Ultrastructural characterization of CD133(+) stem cells bound to superparamagnetic nanoparticles: possible biotechnological applications

CD133 antigen is an integral membrane glycoprotein that can bind with different cells. Originally, however. this cellular surface antigen was expressed in human stem cells and in various cellular progenitors of the haematopoietic system. Human cord blood has been described as an excellent source of CD133(+) haematopoietic progenitor cells with a large application potential. One of the main objectives of the present study is to describe for the first time the ultrastructural characteristics of CD133(+) stem cells using transmission electronic microscopy. Another objective of the manuscript is to demonstrate through transmission electronic microscopy the molecular image of magnetic nanoparticles connected to the stein cells of great biotechnological importance, as well as demonstrating the value of this finding for electronic paramagnetic resonance and its related nanobioscientific value. Ultrastructural results showed the monoclonal antibody anti-CD133 bound to the superparamagnetic nanoparticles by the presence of electrondense granules in cell membrane, as well as in the cytoplasm, revealing the ultrastructural characteristics of CD133(+) cells, exhibiting a round morphology with discrete cytoplasmic projections, having an active nucleus that follows this morphology. The cellular cytoplasm was filled up with mitochondrias, as well as microtubules and vesicles pinocitic. characterizing the process as being related to internalization of the magnetic nanoparticles that were endocyted by the cells in question. Electronic paramagnetic resonance analysis of the CD133(+) stem cells detected that the small (spectrum) generated by the labelled cells comes from the superparamagnetic nanoparticles that are bound to them. These results strongly suggest that these CD133(+) cells can be used in nanobiotechnology applications, with benefits in different biomedical areas.

Microvascular Lymphatic Density Analysis in Cutaneous Regressive and Nonregressive Superficial Spreading Melanomas Using the Lymphatic Marker D2-40

Background: The prognostic significance of spontaneous regression in melanoma, especially thin lesions, has been a controversial issue for the past 20 years, although recent studies suggest that extensive and late regression may be related to worse prognosis. Many data suggest that lymphangiogenesis predicts metastatic spread in melanoma. Methods: We have quantified lymphatic microvascular density (LMVD) in thin (<= 1.0 mm) superficial spreading melanomas comparing regressive and nonregressive melanomas, regressive and nonregressive areas from the same tumor, and early and late histological stages of regression in the same tumor. In addition, we tried to correlate lymphangiogenesis and tumor growth phase. We conducted histological examinations and immunohistochemical analyses using monoclonal antibody D2-40 with subsequent quantification by image analysis of 37 melanomas, 16 regressive and 21 nonregressive (controls). Results: We found higher LMVD in the late stage of regression compared with nonregressive area (internal control) of regressive melanomas. Conclusions: Our study suggest that the late stage of spontaneous regression in thin melanomas may be related to worse prognosis as it showed higher LMVD, and evidence shows that this is related with increased risk of metastatic spread. But this supposition must be confirmed by a longer follow-up for detection of lymph node metastases.

Atopic dermatitis in adults: evaluation of peripheral blood mononuclear cells proliferation response to Staphylococcus aureus enterotoxins A and B and analysis of interleukin-18 secretion

Atopic dermatitis (AD) is a chronic, inflammatory skin disease with a high prevalence and complex pathogenesis. The skin of AD patients is usually colonized by Staphylococcus aureus (S. aureus); its exotoxins may trigger or enhance the cutaneous inflammation. Several mediators are related to the AD immune imbalance and interleukin-18 (IL-18), an inflammatory cytokine, may play a role in the atopic skin inflammation. To evaluate peripheral blood mononuclear cells (PBMC) proliferation response to staphylococcal enterotoxins A (SEA) and B (SEB) and the levels of IL-18 in adults with AD. Thirty-eight adult patients with AD and 33 healthy controls were analysed. PBMC were stimulated with SEA and SEB, phytohemaglutinin (PHA), pokeweed (PWM), tetanus toxoid (TT) and Candida albicans (CMA). IL-18 secretion from PBMC culture supernatants and sera were measured by ELISA. A significant inhibition of the PBMC proliferation response to SEA, PHA, TT and CMA of AD patients was detected (P <= 0.05). Furthermore, increased levels of IL-18 were detected both in sera and non-stimulated PBMC culture supernatants from AD patients (P <= 0.05). A decreased PBMC proliferation response to distinct antigens and mitogens (TT, CMA, SEA and PHA) in adults with AD suggest a compromised immune profile. IL-18 secretion from AD upon stimulation was similar from controls, which may indicate a diverse mechanism of skin inflammation maintained by Staphylococcus aureus. On the other hand, augmented IL-18 secretion from AD sera and non-stimulated cell culture may enhance the immune dysfunction observed in AD, leading to constant skin inflammation.

