significantly higher than reference values, suggesting their use as diagnostic markers and indicators of treatment. The CRP decreasing values along treatment could be taken as a marker of the regression of inflammatory process and of response to treatment in patients with pulmonary tuberculosis.Regarding cytokines, there was significant increase in TNF-alpha, IFN-gamma, IL-10, and TGF-alpha levels before and at three months treatment, with and without stimulus; in TNF-a and IL-10 lvels, with and without stimulus, as well as in TGF-alpha levels without stimulus at six months. Patients had higher levels of all studied cytokines than controls before treatment, and these values decreased along treatment. In this study, pulmonary tuberculosis patients showed a Th0 cytokine profile before treatment, with the production of both Th1 (IFN-gamma) and Th2 (IL-10) cytokines, in addition to TNF-alpha inflammatory and TGF-alpha regulatory and fibrosis-inducer cytokines. At the end of treatment, all had evolved to Th2 profile, probably in an attempt to reduce the harmful effects of the proinflammatory activity of the Th1 cytokine profile and of the still above-normal levels of TNF-alpha. The high levels of TGF-alpha, also found in these patients, are related to its important role in the extracellular matrix deposition and fibrosis induction that characterize tuberculosis healing process. IFN-gamma was the only cytokine reaching normal levels at the end of treatment, which suggests its use as a marker of response to treatment.

Imunoexpressão do c-erbB-2 nas lesões epiteliais proliferativas intraductais da mama de mulheres

OBJETIVOS: Alterações genéticas são relacionadas à gênese e progressão do câncer. Neoplasias de vários órgãos expressam o oncogene c-erbB-2. Nas proliferações intraductais da mama tem sido avaliado como fator de risco para o desenvolvimento de câncer. Foram avaliadas a imunoexpressão do c-erbB-2 em lesões epiteliais proliferativas intraductais e as possíveis correlações com características anatomopatológicas do carcinoma ductal in situ (CDIS). MÉTODOS: Foi utilizado material de arquivo, amostras teciduais fixadas em formalina e incluídas em blocos de parafina de 88 mulheres. Destas, 51 com CDIS e 37 com hiperplasia ductal sem atipias (HDT). A idade variou de 35 a 76 anos. Revisados todos os casos, verificou-se: o grau nuclear, a presença de necrose, o subtipo histológico predominante e sua extensão. Obteve-se material suficiente para o estudo imunohistoquímico do c-erbB-2 de 84 sujeitos do estudo. RESULTADOS: Não foi observada a expressão do oncogene nas hiperplasias sem atipias e nos tecidos adjacentes a todas amostras teciduais. A expressão do c-erbB-2 foi verificada em nove (19,1%) dos CDIS (p= 0,0001). A imunoexpressão não se relacionou à extensão das lesões. A imunoexpressão do c-erbB-2 no CDIS correlacionou-se com subtipo histológico (p=0,019), com a presença de necrose (p=0,0066), com o grau nuclear (p=0,0084) e com a Classificação de Van Nuys (p=0,039). CONCLUSÕES: A expressão do c-erbB-2 foi estatisticamente significante nas lesões proliferativas de risco (CDIS) e correlacionou-se com características histopatológicas: alto grau nuclear, presença de necrose, subtipo comedo. Não houve expressão nas hiperplasias sem atipias e tecidos adjacentes.

Expression of apoptotic and cell proliferation regulatory proteins in keratoacanthomas and squamous cell carcinomas of the skin

The aim of this study was to investigate the expression of p53, caspase-3, bcl-2, MIB-1, and PCNA to validate more objective methods to differentiate squamous cell carcinoma and keratoacanthoma, as well as to understand their pathogenesis with accuracy. A total of 52 cases of histopathologically diagnosed keratoacanthoma in the proliferative stage and 56 cases of well-differentiated squamous cell carcinoma were selected in this study. The expression was evaluated by means of immunohistochemistry. Bcl-2 immunoreactivity was weak or absent in the majority of cases, either in squamous cell carcinoma or in keratoacanthoma. PCNA-positive cells did not show differences between two lesions evaluated. on the other hand, MIB-1 was statistically significant (p<0.05) between squamous cell carcinomas and keratoacanthomas. Moreover, p53 and caspase-3 were overexpressed in squamous cell carcinomas. Together, these results suggest that the biological behavior of the well-differentiated squanous cell carcinomas of the skin may be associated with cellular proliferation and/or deregulation of cell death, indicated by increased expression of the MIB-1 and apoptotic proteins p53 and caspase-3, respectively. (C) 2007 Elsevier GrnbH. All rights reserved.

