High diagnostic efficiency of IgM-ELISA with the use of multiple antigen peptides (MAP1) from T. gondii ESA (SAG-1, GRA-1 and GRA-7), in acute toxoplasmosis

The main serological marker for the diagnosis of recent toxoplasmosis is the specific IgM antibody, along with IgG antibodies of low avidity. However, in some patients these antibodies may persist long after the acute/recent phase, contributing to misdiagnosis in suspected cases of toxoplasmosis. In the present study, the diagnostic efficiency of ELISA was evaluated, with the use of peptides derived from T. gondii ESA antigens, named SAG-1, GRA-1 and GRA-7. In the assay referred to, we studied each of these peptides individually, as well as in four different combinations, as Multiple Antigen Peptides (MAP), aiming to establish a reliable profile for the acute/recent toxoplasmosis with only one patient serum sample. The diagnostic performance of the assay using MAP1, with the combination of SAG-1, GRA-1 and GRA-7 peptides, demonstrated better discrimination of the acute/recent phase from non acute/recent phase of toxoplasmosis. Our results show that IgM antibodies to MAP1 may be useful as a serological marker, enhancing the diagnostic efficiency of the assay for acute/recent phase of toxoplasmosis.

Apoptosis y expresión de receptores de reconocimiento de patrones en Polimorfonucleares Neutrófilos de individuos con infección VIH/SIDA. Apoptosis and recognition patterns receptors expression in Polymorphonuclear Neutrophils of individuals with HIV/AIDS infection.

El Virus de la Inmunodeficiencia Humana tipo 1 (VIH-1) afecta principalmente a la respuesta inmune específica causando una pérdida progresiva de los linfocitos T CD4+. Sin embargo, este virus también afecta a células del sistema inmune innato, tales como los Polimorfonucleares Neutrófilos (PMN). Existen evidencias de alteraciones funcionales de los PMN durante la progresión de la infección por VIH y una de las explicaciones de estos defectos, la atribuye a una muerte celular programada o apoptosis constitutiva incrementada. El compromiso de la apoptosis de los PMN en la infección por VIH no está totalmente dilucidado, por ello, los objetivos de este proyecto son investigar el efecto de la infección por VIH sobre la apoptosis de PMN, analizar la expresión de moléculas y receptores de patrones de reconocimiento en estas células y evaluar el impacto de la terapia antirretroviral sobre la apoptosis y expresión de moléculas y receptores en PMN. Se incluirán individuos en distintos estadios clínicos e inmunológicos de la infección con o sin tratamiento antirretroviral y se determinarán parámetros hematológicos, inmunológicos y virológicos a fin de correlacionar el nivel de apoptosis y expresión de moléculas y receptores con el nivel de linfocitos T CD4+ y carga viral. La importancia de los PMN en el control de la infección por el VIH es actualmente un área de mucho interés, ya pueden ejercer un efecto anti-VIH directo, y al mismo tiempo, ser blancos de la infección viral. Los mecanismos que conducen a la muerte acelerada de los PMN no han sido totalmente dilucidados, por ello, su estudio permitirá entender las bases bioquímicas de los cambios morfológicos y determinar los mecanismos que definen su iniciación y regulación. En el presente proyecto, el estudio de la apoptosis de PMN de pacientes con infección VIH/SIDA posibilitará caracterizar la sobrevida de éstas células y su relación con el estado inmunológico, virológico y la terapia antirretroviral. Además, el estudio de los receptores reconocedores de patrones moleculares asociados a patógenos permitirá aclarar algunos aspectos de la activación de la respuesta inmune innata y su conexión con la inmunidad adaptativa. Comprender aspectos claves de la cascada de la apoptosis de PMN y de la expresión de receptores reconocedores de patrones moleculares en la infección VIH/SIDA podría en un futuro aportar posibles blancos terapéuticos para restaurar la función de estas células durante esta infección.