Clinicopathological evaluation of in vivo epidermal nuclear fluorescence

Nuclear fluorescence in keratinocytes is an occasional phenomenon, often present in autoimmune diseases, especially in connective-tissue disease (CTD); however, its clinical significance remains unclear. To investigate the profile of patients with positive nuclear staining on direct immunofluorescence (DIF) of skin samples. A retrospective analysis of 28 patient records from our immunodermatology laboratory was performed between May 2003 and June 2006. Inclusion criteria were the presence of autoantibodies (IgG, IgA or IgM) or complement (C3) binding keratinocyte nuclei on DIF. The most prevalent diseases related to the nuclear keratinocyte DIF staining were systemic lupus erythematosus (n = 9), mixed CTD (n = 3), overlap syndrome (n = 3), Sjogren`s syndrome (n = 1), and CREST (calcinosis, Raynaud`s phenomenon, oesophageal dysmotility, sclerodactyly and telangiectasia) syndrome (n = 1). Serum antinuclear antibody (ANA) was positive in 20 of 28 patients, with titres varying from 1 : 160 to 1 : 1280. Of the 20 patients with positive anti-nuclear antibodies (ANA), 17 were positive for anti-extractable nuclear antigen antibodies, 12 had anti-SSA/Ro, 11 had anti-SSB/La and 8 had anti-ribonucleoprotein. Eight patients were negative for ANA. Positive predictive value of in vivo ANA for systemic CTDs was 75%. The present data suggest that in vivo ANA evaluation is an additional and feasible auxiliary tool for diagnosing CTDs.

Phylogenetic Analysis of Hepatitis B Virus Genotype F Complete Genome Sequences From Chilean Patients With Chronic Infection

Molecular epidemiological data concerning the hepatitis B virus (HBV) in Chile are not known completely. Since the HBV genotype F is the most prevalent in the country, the goal of this study was to obtain full HBV genome sequences from patients infected chronically in order to determine their subgenotypes and the occurrence of resistance-associated mutations. Twenty-one serum samples from antiviral drug-naive patients with chronic hepatitis B were subjected to full-length PCR amplification, and both strands of the whole genomes were fully sequenced. Phylogenetic analyses were performed along with reference sequences available from GenBank (n = 290). The sequences were aligned using Clustal X and edited in the SE-AL software. Bayesian phylogenetic analyses were conducted by Markov Chain Monte Carlo simulations (MCMC) for 10 million generations in order to obtain the substitution tree using BEAST. The sequences were also analyzed for the presence of primary drug resistance mutations using CodonCode Aligner Software. The phylogenetic analyses indicated that all sequences were found to be the HBV subgenotype F1b, clustered into four different groups, suggesting that diverse lineages of this subgenotype may be circulating within this population of Chilean patients. J. Med. Virol. 83: 1530-1536, 2011. (C) 2011 Wiley-Liss, Inc.

Diminished CD4+/CD25+T cell and increased IFN-gamma levels occur in dogs vaccinated with Leishmune (R) in an endemic area for visceral leishmaniasis

The Leishmune (R) vaccine has been used in endemic areas to prevent canine visceral leishmaniasis in Brazil, but cytokine production induced by vaccination has rarely been investigated in dogs. This study aimed to evaluate the immune response of dogs vaccinated with Leishmune FML vaccine (Fort Dodge) against total antigen of Leishmania (Leishmania) chagasi (TAg) and FML. Twenty healthy dogs from Aracatuba, Sao Paulo, Brazil, an endemic leishmaniasis area, received three consecutive subcutaneous injection of Leishmune vaccine at 21-day intervals. PBMC were isolated before and 10 days after completing vaccination and lymphoproliferative response and antibody production against FML or total promastigote antigen were tested. Cytokines IFN-gamma, IL-4 and TNF-alpha were measured in culture supernatant and CD4+/CD25+ and CD8+/CD25+ T cell presence was determined. Analysis of the data indicated that the vaccine conferred humoral responses (100%) against both antigens and cellular immunity to FML (85%) and total antigen (80%), the supernatant of cultured cells stimulated with TAg and FML showed an increase in IFN-gamma (P < 0.05), and the vaccine reduced CD4+/CD25+ T cell presence compared to that observed before vaccination. These responses may constitute part of the immune mechanism induced by Leishmune. (C) 2010 Elsevier B.V. All rights reserved.

