Persistent T cell unresponsiveness associated with chronic visceral leishmaniasis in HIV-coinfected patients.

A large proportion of HIV-coinfected visceral leishmaniasis (VL-HIV) patients exhibit chronic disease with frequent VL recurrence. However, knowledge on immunological determinants underlying the disease course is scarce. We longitudinally profiled the circulatory cellular immunity of an Ethiopian HIV cohort that included VL developers. We show that chronic VL-HIV patients exhibit high and persistent levels of TIGIT and PD-1 on CD8+/CD8- T cells, in addition to a lower frequency of IFN-γ+ TIGIT- CD8+/CD8- T cells, suggestive of impaired T cell functionality. At single T cell transcriptome and clonal resolution, the patients show CD4+ T cell anergy, characterised by a lack of T cell activation and lymphoproliferative response. These findings suggest that PD-1 and TIGIT play a pivotal role in VL-HIV chronicity, and may be further explored for patient risk stratification. Our findings provide a strong rationale for adjunctive immunotherapy for the treatment of chronic VL-HIV patients to break the recurrent disease cycle.

Cellular Internalization and Toxicity of Chitosan Nanoparticles Loaded with Nobiletin in Eukaryotic Cell Models (Saccharomyces cerevisiae and Candida albicans).

This study involved the synthesis and characterization of chitosan nanoparticles loaded with nobiletin (CNpN) and assessed their toxicity and cellular internalization in eukaryotic cell models (Saccharomyces cerevisiae and Candida albicans). Nanoparticles were prepared via the nanoprecipitation method and physicochemically characterized to determine their hydrodynamic diameter using dynamic light scattering (DLS), their surface charge through ζ-potential measurements, and their chemical structure via Fourier-transform infrared spectroscopy (FTIR). The hydrodynamic diameter and ζ-potential of chitosan nanoparticles (CNp) and CNpN were found to be 288.74 ± 2.37 nm and 596.60 ± 35.49 nm, and 34.51 ± 0.66 mV and 37.73 ± 0.19 mV, respectively. The scanning electron microscopy (SEM) images displayed a particle size of approximately 346 ± 69 nm, with notable sphericity for CNpN. FTIR analysis provided evidence of potential imine bonding between chitosan and nobiletin. Membrane integrity damage could be observed in both S. cerevisiae and C. albicans yeast stained with propidium iodide, demonstrating membrane integrity damage caused by CNp and CNpN, where higher concentration treatments inhibited the development of yeast cells. These findings suggest a selective therapeutic potential of CNpN, which could be promising for the development of antifungal and anticancer therapies. This study contributes to understanding the interaction between nanoparticles and eukaryotic cells, offering insights for future biomedical applications.

Synthesis and toxicological study of chitosan-pirul (Schinus molle L.) essential oil nanoparticles on Aspergillus flavus.

In recent years, the study of essential oils as antifungal alternatives and their encapsulation to increase their properties for greater effects has been tested. In this work, nanoparticles of chitosan-Schinus molle L. essential oil (CS-PEO-Np) with a size of 260 ± 31.1 nm were obtained by ionic gelation and evaluated in some growth phases of Aspergillus flavus, a toxigenic fungus. At a concentration of 250 µg/mL of CS-PEO-Np, the A. flavus mycelial growth was inhibited at 97.1% with respect to control, at 96 h of incubation; the germination and viability of spores were inhibited at 74.8 and 40%, respectively, after exposure to 500 µg/mL of these nanomaterials, at 12 h of incubation. The fluorescence images of stained spores with DAPI showed the affectations caused by nanoparticles in the cell membrane, vacuoles and vacuolar content, cell wall, and nucleic acids. For both nanoparticles, CS-Np and CS-PEO-Np, no mutagenic effect was observed in Salmonella Typhimurium; also, the phytotoxic assay showed low-to-moderate toxicity toward seeds, which was dependent on the nanoparticle's concentration. The acute toxicity of CS-PEO-Np to A. salina nauplii was considered low in comparison to CS-Np (control), which indicates that the incorporation of Schinus molle essential oil into nanoparticles of chitosan is a strategy to reduce the toxicity commonly associated with nanostructured materials. The nanoparticulated systems of CS-PEO-Np represent an effective and non-toxic alternative for the control of toxigenic fungi such as A. flavus by delaying the initial growth stage.

Comparison of serum cytokine levels in symptomatic and asymptomatic HIV-Leishmania coinfected individuals from a Brazilian visceral leishmaniasis endemic area.