Role Of MMP-2 and MMP-9 in Resistance to Drug Therapy in Patients with Resistant Hypertension

AbstractBackground:Despite the increased evidence of the important role of matrix metalloproteinases (MMP-9 and MMP‑2) in the pathophysiology of hypertension, the profile of these molecules in resistant hypertension (RHTN) remains unknown.Objectives:To compare the plasma levels of MMP-9 and MMP-2 and of their tissue inhibitors (TIMP-1 and TIMP-2, respectively), as well as their MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios, between patients with controlled RHTN (CRHTN, n=41) and uncontrolled RHTN (UCRHTN, n=35). In addition, the association of those parameters with clinical characteristics, office blood pressure (BP) and arterial stiffness (determined by pulse wave velocity) was evaluate in those subgroups.Methods:This study included 76 individuals diagnosed with RHTN and submitted to physical examination, electrocardiogram, and laboratory tests to assess biochemical parameters.Results:Similar values of MMP-9, MMP-2, TIMP-1, TIMP-2, and MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios were found in the UCRHTN and CRHTN subgroups (P>0.05). A significant correlation was found between diastolic BP (DBP) and MMP-9/TIMP-1 ratio (r=0.37; P=0.02) and DPB and MMP-2 (r=-0.40; P=0.02) in the UCRHTN subgroup. On the other hand, no correlation was observed in the CRHTN subgroup. Logistic regression models demonstrated that MMP-9, MMP-2, TIMP-1, TIMP-2 and their ratios were not associated with the lack of BP control.Conclusion:These findings suggest that neither MMP-2 nor MMP-9 affect BP control in RHTN subjects.

Analysis of functional impairments of the human P2Y 11 nucleotide receptor with the alanine-87 - threonine mutation, and development of novel agonists specific for the human P2Y 11 and P2Y 6 receptors

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2015

Monoclonal antibodies to murine lipopolysaccharide (LPS)-binding protein (LBP) protect mice from lethal endotoxemia by blocking either the binding of LPS to LBP or the presentation of LPS/LBP complexes to CD14.

Cellular responses to LPS, the major lipid component of the outer membrane of Gram-negative bacteria, are enhanced markedly by the LPS-binding protein (LBP), a plasma protein that transfers LPS to the cell surface CD14 present on cells of the myeloid lineage. LBP has been shown previously to potentiate the host response to LPS. However, experiments performed in mice with a disruption of the LBP gene have yielded discordant results. Whereas one study showed that LBP knockout mice were resistant to endotoxemia, another study did not confirm an important role for LBP in the response of mice challenged in vivo with low doses of LPS. Consequently, we generated rat mAbs to murine LBP to investigate further the contribution of LBP in experimental endotoxemia. Three classes of mAbs were obtained. Class 1 mAbs blocked the binding of LPS to LBP; class 2 mAbs blocked the binding of LPS/LBP complexes to CD14; class 3 mAbs bound LBP but did not suppress LBP activity. In vivo, class 1 and class 2 mAbs suppressed LPS-induced TNF production and protected mice from lethal endotoxemia. These results show that the neutralization of LBP accomplished by blocking either the binding of LPS to LBP or the binding of LPS/LBP complexes to CD14 protects the host from LPS-induced toxicity, confirming that LBP is a critical component of innate immunity.

Autofluorescence Findings Of A Novel Maculopathy In Patient With Spinocerebellar Ataxia Type 1 And Of A Cone - Rod Type Retinal Dysfunction In Three Generation Family With Spinocerebellar Ataxia Type 7

Purpose: To report a novel maculopathy in a patient with SCA1. To describe autofluorescence findings in family with SCA7 and associated cone-rod retinal dysfunction.Methods: 4 affected patients from two families were assessed to investigate a progressive loss of visual acuity (VA). Examinations included fundus photography, autofluorescence (AF) fundus fluorescein angiogragraphy (FFA) and optical coherence tomography. Electroretinogram (full-field) was performed in 2 affected patients. All patients had color vision testing using Ishihara pseudoisochromatic plates. Molecular analysis was performed in family 2.Results: The patient with known diagnosis of SCA1 had a visual acuity of 20/200 bilaterally and dyschromatopsia. He had saccadic pursuit. Fundus examination showed mild retinal pigment epithelium (RPE) changes at the macula. OCT showed bilateral macular serous detachment, which was not obvious at the FFA and explained his VA. AF imaging showed a central hyperfluorescence. The 45 year old proband from family 2 had a visual acuity of 200/20 and dyschromatopsia. ERG testing showed cone type dysfunction of photoreceptors. Her daughter affected at a younger age had the same ERGs findings. Fundus examination showed mild RPE changes in proband, normal findings in her daughter. AF imaging of both patients showed a ring of high density AF around the fovea. The ring was also obvious on near infrared AF. Later onset of gait imbalance led to the diagnosis of SCA7Conclusions: Within the group of spinocerebellar ataxias, only the type 7 is associated with retinal dysfunction. We present the first report of maculopathy associated with SCA1 causing severe vision loss. The ring of high density AF in SCA7 confirmed an early retinal photoreceptor dysfunction in patient with normal fundus.

