EVALUATION OF IMMUNE MECHANISMS INVOLVED IN THE LACK OF CD4+ T CELL RECOVERY IN HIV-INFECTED PATIENTS NON RESPONDER TO ANTIRETROVIRAL THERAPY

INTRODUCTION HIV infection is characterized by a profound impairment of CD4+ T cell functionality with an immunological unbalance toward Th2 response during progression to AIDS. Antiretroviral therapy HAART suppresses viral replication and leads to the recovery of CD4+ T lymphocytes. 15-30% of HIV-infected HAART-treated patients are immunological non responder (INR) to therapy. Several factors are involved in this lack in CD4+ T cell recovery, our attention was focused on the excessive depletion of CD4+ T cells. Recently, hydroxychloroquine (HCQ), an anti-malarian drug, demonstrates to have antiviral and immune modulating effects. Aim of this study was to identify immune mechanisms involved in the lack of CD4+ T cell recovery observed in INR patients and to verify if HCQ might reduce immune activation typical of HIV infection thus leading to a recovery in CD4+ T cells. MATERIAL AND METHODS In the first part of this study, 67 HAART-treated HIV-infected patients were enrolled and stratified into two groups of the basis of CD4+ T cell counts (500cells/\ub5l) while in the second part of this study, analyses were conducted on 20 HAART-treated HIV-infected patients with an absolute CD4 count less than 200 cells/\ub5l during the last 12 months of therapy. Patients were treated with 400mg/die of HCQ and analyses were conducted at baseline, 6 months of therapy and 2 months after HCQ suspension. Immune suppression, immune activation and apoptosis were evaluated in flow cytometry. To identify microbial translocation, plasma LPS was measured with immunoenzymatic assay and TLR signaling pathway was analyzed in Real-Time PCR. Analyses were conducted in both unstimulated and stimulated (HIV-, CMV-, LPS-, ssRNA-specific) conditions. RESULTS AND DISCUSSION Patients characterized by CD4+ cell counts less than 500cells/\ub5l compared with individuals characterized by CD4+ cell counts more than 500cells/\ub5l showed: 1) increase percentage of Treg cells subpopulations and 2) apoptosis, 3) higher percentage of immune suppressive cytokine-expressing CD4+ T cells, 4) a higher percentage of KI67+ CD4+ T cells in both unstimulated and stimulated conditions. 5) an increased percentage of TLR expression on Treg cells and 6) a higher concentrations in plasma LPS. Data herein indicate that defective CD4+ cell counts recovery observed in patients characterized by CD4+ cell counts less than 500cells/\ub5l is associated with lower CD4+ nadir, gut microbial translocation and immune activation, augmented percentage and activity of Treg cells, and higher susceptibility to apoptosis. In the second part of the study, data showed a reduction in immune activation of CD4+ T cells, CD8+ T cells and monocytes. which was coupled with a reduction in plasma LPS concentration and of IL6 pro-inflammatory cytokines production. Moreover it has been shown the pivotal role played by plasmacytoid dendritic cells in the control of infection. Immune activation and immune suppression are strictly correlated, presence of immune suppressor state was well studied thanks to the analyses of both na\uefve and activated Treg lymphocytes, and TLR-expressing Treg cells: these populations were increased in percentage after 6 months of therapy. Several studies put in evidence HCQ effectiveness on TLR signaling pathway which influences immune activation. Flow cytometric analyses elucidate the reduction in TLR expressions by monocytes even in the presence of TLR4 and TLR7/8 agonists (LPS and ssRNA respectively). TLR signaling pathway was studied following PBMC stimulation with specific TLR agonists by Real-Time PCR: 6 months of HCQ treatment resulted in a reduction of TLR responsiveness and the effect was maintained also 2 months after HCQ suspension. CONCLUSIONS The lack in the recovery of CD4+ T lymphocytes characterizing immunological non responder patients to HAART is associated with a low CD4+ nadir, with immune activation due to an increase in microbial translocation and with immune suppression conducted by Treg cells through the induction of apoptosis mechanism and immune suppressor environment. The augment in Treg population might play a pivotal role in reducing immune activation and the increase of TLR-expressing Treg cells supports the effectiveness of a strong immune suppressive activity in controlling immune activation. Preliminary data suggest that HCQ plays a positive effect on CD4+ T cell recovery in immunological non responder patients to HAART and a control of immune activation, sustaining an application of this drug as immune modulating agent, in the treatment of HIV infection

Thermal imaging in the 3-5 micron range for precise localization of defects: Application on frescoes at the Sforza Castle

Infrared methods are of great importance in nondestructive testing of artworks, allowing a remote and wide-field imaging of interesting hidden features. Here we discuss a workflow based on thermal imaging in the mid infrared 3-5 micron range for the evaluation of subsurface defects in frescoes. Particular attention is payed to obtaining a high resolution (submillimetric) localization of the defects. The transfer of diagnostics techniques into real world applications, is discussed through the proof of concept of the proposed workflow on frescoes at the Sforza Castle (Milan, Italy)