BACKGROUND: Visceral leishmaniasis (VL) remains an important infectious disease worldwide. VL-HIV coinfected individuals can present with atypical clinical forms of VL and have a high risk of VL relapse. Some cytokines have been described as potential markers to diagnose active VL and to predict the severity of the cases. However, few studies have included VL-HIV coinfected patients. We aimed to characterize the levels of several cytokines among VL-HIV coinfected individuals living in a VL-endemic area in Northeast Brazil. METHODS: This was a retrospective, cross-sectional study, aiming to estimate the levels of various cytokines in symptomatic and asymptomatic VL-HIV coinfected individuals. There were 134 study participants (35 symptomatic VL-HIV, 75 asymptomatic VL-HIV, and 24 healthy controls), all ≥ 18 years-old. Serum cytokine levels (interferon-γ, tumor necrosis factor, and interleukins 2, 4, 6, 10, and 17A) were quantified using the Becton Dickinson-BD's Cytometric Bead Array (CBA) system. RESULTS: The population mainly consisted of men (64.9%), with a median age of 35 (27-41) years. Asymptomatic individuals were younger (p = 0.013), with more years of education (p < 0.001), and were more often on antiretroviral therapy (p < 0.001) than those in the symptomatic group. Hemoglobin levels (p < 0.001), lymphocytes (p < 0.001) and CD4 count (p < 0.001) were lower in symptomatic individuals, while HIV viral loads were higher (p < 0.001). In the symptomatic VL-HIV coinfected group, we observed increased serum levels of IL-17A, IL-6, and IL-10 compared to asymptomatic patients and the healthy controls. There were no differences in the levels of all cytokines between asymptomatic VL-HIV coinfected individuals and the healthy controls. CONCLUSIONS: Higher serum levels of IL-17A, IL-6, and IL-10 cytokines were observed in symptomatic coinfected individuals but not in asymptomatically infected individuals. More studies among HIV-positive persons are needed to better understand the role of serum cytokines for prognosis, to define cure and predict VL relapses in VL-HIV coinfected individuals.

HIF2α regulates the synthesis and release of epinephrine in the adrenal medulla.

The adrenal gland and its hormones regulate numerous fundamental biological processes; however, the impact of hypoxia signaling on adrenal function remains poorly understood. Here, we reveal that deficiency of HIF (hypoxia inducible factors) prolyl hydroxylase domain protein-2 (PHD2) in the adrenal medulla of mice results in HIF2α-mediated reduction in phenylethanolamine N-methyltransferase (PNMT) expression, and consequent reduction in epinephrine synthesis. Simultaneous loss of PHD2 in renal erythropoietin (EPO)-producing cells (REPCs) stimulated HIF2α-driven EPO overproduction, excessive RBC formation (erythrocytosis), and systemic hypoglycemia, which is necessary and sufficient to enhance exocytosis of epinephrine from the adrenal medulla. Based on these results, we propose that the PHD2-HIF2α axis in the adrenal medulla regulates the synthesis of epinephrine, whereas in REPCs, it indirectly induces the release of this hormone. Our findings are also highly relevant to the testing of small molecule PHD inhibitors in phase III clinical trials for patients with renal anemia. KEY MESSAGES: HIF2α and not HIF1α modulates PNMT during epinephrine synthesis in chromaffin cells. The PHD2-HIF2α-EPO axis induces erythrocytosis and hypoglycemia. Reduced systemic glucose facilitates exocytosis of epinephrine from adrenal gland.

HIF1α is a direct regulator of steroidogenesis in the adrenal gland.

Endogenous steroid hormones, especially glucocorticoids and mineralocorticoids, derive from the adrenal cortex, and drastic or sustained changes in their circulatory levels affect multiple organ systems. Although hypoxia signaling in steroidogenesis has been suggested, knowledge on the true impact of the HIFs (Hypoxia-Inducible Factors) in the adrenocortical cells of vertebrates is scant. By creating a unique set of transgenic mouse lines, we reveal a prominent role for HIF1α in the synthesis of virtually all steroids in vivo. Specifically, mice deficient in HIF1α in adrenocortical cells displayed enhanced levels of enzymes responsible for steroidogenesis and a cognate increase in circulatory steroid levels. These changes resulted in cytokine alterations and changes in the profile of circulatory mature hematopoietic cells. Conversely, HIF1α overexpression resulted in the opposite phenotype of insufficient steroid production due to impaired transcription of necessary enzymes. Based on these results, we propose HIF1α to be a vital regulator of steroidogenesis as its modulation in adrenocortical cells dramatically impacts hormone synthesis with systemic consequences. In addition, these mice can have potential clinical significances as they may serve as essential tools to understand the pathophysiology of hormone modulations in a number of diseases associated with metabolic syndrome, auto-immunity or even cancer.

