Trypanosoma cruzi: identification of specific epimastigote antigens by human immune sera

Soluble antigens from epimastigotes of Trypanosoma cruzi were analyzed by western blot in terms of their reactivity with sera from patients with Chagas' disease. In addition, sera from patients with visceral (AVL) and tegumental leishmaniasis (ATL) were also tested in order to identify cross-reactivities with Trypanosoma cruzi antigens. Twenty eight polypeptides with molecular weights ranging from 14 kDa to 113 kDa were identified with sera from Chagas' disease patients. An extensive cross-reactivity was observed when sera from human visceral leishmaniasis were used, while only a slight cross-reaction was observed with sera from tegumental leishmaniasis. On the other hand, 10 polypeptides specifically reacting with sera from Chagas' disease patients were identified. Among them, the antigens with molecular weights of 46 kDa and 25 kDa reacted with all sera tested and may be good candidates for specific immunodiagnosis of Chagas' disease.     

蚕豆叶片发育与衰老过程中超氧物歧化酶活性与丙二醛含量变化

蚕豆植株叶片随茎节自上而下表现出明显的发育与衰老顺序,可作为衰老特征的是叶绿素和蛋白质含量明显下降。蚕豆叶中SOD活性主要定位于12 000× g离心后所得的上清液和叶绿体组分。衰老叶片的SOD总活性和叶绿体组分的相对活性都有所下降,SOD同工酶谱也发生了改变。O_2~ 产生速率随叶龄增大而稍上升;而MDA含量在叶片外观表现枯黄衰老征兆前就急剧上升。可能因为衰老叶片过氧化氢酶活性大幅度下降与SOD之间的不平衡,致使O_2~ 代谢中间产物累积而引起膜的损伤.     

Prevalence, biotypes, plasmid profile and antimicrobial resistance of Campylobacter isolated from wild and domestic animals from northeast Portugal.

The incidence of Campylobacter jejuni and Campylobacter coli in wild and producing animals has been studied to evaluate their importance as potential reservoirs of campylobacter infection. These organisms were isolated from: 59 chicken (60.2%), 65 swine (59.1%), 31 black rats (57.4%), 61 sparrows (45.5%), 21 ducks (40.5%), 32 cows (19.5%) and 27 sheep (15.3%). Biotypes, plasmid and resistance profiles were studied in order to characterize the isolates. Biotypes I and II of C. jejuni were predominant in all reservoirs except swine, where C. coli I was more frequent. Plasmid prevalence was higher in strains isolated from swine (53.8%) and rats (45.5%). The size of the plasmids ranged from 1.3 to 82 MDa. A 2.3 MDa plasmid was the most frequent, detected in all the reservoirs except ducks. Antimicrobial susceptibility testing revealed that 5.5% of the strains were resistant to ampicillin, 5.5% to tetracycline, 12.6% to erythromycin and 23.5% to streptomycin. Resistance to erythromycin (26.2%) and to streptomycin (58.4%) was particularly high in isolates from swine. Tetracycline resistance was encoded by a 33 or a 41 MDa plasmid and transferred by conjugation.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

TLR and NKG2D Signaling Pathways Mediate CS-Induced Pulmonary Pathologies

Long-term exposure to cigarette smoke (CS) can have deleterious effects on lung epithelial cells including cell death and the initiation of inflammatory responses. CS-induced cell injury can elaborate cell surface signals and cellular byproducts that stimulate immune system surveillance. Our previous work has shown that the expression of ligands for the cytotoxic lymphocyte activating receptor NKG2D is enhanced in patients with COPD and that the induction of these ligands in a mouse model can replicate COPD pathologies. Here, we extend these findings to demonstrate a role for the NKG2D receptor in CS-induced pathophysiology and provide evidence linking nucleic acid-sensing endosomal toll-like receptor (TLR) signaling to COPD pathology through NKG2D activation. Specifically, we show that mice deficient in NKG2D exhibit attenuated pulmonary inflammation and airspace enlargement in a model of CS-induced emphysema. Additionally, we show that CS exposure induces the release of free nucleic acids in the bronchoalveolar lavage and that direct exposure of mouse lung epithelial cells to cigarette smoke extract similarly induces functional nucleic acids as assessed by TLR3, 7, and 9 reporter cell lines. We demonstrate that exposure of mouse lung epithelial cells to TLR ligands stimulates the surface expression of RAET1, a ligand for NKG2D, and that mice deficient in TLR3/7/9 receptor signaling do not exhibit CS-induced NK cell hyperresponsiveness and airspace enlargement. The findings indicate that CS-induced airway injury stimulates TLR signaling by endogenous nucleic acids leading to elevated NKG2D ligand expression. Activation of these pathways plays a major role in the altered NK cell function, pulmonary inflammation and remodeling related to long-term CS exposure.     

T Cell Activation and Cytokine Profile of Tuberculosis and HIV-Positive Individuals during Antituberculous Treatment and Efavirenz-Based Regimens

Introduction

The profile of immune activation markers in tuberculosis and HIV-infected patients is already known. The impact of simultaneous infections on the immune parameters is still not fully explored.