Post-transplant recurrent hepatitis C: immunohistochemical detection of hepatitis C virus core antigen and possible pathogenic implications

Introduction: The mechanisms by which severe cholestatic hepatitis develops after liver transplantation are not fully understood. Reports on immunohistochemical distribution of hepatitis C virus (HCV) antigens are still scarce, but recently, HCV immunostaining was suggested for early diagnosis of cholestatic forms of recurrent hepatitis C in liver grafts. After purification, Rb246 pab anticore (aa1-68) yielded specific, granular cytoplasmic staining in hepatocytes. Signal amplification through the Envision-Alkaline Phosphatase System avoided endogenous biotin and peroxidase. Aims/Methods: Rb246 was applied to liver samples of explants of 12 transplant recipients, six with the most severe form of post-transplantation recurrence, severe cholestatic hepatitis (group 1) and six with mild recurrence (group 2). We also assessed immuno-reactivity at two time-points post-transplantation (median 4 and 22 months) in both groups. HCV-core Ag was semiquantified from 0 to 3+ in each time point. Serum HCV-RNA was also measured on the different time points by branched DNA. Results: In the early post-transplant time point, one patient had a mild staining (1+), two patients had a moderate staining (2+) and the other three had no staining in group 1, compared with five patients with no staining (0) and one patient with mild staining (1+) in group 2. Late post-transplant liver samples were available in nine patients, and two out of four samples in group 1 showed a mild staining, compared with no staining patients in five patients in group 2. Strikingly, on the explant samples, HCV immunostaining was strongly positive in group 1, and mildly positive in group 2. Two out of five samples showed 3+ staining, and three samples showed 2+ staining in group 1; two out of five samples showed no staining, two samples showed 1+ staining and one sample showed 2+ staining in group 2. Serum HCV-RNA was significantly higher in group 1, on both time-points post-transplantation. HCV-core Ag was not directly associated with serum HCV-RNA on the different time points. Conclusion: These preliminary results suggest that strong HCV immunostaining in the explant is predictive of more severe disease recurrence.

Atmospheric pollutants monitoring by analysis of epiphytic lichens

The Canoparmelia texana epiphytic lichenized fungi was used to monitor atmospheric pollution in the Sao Paulo metropolitan region, SP, Brazil. The cluster analysis applied to the element concentration values confirmed the site groups of different levels of pollution due to industrial and vehicular emissions. In the distribution maps of element concentrations, higher concentrations of Ba and Mn were observed in the vicinity of industries and of a petrochemical complex. The highest concentration of Co found in lichens from the Sao Miguel Paulista site is due to the emissions from a metallurgical processing plant that produces this element. For Br and Zn, the highest concentrations could be associated both to vehicular and industrial emissions. Exploratory analyses revealed that the accumulation of toxic elements in C. texana may be of use in evaluating the human risk of cardiopulmonary mortality due to prolonged exposure to ambient levels of air pollution. (c) 2007 Elsevier Ltd. All rights reserved.

Exacerbation of Leishmania (Viannia) shawi infection in BALB/c mice after immunization with soluble antigen from amastigote forms

The present study aimed to evaluate the effects of immunization with soluble amastigote (AmaAg) and promastigote (ProAg) antigens from Leishmania (Viannia) shawi on the course of infection in BALB/c mice. After immunization with AmaAg, the challenged group showed greater lesion size and parasite load in the skin and lymph nodes, associated with diminished interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-gamma and nitrate levels in the supernatant of lymph node cell cultures, together with increases in transforming growth factor (TGF)-beta concentrations and humoral immune response. In contrast, immunization with ProAg led to smaller lesion size with reduced numbers of viable parasites in the skin. Protection was associated with increases in IL-12, IFN-gamma, TGF-beta and nitrates and decreases in IL-4 and IL-10 levels. Concerning humoral immune response, a significant reduction in anti-leishmania immunoglobulin G was verified in the ProAg-challenged group. Analysis of these results suggests that AmaAg induced a suppressive cellular immune response in mice, favouring the spread of infection, whereas ProAg induced partial protection associated with increased cellular immune response.