Asymptomatic immune responders to Leishmania among HIV positive patients.

Concomitant infection with human immunodeficiency virus (HIV) and the Leishmania parasite is a growing public health problem, the result of the former spreading to areas where the latter is endemic. Leishmania infection is usually asymptomatic in immunocompetent individuals, but the proportion of HIV+ individuals in contact with the parasite who remain asymptomatic is not known. The aim of the present work was to examine the use of cytokine release assays in the detection of asymptomatic immune responders to Leishmania among HIV+ patients with no previous leishmaniasis or current symptomatology. Eighty two HIV+ patients (all from Fuenlabrada, Madrid, Spain, where a leishmaniasis outbreak occurred in 2009) were examined for Leishmania infantum infection using molecular and humoral response-based methods. None returned a positive molecular or serological result for the parasite. Thirteen subjects showed a positive lymphoproliferative response to soluble Leishmania antigen (SLA), although the mean CD4+ T lymphocyte counts of these patients was below the normal range. Stimulation of peripheral blood mononuclear cells (PBMC) or whole blood with SLA (the lymphoproliferative assay and whole blood assay respectively), led to the production of specific cytokines and chemokines. Thus, despite being immunocompromised, HIV+ patients can maintain a Th1-type cellular response to Leishmania. In addition, cytokine release assays would appear to be useful tools for detecting these individuals via the identification of IFN-γ in the supernatants of SLA-stimulated PBMC, and of IFN-γ, MIG and IL-2 in SLA-stimulated whole blood. These biomarkers appear to be 100% reliable for detecting asymptomatic immune responders to Leishmania among HIV+ patients.

Short-Term Hypoxia Dampens Inflammation in vivo via Enhanced Adenosine Release and Adenosine 2B Receptor Stimulation.

Hypoxia and inflammation are closely intertwined phenomena. Critically ill patients often suffer from systemic inflammatory conditions and concurrently experience short-lived hypoxia. We evaluated the effects of short-term hypoxia on systemic inflammation, and show that it potently attenuates pro-inflammatory cytokine responses during murine endotoxemia. These effects are independent of hypoxia-inducible factors (HIFs), but involve augmented adenosine levels, in turn resulting in an adenosine 2B receptor-mediated post-transcriptional increase of interleukin (IL)-10 production. We translated our findings to humans using the experimental endotoxemia model, where short-term hypoxia resulted in enhanced plasma concentrations of adenosine, augmentation of endotoxin-induced circulating IL-10 levels, and concurrent attenuation of the pro-inflammatory cytokine response. Again, HIFs were shown not to be involved. Taken together, we demonstrate that short-term hypoxia dampens the systemic pro-inflammatory cytokine response through enhanced purinergic signaling in mice and men. These effects may contribute to outcome and provide leads for immunomodulatory treatment strategies for critically ill patients.

PHD2: from hypoxia regulation to disease progression.

Oxygen represents one of the major molecules required for the development and maintenance of life. An adequate response to hypoxia is therefore required for the functioning of the majority of living organisms and relies on the activation of the hypoxia-inducible factor (HIF) pathway. HIF prolyl hydroxylase domain-2 (PHD2) has long been recognized as the major regulator of this response, controlling a myriad of outcomes that range from cell death to proliferation. However, this enzyme has been associated with more pathways, making the role of this protein remarkably complex under distinct pathologies. While a protective role seems to exist in physiological conditions such as erythropoiesis; the picture is more complex during pathologies such as cancer. Since the regulation of this enzyme and its closest family members is currently considered as a possible therapy for various diseases, understanding the different particular roles of this protein is essential.

Lymphoproliferative response after stimulation with soluble leishmania antigen (SLA) as a predictor of visceral leishmaniasis (VL) relapse in HIV+ patients.

The introduction of HAART resulted in the decrease of Leishmania/HIV co-infection cases; nevertheless, the number of relapses remains high and secondary prophylaxis is recommended. However, secondary prophylaxis is not necessary in all patients, and presents a high risk of toxicity and an elevated cost. Our aim was to study whether specific cellular response to Leishmania infantum (measured by cell proliferation response after stimulation with soluble Leishmania antigen (SLA)), could be a useful tool to attempt a secondary prophylaxis withdrawal. In June 2009 an outbreak of leishmaniasis by Leishmania infantum was declared in the southeast of Madrid, and since January 2013, we recruited 10 HIV+ patients that had been treated for visceral leishmaniasis. 6 patients had positive SLA-cell proliferation test. The mean CD4 cell counts of those patients with positive SLA were 140 cel/mm3 and 40 cel/mm3 in those with negative SLA test. 3 patients with positive SLA-cell proliferation test (CD4 count: 336, 307, 625) were not on prophylaxis, and the other 3 patients (CD4 count: 152, 189, 359) were on secondary prophylaxis that was withdrawn after the positive SLA-cell proliferation test with no posterior relapses (mean follow up 60 weeks). From the 4 patients, which had negative SLA-cell proliferation test and continued on prophylaxis, 3 had positive PCR for Leishmania at the end of the follow-up and 2 presented clinical relapses. The performance of SLA-cell proliferation test can be a useful tool that can permit us to try withdrawal of the prophylaxis in Leishmania/HIV co-infected patients with low CD4+ counts under clinical supervision, diminishing risk of toxicity and cost.