Low-frequency drug-resistant HIV-1 and risk of virological failure to first-line NNRTI-based ART: a multicohort European case-control study using centralized ultrasensitive 454 pyrosequencing.

OBJECTIVES: It is still debated if pre-existing minority drug-resistant HIV-1 variants (MVs) affect the virological outcomes of first-line NNRTI-containing ART. METHODS: This Europe-wide case-control study included ART-naive subjects infected with drug-susceptible HIV-1 as revealed by population sequencing, who achieved virological suppression on first-line ART including one NNRTI. Cases experienced virological failure and controls were subjects from the same cohort whose viraemia remained suppressed at a matched time since initiation of ART. Blinded, centralized 454 pyrosequencing with parallel bioinformatic analysis in two laboratories was used to identify MVs in the 1%-25% frequency range. ORs of virological failure according to MV detection were estimated by logistic regression. RESULTS: Two hundred and sixty samples (76 cases and 184 controls), mostly subtype B (73.5%), were used for the analysis. Identical MVs were detected in the two laboratories. 31.6% of cases and 16.8% of controls harboured pre-existing MVs. Detection of at least one MV versus no MVs was associated with an increased risk of virological failure (OR = 2.75, 95% CI = 1.35-5.60, P = 0.005); similar associations were observed for at least one MV versus no NRTI MVs (OR = 2.27, 95% CI = 0.76-6.77, P = 0.140) and at least one MV versus no NNRTI MVs (OR = 2.41, 95% CI = 1.12-5.18, P = 0.024). A dose-effect relationship between virological failure and mutational load was found. CONCLUSIONS: Pre-existing MVs more than double the risk of virological failure to first-line NNRTI-based ART.

Naturally acquired MAGE-A10- and SSX-2-specific CD8+ T cell responses in patients with hepatocellular carcinoma.

Immunotherapy is being proposed to treat patients with hepatocellular carcinoma (HCC). However, more detailed knowledge on tumor Ag expression and specific immune cells is required for the preparation of highly targeted vaccines. HCC express a variety of tumor-specific Ags, raising the question whether CTL specific for such Ags exist in HCC patients. Indeed, a recent study revealed CTLs specific for two cancer-testis (CT) Ags (MAGE-A1 and MAGE-A3) in tumor infiltrating lymphocytes of HCC patients. Here we assessed the presence of T cells specific for additional CT Ags: MAGE-A10, SSX-2, NY-ESO-1, and LAGE-1, which are naturally immunogenic as demonstrated in HLA-A2(+) melanoma patients. In two of six HLA-A2(+) HCC patients, we found that MAGE-A10- and/or SSX-2-specific CD8(+) T cells naturally responded to the disease, because they were enriched in tumor lesions but not in nontumoral liver. Isolated T cells specifically and strongly killed tumor cells in vitro, providing evidence that these CTL were selected in vivo for high avidity Ag recognition. Therefore, besides melanoma, HCC is the second solid human tumor with clear evidence for in vivo tumor recognition by T cells, providing the rational for specific immunotherapy, based on immunization with CT Ags such as MAGE-A10 and SSX-2.

Endothelial cell integrins and COX-2: mediators and therapeutic targets of tumor angiogenesis.

Vascular integrins are essential regulators and mediators of physiological and pathological angiogenesis, including tumor angiogenesis. Integrins provide the physical interaction with the extracellular matrix (ECM) necessary for cell adhesion, migration and positioning, and induce signaling events essential for cell survival, proliferation and differentiation. Integrins preferentially expressed on neovascular endothelial cells, such as alphaVbeta3 and alpha5beta1, are considered as relevant targets for anti-angiogenic therapies. Anti-integrin antibodies and small molecular integrin inhibitors suppress angiogenesis and tumor progression in many animal models, and are currently tested in clinical trials as anti-angiogenic agents. Cyclooxygense-2 (COX-2), a key enzyme in the synthesis of prostaglandins and thromboxans, is highly up-regulated in tumor cells, stromal cells and angiogenic endothelial cells during tumor progression. Recent experiments have demonstrated that COX-2 promotes tumor angiogenesis. Chronic intake of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors significantly reduces the risk of cancer development, and this effect may be due, at least in part, to the inhibition of tumor angiogenesis. Endothelial cell COX-2 promotes integrin alphaVbeta3-mediated endothelial cell adhesion, spreading, migration and angiogenesis through the prostaglandin-cAMP-PKA-dependent activation of the small GTPase Rac. In this article, we review the role of integrins and COX-2 in angiogenesis, their cross talk, and discuss implications relevant to their targeting to suppress tumor angiogenesis.