Methods

We conducted a prospective study to estimate trajectories of activated T cell subsets and the profile of anti- and pro-inflammatory cytokines in a group of HIV-TB individuals, previously naïve for HAART, recruited from a randomized clinical trial during TB treatment and first antiretroviral therapy with efavirenz. Patients were evaluated according to the immunosuppression levels at baseline as group 1 (CD4<200 cells/mm³) and group 2 (CD4>200 cells/mm³). These parameters were measured at the time of HAART initiation (started about 30 days after the onset of TB treatment) and at the follow-up visits after 30, 60, 90 and 180 days. Trajectories were estimated using least squares estimates of the coefficients of a restricted cubic spline function in time after adjusting for subject effects, bootstrapping it 500 times.

Results

Increase of CD4 T cell counts and suppression of HIV viral load were observed for all patients under HAART and TB treatment. Descendent trajectories were observed for the activated CD8⁺/CD38⁺ and CD3⁺/HLA-DR⁺ T cell subsets, and for plasma concentration of gamma- interferon (IFN-γ). Except for TNF-α and IL-2 discrete variations were observed for the other cytokines. Differences in the trajectories of these parameters were observed for groups 1 and 2. Higher values of IFN-γ, IL-2, IL-6 and IL-10 were observed for group 1 from the baseline to two months after treatment initiation, whereas reduced levels of TNF-α were observed for this group between 60 and 120 days of HAART.

Conclusion

Independent of the immunosuppression profile at baseline, HIV-TB patients under HAART were able to recover the CD4⁺ T cell counts, and control viral replication and immune activation parameters over time.     

Death receptors and mitochondria: Two prime triggers of neural apoptosis and differentiation

Background

Stem cell therapy is a strategy far from being satisfactory and applied in the clinic. Poor survival and differentiation levels of stem cells after transplantation or neural injury have been major problems. Recently, it has been recognized that cell death-relevant proteins, notably those that operate in the core of the executioner apoptosis machinery are functionally involved in differentiation of a wide range of cell types, including neural cells.

Scope of review

This article will review recent studies on the mechanisms underlying the non-apoptotic function of mitochondrial and death receptor signaling pathways during neural differentiation. In addition, we will discuss how these major apoptosis-regulatory pathways control the decision between differentiation, self-renewal and cell death in neural stem cells and how levels of activity are restrained to prevent cell loss as final outcome.

Major conclusions

Emerging evidence suggests that, much like p53, caspases and Bcl-2 family members, the two prime triggers of cell death pathways, death receptors and mitochondria, may influence proliferation and differentiation potential of stem cells, neuronal plasticity, and astrocytic versus neuronal stem cell fate decision.

General significance

A better understanding of the molecular mechanisms underlying key checkpoints responsible for neural differentiation as an alternative to cell death will surely contribute to improve neuro-replacement strategies.     

Dual on-off and off-on switchable oligoaziridine biosensor

A water-soluble biocompatible aziridine-based biosensor with pendant anthracene units was synthesized by radicalar polymerization of N-substituted aziridines in supercritical carbon dioxide. The binding ability of the sensor towards a series of metal ions was examined by comparing the fluorescence intensities of the solutions before and after the addition of 100 equivalents of a solution of the metal ion chloride salt. A fast, simple and highly optical sensitive dual behavior, "off-on" and "on-off" response, was observed after the biosensor was exposed to the metal cations in aqueous solution. Zinc presented the highest fluorescence enhancement (turn-on) and copper presented the highest fluorescence quenching (turn-off). The response time was found to be instantaneous and the detection limit was achieved even in the presence of excess metal cation competitors. By using immunofluorescence microscopy it was also shown that oligoaziridine acts as an "on-off" probe through highly sensitive (detection limit of 1.6nM), selective and reversible binding to copper anions under physiologic conditions using living Human Fibroblast cells. The stoichiometry for the reaction of the biosensor with Cu(2+) was determined by a Job plot and indicates the formation of an oligoaziridine-Cu(2+) 1:2 adduct.     

Polymorphism of the Human Immunodeficiency Virus Type 1 in Brazil: genetic characterization of the nef gene and implications for vaccine design

Most of the Brazilian HIV-1 samples have been characterized based on the structural genes (env, gag and pol) and no data concerning the variability of the accessory genes such as nef have been available so far. Considering the role of the nef on virus biology and the inclusion of this region in some HIV/AIDS vaccine products under testing, the purpose of this study was to document the genetic diversity of the nef gene in third-four HIV-1 Brazilian samples previously subtyped based on the env C2-V3 region. Although only few non-subtype B samples have already been analyzed so far, the cytotoxic Tlymphocyte epitopes encoded in this region were relatively conserved among the subtypes, with some amino acid signatures mainly in the subtype C samples. Considering the increasing of the non-B HIV-1 subtypes worldwide, in special the subtype C, more data should be generated concerning the genetic and antigenic variability of these subtypes, as well as the study of the impact of such polymorphism in HIV/AIDS vaccine design and testing.