Characteristics of seminal plasma proteins and their correlation with canine semen analysis

The objectives were to separate canine seminal plasma proteins (with SDS-PAGE) and to determine the correlation between specific proteins and semen characteristics. Three ejaculates from 20 mixed-breed dogs, of unknown fertility, were collected by digital manipulation. Ejaculate volume and color, sperm motility, sperm vigor, percentage of morphologically normal spermatozoa, and membrane integrity (hypoosmotic swelling test and fluorescent staining) were assessed. For each dog, seminal plasma was pooled from all three ejaculates and proteins were separated with SDS-PAGE, using polyacrylamide concentrations of 13% and 22% in the separation gels. After staining, gel images were digitized to estimate molecular weights (MW) and integrated optical density (IOD) of each lane and of individual bands. Total seminal plasma protein concentration was 2.19 +/- 1.56 g/dL (mean +/- SD; range 1.12-5.19 g/dL). A total of 37 protein bands were identified (although no dog had all 37 bands). In the 13% gel, molecular weights ranged from 100.6 to 17.1 kDa, with four bands (49.7, 33.2, 26.4, and 19.5 kDa) present in samples from all dogs. In the 22% gel, molecular weights ranged from 15.6 to 3.6 kDa, with nine bands (15.6, 13.5, 12.7, 11.7, 10.5, 8.7, 7.8, 5.6, and 4.9 kDa) present in samples from all dogs. Combined for both gels, the majority of bands (85%) had molecular weights < 17 kDa, with B20 (15.6 kDa) in high concentrations in samples from all dogs. There were positive correlations (P <= 0.01) between two bands, 134 (67 kDa) and B5 (58.6 kDa), and sperm motility (r = 0.66 and r = 0.46), sperm vigor (r = 0.56 and r = 0.66), percentage of morphologically normal spermatozoa (r = 0.55 and r = 0.59), the hypoosmotic swelling test (r = 0.76 and r 0.68), and fluorescent staining (r = 0.56 and r = 0.59), respectively. In conclusion, 37 proteins were identified in seminal plasma; two were significantly correlated with semen characteristics. (c) 2007 Elsevier B.V. All rights reserved.

Heparin-binding proteins of canine seminal plasma

Heparin-binding proteins (HBP) from seminal plasma have been expected to participate in modulation of the acrosomal reaction, and have been correlated with fertility in some species. However, they have not been described in the dog. The aim of this study was to document the HBPs of canine seminal plasma. Six pooled samples of seminal plasma from three crossbred dogs were used. The HBPs were isolated by heparin affinity chromatography and the fractions recovered were pooled. One-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on 12 and 18% vertical minigels. The stained gels were scanned and the molecular weight (kDa) values for each band within a lane were calculated by image analysis software. The electrophoresis analysis of the pooled eluded fractions identified 19 bands, with molecular weights varying from 61.5 to 5.2 kDa. Previous studies, using one-dimensional SDS-PAGE, identified two bands (67 and 58.6 kDa), which were positively correlated with some semen parameters (sperm motility, sperm vigor, percentage of morphologically normal sperm and plasma membrane integrity). The 61.5 kDa band detected in the present study apparently corresponded to the 58.6 kDa band identified previously. Canine seminal plasma contained HBP; since HBP modulate the acrosome reaction in other species, they may have the same function in the dog. Further studies are necessary to better characterize this protein and determine if it is associated with fertility in the dog. (c) 2006 Elsevier B.V. All rights reserved.

Abnormal cell-cycle expression of the proteins p27, mdm2 and cathepsin B in oral squamous-cell carcinoma infected with human papillomavirus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Human Platelet Antigen Genotype Is Associated With Progression of Fibrosis in Chronic Hepatitis C

Although progression of fibrosis in the chronic hepatitis C depends on environmental, viral, and host factors, genetic polymorphisms have been associated recently with this progression, including the expression of integrins, adhesion proteins. Some integrins expressed on the platelet membrane show polymorphic antigenic determinants called human platelet antigens (HPA), where the major ones are HPA-1, -3, -5. The association between HCV infection and HPA-5b has been demonstrated. Similarly, the HPA profile could determine if HPA is related to progression of fibrosis. The goal of this study was to evaluate the association between the frequencies of HPA-1, -3, and -5 and degree of fibrosis in HCV-infected patients. Genomic DNA from 143 HCV-infected patients was used as the source for HPA genotyping by PCR-SSP or PCR-RFLP. Progression of fibrosis was evaluated using the METAVIR scoring system, and the patients were grouped according to degree of fibrosis into G1 (n = 81, with F1, portal fibrosis without septa or F2, few septa) and G2 (n = 62, with F3, numerous septa, or F4, cirrhosis). Statistical analysis was performed using the proportional odds model. The genotypic frequency of HPA-1a/1b was significantly higher in the patients in G2. To evaluate the influence of the time of infection to the development of fibrosis and its effect on the genetic factor HPA-1, 96 patients from 143 studied were evaluated considering the time of HCV infection, and these results suggest that the HPA-1a/1b genotype promotes the development of fibrosis in HCV infection with time. J. Med. Virol. 84: 56-60, 2012. (C) 2011 Wiley Periodicals, Inc.