Genetic Analysis of Age-at-Onset for Cardiovascular Risk Factors in a Brazilian Family Study

Background/Aims: Statistical analysis of age-at-onset involving family data is particularly complicated because there is a correlation pattern that needs to be modeled and also because there are measurements that are censored. In this paper, our main purpose was to evaluate the effect of genetic and shared family environmental factors on age-at-onset of three cardiovascular risk factors: hypertension, diabetes and high cholesterol. Methods: The mixed-effects Cox model proposed by Pankratz et al. [2005] was used to analyze the data from 81 families, involving 1,675 individuals from the village of Baependi, in the state of Minas Gerais, Brazil. Results: The analyses performed showed that the polygenic effect plays a greater role than the shared family environmental effect in explaining the variability of the age-at-onset of hypertension, diabetes and high cholesterol. The model which simultaneously evaluated both effects indicated that there are individuals which may have risk of hypertension due to polygenic effects 130% higher than the overall average risk for the entire sample. For diabetes and high cholesterol the risks of some individuals were 115 and 45%, respectively, higher than the overall average risk for the entire population. Conclusions: Results showed evidence of significant polygenic effects indicating that age-at-onset is a useful trait for gene mapping of the common complex diseases analyzed. In addition, we found that the polygenic random component might absorb the effects of some covariates usually considered in the risk evaluation, such as gender, age and BMI. Copyright (C) 2008 S. Karger AG, Basel

Simplified criteria for the diagnosis of autoimmune hepatitis

Diagnosis of autoimmune hepatitis (AIH) may be challenging. However, early diagnosis is important because immunosuppression is life-saving. Diagnostic criteria of the International Autoimmune Hepatitis Group (IAIHG) were complex and purely meant for scientific purposes. This study of the IAIHG aims to define simplified diagnostic criteria for routine clinical practice. Candidate criteria included sex, age, autoantibodies, immunoglobutins, absence of viral hepatitis, and histology. The training set included 250 AIH patients and 193 controls from 11 centers worldwide. Scores were built from variables showing predictive ability in univariate analysis. Diagnostic value of each score was assessed by the area under the receiver operating characteristic (ROC) curve. The best score was validated using data of an additional 109 AIH patients and 284 controls. This score included autoantibodies, immunoglobulin G, histology, and exclusion of viral hepatitis. The area under the curve for prediction of AIH was 0.946 in the training set and 0.91 in the validation set. Based on the ROC curves, two cutoff points were chosen. The score was found to have 88% sensitivity and 97% specificity (cutoff >= 6) and 81% sensitivity and 99% specificity (cutoff 2:7) in the validation set. Conclusion: A reliable diagnosis of AIH can be made using a very simple diagnostic score. We propose the diagnosis of probable AIH at a cutoff point greater than 6 points and definite AIH 7 points or higher.

Antigen-presenting cells in draining lymph nodes of goats repeatedly infested by the Cayenne tick Amblyomma cajennense nymphs

Resistance to tick feeding has been previously shown to be an acquired, immunologically mediated phenomenon in goats, associated with cutaneous basophilia to nymphs of Amblyomma cajennense, the Cayenne tick, after repeated infestations. On the other hand, it is well known that antigen-presenting cells (APCs) play an important role in the host immune reaction to tick infestations. The most able APCs for Th cells are the well defined dendritic cells, mononuclear phagocytes and B-lymphocytes. Immunohistochemical analysis of draining lymph nodes of goats repeatedly infested with nymphs of the ixodid tick A. cajennense to search for APCs was done. Pre-scapular lymph nodes draining the tick attachment sites were collected 15 days after both the first and third infestations. Tick infestations resulted in increased number of CD21(+) B lymphocytes in lymph nodes after the tertiary infestation. However, the number of CD11b(+) and CD11c(+) cells were not altered after the successive infestations. Lower numbers of CD11c(+) cells had infiltrated lymph nodes responsible for draining the tick infested skin. These findings suggest that acquired immunity of goats against nymphs of A. cajennense is possibly established by B lymphocytes during the first infestation and that APCs may play a key role in this mechanism.