Anisotropic osmosis filtering for shadow removal in images

We present an anisotropic extension of the isotropic osmosis model that has been introduced by Weickert et al.~(Weickert, 2013) for visual computing applications, and we adapt it specifically to shadow removal applications. We show that in the integrable setting, linear anisotropic osmosis minimises an energy that involves a suitable quadratic form which models local directional structures. In our shadow removal applications we estimate the local structure via a modified tensor voting approach (Moreno, 2012) and use this information within an anisotropic diffusion inpainting that resembles edge-enhancing anisotropic diffusion inpainting (Weickert, 2006, Gali\'c, 2008). Our numerical scheme combines the nonnegativity preserving stencil of Fehrenbach and Mirebeau (Fehrenbach, 2014) with an exact time stepping based on highly accurate polynomial approximations of the matrix exponential. The resulting anisotropic model is tested on several synthetic and natural images corrupted by constant shadows. We show that it outperforms isotropic osmosis, since it does not suffer from blurring artefacts at the shadow boundaries

Alternating Direction Implicit (ADI) schemes for a PDE-based image osmosis model

We consider \emph{Alternating Direction Implicit} (ADI) splitting schemes to compute efficiently the numerical solution of the PDE osmosis model considered by Weickert et al. for several imaging applications. The discretised scheme is shown to preserve analogous properties to the continuous model. The dimensional splitting strategy traduces numerically into the solution of simple tridiagonal systems for which standard matrix factorisation techniques can be used to improve upon the performance of classical implicit methods, even for large time steps. Applications to the shadow removal problem are presented

Hydroxychloroquine drastically reduces immune activation in HIV-infected, antiretroviral therapy-treated immunologic nonresponders

Despite optimal suppression of HIV replication, restoration of CD4(+) T cells is not always achieved in antiretroviral therapy-treated individuals. Defective CD4 recovery in immunologic nonresponders is possibly associated with TLR-mediated immune activation driven by alterations of gut permeability. Hydroxychloroquine (HCQ) reduces endosomal TLR signaling; thus, we verified whether HCQ could dampen immune activation and be associated with an increase in CD4(+) T cells. To this end, we enrolled in a prospective study 20 HIV-infected immunologic nonresponders (CD4 count < 200 cells/mL or CD4 increase < 5% in the last 12 months) who received 400 mg/day HCQ for 6 months. HCQ had a notable impact on immune activation as shown by significant modifications of the following parameters: (1) reduced plasma lipopolysaccharide; (2) decreased TLR4-expressing CD14(+) cells, TLR4-mediated signal transduction, and mRNA synthesis; (3) reduced percentages of activated CD4(+) (CD4(+)/Ki67(+)) and CD14(+) (CD14(+)/CD69(+)) cells; (4) increased T-regulatory cells (Tregs), naive Tregs, and TLR4-expressing Tregs; (5) augmented plasmacytoid dendritic cells and reduced IFN alpha-secreting plasmacytoid dendritic cells; and (6) reduced IL-6 and TNF alpha production. HCQ-induced immune modulation was associated with increased percentages of circulating CD4(+) T cells and was mostly retained 2 months after therapy interruption. HCQ reduces lipopolysaccharide/TLR-mediated immune activation; this compound could be a useful immunomodulant in HIV-infected patients. This study is registered at EutraCT as 2009-012499-28 with study number HLS01/2009-1-16-03-2009. (Blood. 2011;118(12):3263-3272

Cárie dentária e fatores relacionados em crianças Brasileiras de região com água fluoretada e não fluoretada