Storage sites in seeds of Caesalpinia echinata and C. ferrea (Leguminosae) with considerations on nutrients flow

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bothrops moojeni myotoxin-II, a Lys49-phospholipase A(2) homologue: An example of function versatility of snake venom proteins

MjTX-II, a myotoxic phospholipase A(2) (PLA(2)) homologue from Bothrops moojeni venom, was functionally and structurally characterized. The MjTX-II characterization included: (i) functional characterization (antitumoral, antimicrobial and antiparasitic effects); (ii) effects of structural modifications by 4-bromophenacyl bromide (BPB), cyanogen bromide (CNBr), acetic anhydride and 2-nitrobenzenesulphonyl fluoride (NBSF); (iii) enzymatic characterization: inhibition by low molecular weight heparin and EDTA; and (iv) molecular characterization: cDNA sequence and molecular structure prediction. The results demonstrated that MjTX-II displayed antimicrobial activity by growth inhibition against Escherichia coli and Candida albicans, antitumoral activity against Erlich ascitic tumor (EAT), human breast adenocarcinoma (SK-BR-3) and human T leukemia cells (JURKAT) and antiparasitic effects against Schistosoma mansoni and Leishmania spp., which makes MjTX-II a promising molecular model for future therapeutic applications, as well as other multifunctional homologous Lys49-PLA(2)S or even derived peptides. This work provides useful insights into the structural determinants of the action of Lys49-PLA2 homologues and, together with additional strategies, supports the concept of the presence of others bioactive sites distinct from the catalytic site in snake venom myotoxic PLA(2)s. (c) 2005 Elsevier B.V. All rights reserved.

Indigofera suffruticosa Mill as new source of healing agent: Involvement of prostaglandin and mucus and heat shock proteins

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Protection of calves against Haemonchus placei and Haemonchus contortus after immunization with gut membrane proteins from H. contortus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fat body in some genera of leaf-cutting ants (Hymenoptera : Formicidae). Proteins, lipids and polysaccharides detection

The comparative histochemical analysis of the fat body of workers belonging to the basal species Cyphomyrmex rimosus and Mycetarotes parallelus and to derived species Acromyrmex disciger and Atta laevigata revealed that this tissue is constituted mainly by cells denominated trophocytes and oenocytes. The trophocytes of all species studied here were characterized mainly by the proteins and lipids synthesis and storage, being the derived species the ones who have presented higher quantity of lipids in the trophocytes when compared to the trophocytes of basal species. In workers M. parallelus and A. laevigata, besides proteins and lipids, there has being observed the presence of polysaccharides, however, in C. rimosus and A. disciger these elements were detected in lower quantities. The histochemical studies of the oenocytes of basal and derived species revealed significant presence of proteins as well as lipids in these cells. In the oenocytes of derived species A. disciger and A. laevigata a higher quantity of lipidic inclusions has being observed, when compared to the basal species. (c) 2005 Elsevier Ltd. All rights reserved.

In situ localization of heat-shock proteins and cell death labelling in the salivary gland of acaricide-treated honeybee larvae

The effects of the acaricides, rotenone and oxalic acid (OA) on salivary glands of honeybee larvae were evaluated. Immunohistochemical methods were used to detect cell death and heat-shock protein (HSP70 and 90) localizations. Heat-shock proteins (HSP70 and 90) were localized in the cytoplasm and/or the nuclei of secretory gland cells, both under stress and in normal conditions. In rotenone-treated larvae, there were no changes in the normal level of cell death and also there were no morphological alterations in the secretory cells. In the larvae treated with oxalic acid, the salivary gland showed varying degrees of morphological cellular alteration and an increase in the cell death level. The present data suggest that stress-induced HSP70 might have an antiapoptotic effect while the stress-induced HSP90 might have a chaperone function in the larval salivary glands.

Ultrastructural detection of proteins, lipids and carbohydrates in oocytes of Amblyomma triste (Koch, 1844) (Acari; Ixodidae) during the vitellogenesis process

The ovary of the tick Ainblyomma triste is classified as panoistic, which is characterized by the presence of oogonia without nurse and follicular cells. The present study has demonstrated that the oocytes in all developmental stages (I-IV) are attached to the ovary through a pedicel, a cellular structure that synthesizes and provides carbohydrate, lipids and proteins supplies for the oocytes during the vitellogenesis process. The lipids are deposited during all oocyte stages; they are freely distributed as observed in stages II, III and IV or they form complexes with other elements. The proteins are also deposited in all stages of the oocytes, however, in lower concentration in the stage IV. There is carbohydrate deposition from oocytes in the stage II as well as in stages III and IV. In addition, the present work has demonstrated that the oocyte yolk of A. triste has a glycolipoprotein nature and the elements are deposited in the following sequence: firstly the lipids and proteins, and finally the carbohydrates. (c) 2007 Elsevier Ltd. All rights reserved.

Chemical detection of the proteins and lipids in the fat body cells from workers of Attini ants (Hymenoptera: Formicidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)