Regulation of GLUT3 and glucose uptake by the cAMP signalling pathway in the breast cancer cell line ZR-75.

Increased glucose uptake as a principal energy source is a requirement for the continued survival of tumour cells. Facilitative glucose transporter-1 (GLUT1) and -3 (GLUT3) have been previously shown to be present and regulated in breast cancer cells and are associated with poor patient prognosis. In cancer cells, the cAMP secondary messenger pathway is known to potentiate described glucose transporter activators and regulate cell fate. However, no regulation of the glucose transporters in breast cancer cells by cAMP has previously been examined. Herein, we determined in the well-characterized breast cancer cell line ZR-75, if the cAMP analogue 8-br-cAMP was capable of regulating GLUT1 and GLUT3 expression and thus glucose uptake. We demonstrated that 8-br-cAMP transiently up-regulates GLUT3 mRNA levels. The use of actinomycin-D and the cloning of 1,200 bp upstream of the human GLUT3 promoter demonstrated that this regulation was transcriptional. Immunocytochemistry and Western blotting confirmed that the increase in mRNA was reflected by an increase in protein levels. No notable regulation of GLUT1 in the presence of 8-br-cAMP was detected. Finally, we determined using the non-metabolizable glucose analogue 2-DOG if this up-regulation in GLUT3 increased glucose uptake. We observed the presence of two uptake components, one corresponding to the Km of GLUT1/4 and the other to GLUT3. A doubling in the uptake velocity was observed only at the Km corresponding to GLUT3. In conclusion, we demonstrate and characterize for the first time, an up-regulation of GLUT3 mRNA, protein and glucose uptake by the cAMP pathway in breast cancer cells.

Crianças e adolescentes vivendo com HIV/Aids e suas famílias: aspectos psicossociais e enfrentamento / Children and adolescents living with HIV/Aids and their families: psychosocial aspects and coping

O estudo teve por objetivos: descrever aspectos sociodemográficos, médico-clínicos e da organização familiar de crianças e adolescentes soropositivos infectados pela transmissão vertical; descrever dificuldades e estressores percebidos pelos cuidadores sobre aspectos psicossociais e do tratamento para o HIV e analisar estratégias de enfrentamento utilizadas. Participaram 43 cuidadores primários, a maioria (N = 24) mães soropositivas; a idade variou de 18 a 68 anos. Os instrumentos incluíram entrevista semi-estruturada e a Escala Modos de Enfrentamento de Problemas (EMEP). Os resultados revelaram a presença de dificuldades em áreas como adesão ao tratamento, revelação do diagnóstico para a criança/adolescente e informação sobre o diagnóstico na escola. Quanto às estratégias de enfrentamento, houve predomínio de busca de práticas religiosas/pensamento fantasioso e focalização no problema, segundo escores da EMEP. O estudo indica a necessidade das equipes de saúde se qualificarem para atendimento a demandas psicossociais, visando atenção integral e interdisciplinar a familiares e crianças/adolescentes vivendo com HIV/aids.

The objectives of this study were to describe socio-demographic and medical aspects of children and adolescents who acquired HIV infection during the prenatal period. It also intends to describe de organizational aspects of their families; to delineate difficulties and stressors perceived by caregivers based upon psychosocial and HIV-related issues, so that it became able to analyze coping strategies utilized, one of the main objectives of this paper. Forty-three primary caregivers took part in this research, most of them were mothers infected with HIV (24), between the ages of 18 and 68. The instruments used included a semi-structured interview and the Ways of Coping with Problems Scale (WCPS). The results revealed difficulties in areas such as adherence to treatment, disclosure of diagnosis to children/adolescents and providing of information about the diagnosis at school. With reference to the coping strategies, there was a predominance of search for religious practices/fantastical thoughts and problem focusing, according to the scores of the WCPS. The study corroborates the necessity for health care professionals to strive to sharpen their professional expertise to attend psychosocial demands, endeavoring to devote interdisciplinary attention to children and adolescents living with HIV/AIDS, as well as their family members.