Association of Lifecourse Socioeconomic Status with Chronic Inflammation and Type 2 Diabetes Risk: The Whitehall II Prospective Cohort Study.

BACKGROUND: Socioeconomic adversity in early life has been hypothesized to "program" a vulnerable phenotype with exaggerated inflammatory responses, so increasing the risk of developing type 2 diabetes in adulthood. The aim of this study is to test this hypothesis by assessing the extent to which the association between lifecourse socioeconomic status and type 2 diabetes incidence is explained by chronic inflammation. METHODS AND FINDINGS: We use data from the British Whitehall II study, a prospective occupational cohort of adults established in 1985. The inflammatory markers C-reactive protein and interleukin-6 were measured repeatedly and type 2 diabetes incidence (new cases) was monitored over an 18-year follow-up (from 1991-1993 until 2007-2009). Our analytical sample consisted of 6,387 non-diabetic participants (1,818 women), of whom 731 (207 women) developed type 2 diabetes over the follow-up. Cumulative exposure to low socioeconomic status from childhood to middle age was associated with an increased risk of developing type 2 diabetes in adulthood (hazard ratio [HR] = 1.96, 95% confidence interval: 1.48-2.58 for low cumulative lifecourse socioeconomic score and HR = 1.55, 95% confidence interval: 1.26-1.91 for low-low socioeconomic trajectory). 25% of the excess risk associated with cumulative socioeconomic adversity across the lifecourse and 32% of the excess risk associated with low-low socioeconomic trajectory was attributable to chronically elevated inflammation (95% confidence intervals 16%-58%). CONCLUSIONS: In the present study, chronic inflammation explained a substantial part of the association between lifecourse socioeconomic disadvantage and type 2 diabetes. Further studies should be performed to confirm these findings in population-based samples, as the Whitehall II cohort is not representative of the general population, and to examine the extent to which social inequalities attributable to chronic inflammation are reversible. Please see later in the article for the Editors' Summary.

Stimulation-induced increases of astrocytic oxidative metabolism in rats and humans investigated with 1-11C-acetate

The purpose of this study was to investigate astrocytic oxidative metabolism using 1-(11)C-acetate. 1-(11)C-acetate kinetics were evaluated in the rat somatosensory cortex using a beta-scintillator during different manipulations (test-retest, infraorbital nerve stimulation, and administration of acetazolamide or dichloroacetate). In humans a visual activation paradigm was used and kinetics were measured with positron emission tomography. Data were analyzed using a one-tissue compartment model. The following features supported the hypothesis that washout of radiolabel (k(2)) is because of (11)C-CO(2) and therefore related to oxygen consumption (CMRO(2)): (1) the onset of (11)C washout was delayed; (2)k(2) was not affected by acetazolamide-induced blood flow increase; (3)k(2) demonstrated a significant increase during stimulation in rats (from 0.014+/-0.007 to 0.027+/-0.006 per minute) and humans (from 0.016+/-0.010 to 0.026+/-0.006 per minute); and (4) dichloroacetate led to a substantial decrease of k(2). In the test-retest experiments K(1) and k(2) were very stable. In summary, 1-(11)C-acetate seems a promising tracer to investigate astrocytic oxidative metabolism in vivo. If the washout rate indeed represents the production of (11)C-CO(2), then its increase during stimulation would point to a substantially higher astrocytic oxidative metabolism during brain activation. However, the quantitative relationship between k(2) and CMRO(2) needs to be determined in future experiments.

The Marmot Review: Updated data and information 2 years on

To mark the two year anniversary since The Marmot Review ('Fair Society, Healthy Lives') was published, on the 15th of February the UCL Institute of Health Equity published new data on key health inequalities indicators at local authority level in England.Main Findings:Life Expectancy – this has historically been one of the main indicators of health inequalities.The Marmot Indicators from this year’s charts show the average life expectancy for eachlocal authority and the level of inequality within each authority area (7):-While overall life expectancy at birth in England increased by 0.3 years for both menand women between 2007-9 and 2008-10, inequalities in life expectancy betweenneighbourhoods increased by 0.1 years for men and showed no change for women-Among the 150 upper tier local authorities in England, life expectancy improved inthe majority of cases (133 areas saw improvements for men and 125 sawimprovements for women). However inequalities also increased in the majority ofareas (104 for men and 92 for women).-The largest increase in inequality in life expectancy was in West Berkshire for men(2.0 years) and inMiddlesbrough for women (2 years). The largest decreases ininequality were in Kensington and Chelsea for both men and women (1.9 and 1.1years respectively. To find out more, please read: - The press release, including key figures and main findings. - A blog by Michael Marmot about the data and it's implications. - Press coverage of the data in national and local newspapers and websites. - A powerpoint presentation on the key findings.