PURPOSE: To compare the caries prevalence, saliva buffering capacity (SBC), oral hygiene (OH), dietary habits, family income (FI) and frequency of visits to a dental office (Do) between Brazilian children living in areas with and without fluoridated public water supply. METHODS: Forty-six 5-7-year-old preschoolers were selected in Itatiba, SP, Brazil; 19 were from a fluoridated area, and 27 were from a non-fluoridated area. The caries index was determined according to the World Health Organization criteria, and the SBC was assessed by titration with hydrochloric acid. The FI, frequency of OH and visits to Do were estimated by questionnaire. The dietary habits were assessed with a diet chart. The differences between the groups were analyzed with Mann-Whitney-U tests (&#945;=0.05). RESULTS: Children from the non-fluoridated area showed significantly higher dmft/DMFT than those from the fluoridated area, but they showed significantly lower SBC, OH frequency and FI. No significant differences were observed between the areas for dietary habits and visits to Do. CONCLUSION: Children from fluoridated areas showed higher salivary buffering capacity, family income and oral hygiene frequency as well as lower caries prevalence, supporting the beneficial effect of fluoride in the tap water for caries prevention.OBJETIVO: Comparar prevalência de cárie, capacidade tamponante da saliva (CTS), higiene bucal (HB), hábitos dietéticos, renda familiar (RF) e frequência de visita a consultórios odontológicos (Co) entre crianças brasileiras residentes em áreas de água de abastecimento público fluoretadas e não fluoretadas. METODOLOGIA: Quarenta e seis crianças entre 5-7 anos foram selecionadas em Itatiba-SP-Brasil, sendo 19 pertencentes à área fluoretada e 27 à área não fluoretada. O índice de cárie foi determinado de acordo com o critério da Organização Mundial de Saúde e a CTS foi medida por titulação com ácido clorídrico. A RF, frequência de HB e visita ao Co foram estimadas por questionário. Os hábitos dietéticos foram avaliados com diário de dieta. As diferenças entre as variáveis foram analisadas pelo teste de Mann Whitney (&#945;=0,05). RESULTADOS: As crianças da área não fluoretada apresentaram significativamente maior ceod/CPOD que àquelas da área fluoretada, porém significativamente menor CTS, HB e RF. Com relação a hábitos dietéticos e visitas ao Co, não foi observada diferença significativa entre as áreas. CONCLUSÃO: As crianças da área fluoretada apresentaram maior capacidade tamponante da saliva, renda familiar e frequência de higiene bucal, assim como menor prevalência de cárie, reforçando o efeito benéfico do flúor nas águas de abastecimento para prevenir a cárie

Mitochondrial dysfunction mediates neuronal cell response to DMMB photodynamic therapy

Photodynamic therapy (PDT) is a process in which a photosensitizer (PS) is exposed to specific wavelengths and generates reactive oxygen species (ROS) which act within nanometers. The low invasive nature and directed cytotoxicity of this approach render it attractive to the treatment of different conditions, including the ones that affect the central nervous system (CNS). The effect of PDT on healthy neurons is one main concern over its use in the CNS, since neuronal-like cells were shown to be particularly sensitive to certain PSs. Among available PSs, 1,9-dimethyl-methylene blue (DMMB) stands out as being resistant to reduction to its inactive leuco form and by being able to produce high levels of singlet‑oxygen. In this study, we aimed to investigate DMMB photodamage mechanisms in the hippocampal cell line HT22. Our results demonstrate that DMMB-PDT decrease in cell viability was linked with an increase in cell death and overall ROS production. Besides, it resulted in a significant increase in mitochondrial ROS production and decreased mitochondria membrane potential. Furthermore, DMMB-PDT significantly increased the presence of acidic autolysosomes, which was accompanied by an increase in ATG1 and ATG8 homologue GaBarap1 expression, and decreased DRAM1 expression. Taken together our results indicated that mitochondrial and autophagic dysfunction underlie DMMB-PDT cytotoxicity in neuronal cells.</p

Ehrlich ascites tumor-bearing mice treated with aqueous ethanol plant extract from Euphorbia tirucalli showed signs of systemic toxicity

Purpose: To evaluate the antitumor effect of a latex extract from Euphorbia tirucalli Linn. (Euphorbiaceae) and its toxicity.Methods: Aqueous ethanol and petroleum ether extracts were obtained through maceration. .Maximum tolerated dose was determined in healthy mice. Antitumor activity was measured in Ehrlich ascites tumor-bearing mice treated with the extract through intraperitoneal injection (62.5, 125 or 250 mg/kg) every 48 h (four doses). Efficacy was assessed by weight gain, abdominal circumference, volume of ascitic fluid and packed tumor cells, tumor cell viability and survival. Toxicity indicators were serum glucose, triglycerides, total proteins, activity of alanine and aspartate aminotransferases and mass of heart, spleen, kidney and liver. A hemolysis assay was also performed.Results: Doses of 62.5 and 125 mg/kg caused no antitumor activity, while 250 mg/kg dose reduced weight gain (3-fold), abdominal circumference and volume of ascitic fluid (&gt; 50 %) and packed cells (50 %), but lowered tumor cell viability (40 %). However, mice treated with the extract survived for a shorter time than control mice. Furthermore, the 250 mg/kg dose caused cardiac atrophy, splenomegaly and fasting hyperglycemia. The extract caused hemolysis, and the half-maximal effective concentration (EC50) was 1.6 (0.9 – 2.7) mg/mL.Conclusion: Euphorbia tirucalli extract inhibits Ehrlich ascites tumor in mice, but the therapeutic dose is also harmful to non-tumor tissues.Keywords: Euphorbia tirucalli, Ehrlich ascites tumor-bearing mice, Antitumor, Toxicity, Cardiac atrophy, Splenomegal