Differential regulation of glucose transporter expression by estrogen and progesterone in Ishikawa endometrial cancer cells.

Estrogen replacement therapy and other unopposed estrogen treatments increase the incidence of endometrial abnormalities, including cancer. However, this effect is counteracted by the co-administration of progesterone. In the endometrium, glucose transporter (GLUT) expression and glucose transport are known to fluctuate throughout the menstrual cycle. Here, we determined the effect of estrogen and progesterone on the expression of GLUT1-4 and on the transport of deoxyglucose in Ishikawa endometrial cancer cells. Cells were incubated with estrogen, progesterone or combined estrogen and progesterone for 24 h and the effect on the expression of GLUT1-4 and on deoxyglucose transport was determined. We show that GLUT1 expression is upregulated by estrogen and progesterone individually, but that combined estrogen and progesterone treatment reverses this increase. Hormonal treatments do not affect GLUT2, GLUT3 or GLUT4 expression. Transport studies demonstrate that estrogen increases deoxyglucose transport at Michaelis-Menten constants (Kms) corresponding to GLUT1/4, an effect which disappears when progesterone is added concomitantly. These data demonstrate that different hormonal treatments differentially regulate GLUT expression and glucose transport in this endometrial cancer cell line. This regulation mirrors the role played by estrogen and progesterone on the incidence of cancer in this tissue and suggests that GLUT1 may be utilized by endometrial cancer cells to fuel their demand for increased energy requirement.

Estrogen and progesterone up-regulate glucose transporter expression in ZR-75-1 human breast cancer cells.

Breast cancer incidence increases in women receiving combined estrogen and progesterone therapy. Breast tumors show increased expression of the glucose transporter GLUT1. We determined the effect of these hormones on GLUT1-4 expression and deoxyglucose transport in ZR-75-1 breast cancer cells. Immunoblotting, immunocytochemistry, flow cytometry, and RT-PCR showed that GLUT1 expression is up-regulated by progesterone and, to a greater degree, combined therapy. GLUT2 expression is unaffected by hormonal treatment. GLUT3 protein and RNA is up-regulated by progesterone and combined therapy, and GLUT4 protein expression is up-regulated by all hormonal treatments. Deoxyglucose transport studies revealed the presence of three transport components with characteristics corresponding to GLUT1/4, GLUT2, and GLUT3. 17beta-Estradiol produced a slight increase in transport at the Michaelis constant (Km) corresponding to GLUT3. Progesterone produced a small increase in transport at the Km corresponding to GLUT1/4, and combined 17beta-estradiol and progesterone produced a small increase in transport at the Km corresponding to GLUT3 and a large increase in transport at the Km corresponding to GLUT1/4. This indicates that 17beta-estradiol and progesterone differentially regulate GLUT1-4 expression and that these changes correlate to changes in glucose uptake. We postulate that combined hormone replacement therapy provides a survival advantage to developing ZR-75 breast cancer cells.

Effect of hormone replacement therapy on the bone mass and urinary excretion of pyridinium cross-links

CONTEXT: The menopause accelerates bone loss and is associated with an increased bone turnover. Bone formation may be evaluated by several biochemical markers. However, the establishment of an accurate marker for bone resorption has been more difficult to achieve. OBJECTIVE: To study the effect of hormone replacement therapy (HRT) on bone mass and on the markers of bone resorption: urinary excretion of pyridinoline and deoxypyridinoline. DESIGN: Cohort correlational study. SETTING: Academic referral center. SAMPLE: 53 post-menopausal women, aged 48-58 years. MAIN MEASUREMENTS: Urinary pyr and d-pyr were measured in fasting urine samples by spectrofluorometry after high performance liquid chromatography and corrected for creatinine excretion measured before treatment and after 1, 2, 4 and 12 months. Bone mineral density (BMD) was measured by dual energy X-ray absorptiometry (DEXA) before treatment and after 12 months of HRT. RESULTS: The BMD after HRT was about 4.7 percent (P < 0.0004); 2 percent (P < 0.002); and 3 percent (P < 0.01) higher than the basal values in lumbar spine, neck and trochanter respectively. There were no significant correlations between pyridinium cross-links and age, weight, menopause duration and BMD. The decrease in pyr and d-pyr was progressive after HRT, reaching 28.9 percent (P < 0.0002), and 42 percent (P < 0.0002) respectively after 1 year. CONCLUSIONS: Urinary pyridinoline and deoxypyridinoline excretion decreases early in hormone replacement therapy, reflecting a decrease in the bone resorption rate, and no correlation was observed with the bone mass evaluated by densitometry