Epithelial effects of proteinase-activated receptors in the gastrointestinal tract

The intestinal epithelium plays a crucial role in providing a barrier between the external environment and the internal milieu of the body. A compromised mucosal barrier is characteristic of mucosal inflammation and is a key determinant of the development of intestinal diseases such as Crohn's disease and ulcerative colitis. The intestinal epithelium is regularly exposed to serine proteinases and this exposure is enhanced in numerous disease states. Thus, it is important to understand how proteinase-activated receptors (PARs), which are activated by serine proteinases, can affect intestinal epithelial function. This review surveys the data which demonstrate the wide distribution of PARs, particularly PAR-1 and PAR-2, in the gastrointestinal tract and accessory organs, focusing on the epithelium and those cells which communicate with the epithelium to affect its function. PARs have a role in regulating secretion by epithelia of the salivary glands, stomach, pancreas and intestine. In addition, PARs located on subepithelial nerves, fibroblasts and mast cells have important implications for epithelial function. Recent data outline the importance of the cellular site of PAR expression, as PARs expressed on epithelia may have effects that are countered by PARs expressed on other cell types. Finally, PARs and their ability to promote epithelial cell proliferation are discussed in terms of colon cancer.

Re-mapping the molecular features of the human immunodeficiency virus type 1 and human T-cell lymphotropic virus type 1 Brazilian sequences using a bioinformatics unit established in Salvador, Bahia, Brazil, to give support to the viral epidemiology studies

The analysis of genetic data for human immunodeficiency virus type 1 (HIV-1) and human T-cell lymphotropic virus type 1 (HTLV-1) is essential to improve treatment and public health strategies as well as to select strains for vaccine programs. However, the analysis of large quantities of genetic data requires collaborative efforts in bioinformatics, computer biology, molecular biology, evolution, and medical science. The objective of this study was to review and improve the molecular epidemiology of HIV-1 and HTLV-1 viruses isolated in Brazil using bioinformatic tools available in the Laboratório Avançado de Sáude Pública (Lasp) bioinformatics unit. The analysis of HIV-1 isolates confirmed a heterogeneous distribution of the viral genotypes circulating in the country. The Brazilian HIV-1 epidemic is characterized by the presence of multiple subtypes (B, F1, C) and B/F1 recombinant virus while, on the other hand, most of the HTLV-1 sequences were classified as Transcontinental subgroup of the Cosmopolitan subtype. Despite the high variation among HIV-1 subtypes, protein glycosylation and phosphorylation domains were conserved in the pol, gag, and env genes of the Brazilian HIV-1 strains suggesting constraints in the HIV-1 evolution process. As expected, the functional protein sites were highly conservative in the HTLV-1 env gene sequences. Furthermore, the presence of these functional sites in HIV-1 and HTLV-1 strains could help in the development of vaccines that pre-empt the viral escape process.

High proportion of hepatitis C virus genotypes 1 and 3 in a large cohort of patients from Southern Brazil

Hepatitis C virus (HCV) isolates have been divided into six genotypes (1 to 6). The duration of hepatitis C standard treatment is 48 weeks for patients infected with HCV genotype 1 vs 24 weeks for those infected with genotypes 2 and 3. A total of 1544 HCV isolates from chronic patients living in the southern Brazilian states of Rio Grande do Sul (RS, n = 627) and Santa Catarina (SC, n = 917) were genotyped by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products. In RS, 338 (53.9%; 95% CI 50.0 - 57.8%), 34 (5.4%; 95% CI 3.8 - 7.4%) and, 255 (40.7%; 95% CI 36.9 - 44.6%) samples were from genotypes 1, 2, and 3, respectively. In SC, 468 (51%; 95% CI 47.8 - 54.2%), 26 (2.9%; 95% CI 1.9 - 4.1%) and, 423 (46.1%; 95% CI 42.9 - 49.3%) samples were from genotypes 1, 2, and 3, respectively. Genotyping results were confirmed by direct nucleotide sequencing of PCR products derived from 68 samples, without any discrepancy between PCR-RFLP and nucleotide sequencing methods. In conclusion, almost half of the hepatitis C patients from South of Brazil are infected by genotypes 2 and 3 and, these results have important consequential therapeutic implications as they can be treated for only 24 weeks, not 48.