Synthesis, spectroscopic characterization, DFT studies and biological assays of a novel gold(I) complex with 2-mercaptothiazoline

A new gold(I) complex with 2-mercaptothiazoline (MTZ) with the coordination formula [AuCN(C(3)H(5)NS(2))] was synthesized and characterized by chemical and spectroscopic measurements, OFT studies and biological assays. Infrared (IR) and (1)H, (13)C and (15)N nuclear magnetic resonance (NMR) spectroscopic measurements indicate coordination of the ligand to gold(I) through the nitrogen atom. Studies based on OFT confirmed nitrogen coordination to gold(I) as a minimum of the potential energy surface with calculations of the hessians showing no imaginary frequencies. Thermal decomposition starts at temperatures near 160 degrees C, leading to the formation of Au as the final residue at 1000 degrees C. The gold(I) complex with 2-mercaptothiazoline (Au-MTZ) is soluble in dimethyl sulfoxide (DMSO), and is insoluble in water, methanol, ethanol, acetonitrile and hexane. The antibacterial activities of the Au-MTZ complex were evaluated by an antibiogram assay using the disc diffusion method. The compound showed an effective antibacterial activity against Staphylococcus aureus (Gram-positive) and Escherichia coli and Pseudomonas aeruginosa (Gram-negative) bacterial cells. Biological analysis for evaluation of the cytotoxic effect of the Au-MTZ complex was performed using HeLa cells derived from human cervical adenocarcinoma. The complex presented a potent cytotoxic activity, inducing 85% of cell death at a concentration of 2.0 mu mol L(-1). (C) 2011 Elsevier Ltd. All rights reserved.

Molecular cloning, sequencing, and expression analysis of presenilin cDNA from Schistosoma mansoni

Presenilins (PS) are integral membrane proteins involved, among other functions, in regulated intramembrane proteolysis. In this study, we report the identification and characterization of a complementary DNA from Schistosoma mansoni exhibiting a significant homology to human and nonvertebrate presinilins. S. mansoni contained a 1,485 bp open reading frame encoding a predicted protein of 494 amino acids. Alignment of predicted amino acid sequence of S. mansoni with PS (SmPS) from other species revealed up to 40% similarity shared among the investigated organisms. In addition, phylogenetic analyses demonstrated SmPS being closely related to its orthologues found in Schistosoma japonicum and Caenorhabditis elegans. Expression analysis of SmPS using quantitative real-time PCR revealed that the transcript is up-regulated in the egg stage. We hypothesize that the high level of SmPS in the S. mansoni embryo correlates to an important role during cellular signaling associated to larval development. To our knowledge, this study represents the first attempt to investigate the existence and abundance of PS from a helminth parasite.

Major histocompatibility complex (MHC) class II but not MHC class I molecules are required for efficient control of Strongyloides venezuelensis infection in mice

P>Strongyloides stercoralis is an intestinal nematode capable of chronic, persistent infection and hyperinfection of the host; this can lead to dissemination, mainly in immunosuppressive states, in which the infection can become severe and result in the death of the host. In this study, we investigated the immune response against Strongyloides venezuelensis infection in major histocompatibility complex (MHC) class I or class II deficient mice. We found that MHC II(-/-) animals were more susceptible to S. venezuelensis infection as a result of the presence of an elevated number of eggs in the faeces and a delay in the elimination of adult worms compared with wild-type (WT) and MHC I(-/-) mice. Histopathological analysis revealed that MHC II(-/-) mice had a mild inflammatory infiltration in the small intestine with a reduction in tissue eosinophilia. These mice also presented a significantly lower frequency of eosinophils and mononuclear cells in the blood, together with reduced T helper type 2 (Th2) cytokines in small intestine homogenates and sera compared with WT and MHC I(-/-) animals. Additionally, levels of parasite-specific immunoglobulin M (IgM), IgA, IgE, total IgG and IgG1 were also significantly reduced in the sera of MHC II(-/-) infected mice, while a non-significant increase in the level of IgG2a was found in comparison to WT or MHC I(-/-) infected mice. Together, these data demonstrate that expression of MHC class II but not class I molecules is required to induce a predominantly Th2 response and to achieve efficient control of S. venezuelensis infection in mice.