Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

Trypanosoma cruzi, etiological agent of Chagas’ disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas’ disease.     

Interaction of medroxyprogesterone acetate with cytosol androgen receptors in the rat hypothalamus and pituitary

The binding of medroxyprogesterone acetate (MPA) with cytosol androgen receptors from rat pituitary and hypothalamus was studied. The pituitary and hypothalamic cytosol androgen receptors from adult castrated female rats were in vitro labeled using 3H natural (testosterone (T) and 5 alpha-dihydrotestosterone (DHT] and [3H]synthetic (methyltrienolone) androgens as radioligands. The [3H]androgen-receptor complexes sedimented with a coefficient of 8S in linear sucrose gradients. When incubated with an excess of radioinert MPA, specific binding was abolished indicating interaction of MPA with androgen receptors. Furthermore specific [3H]MPA-androgen cytosol receptor complexes could be identified in these neuroendocrine tissues when a post-gradient receptor labeling technique was used in the absence or presence of radioinert MPA, DHT, and triamcinolone acetonide. A study of binding kinetics disclosed that the equilibrium dissociation constant and saturation binding capacity for the MPA binder, were similar to those exhibited by DHT binding to androgen receptors in both studied tissues under identical experimental conditions. The overall results were interpreted as demonstrating that MPA interacts with cytosol steroid receptors other than those of progesterone in the rat hypothalamus and anterior pituitary. The data are consistent with MPA binding to androgen receptors.     

Genetic polymorphism of the human sex hormone-binding globulin: evidence of an isoelectric focusing variant with normal androgen-binding affinities

Human sex hormone-binding globulin (hSHBG) is a plasma glycoprotein composed of two identical subunits. The protein, which has high affinity for testosterone and estradiol has been purified to homogeneity. In this study we have investigated, on neuraminidase-treated serum samples, the presence of genetic variations of hSHBG by polyacrylamide gel isoelectric focusing (IEF). Based on IEF analyses of 110 serum samples from adult Mexican individuals we have identified two distinct IEF-patterns. The most frequent phenotype (95.45%) was characterized by two IEF-bands with pIs of 6.50 and 6.63, respectively. In five serum samples, a different 4-band pattern with pIs of 6.50, 6.63, 6.70 and 6.76 was identified. Family studies showed that this pattern was genetically determined. The frequency of this variant was 4.55%, and the observed phenotypes were consistent with the expression of an autosomal genetic system. The estimated gene frequencies for both alleles were shown to be in genetic equilibrium. Affinity constants, binding kinetics and serum concentrations of hSHBG from individuals having a 4-band pattern were similar to those obtained in individuals with a 2-band pattern, thus suggesting that the mechanism responsible for the generation of polymorphic variants of hSHBG reported herein did not involve the steroid binding site of the molecule. These findings may be of broad interest, as other serum binding proteins express genetic variants, which may permit their further structural and functional subclassification.     

Calcitriol downregulates TNF-α and IL-6 expression in cultured placental cells from preeclamptic women

Placenta is an important source and target of hormones that contribute to immunological tolerance and maintenance of pregnancy. In preeclampsia (PE), placental calcitriol synthesis is low; whereas pro-inflammatory cytokines levels are increased, threatening pregnancy outcome. Previously, we showed that calcitriol inhibits Th-1 cytokines under experimental inflammatory conditions in normal trophoblasts. However, a study of the regulation of inflammatory cytokines by calcitriol in trophoblasts from a natural inflammatory condition, such as PE, is still lacking. Therefore, the aim of the present study was to investigate calcitriol effects upon TNF-α, IFN-γ, IL-6 and IL-1β in cultured placental cells from preeclamptic women by using qPCR and ELISA. Placentas were collected after cesarean section from preeclamptic women and enriched trophoblastic preparations were cultured in the absence or presence of different calcitriol concentrations during 24 h. In these cell cultures, pro-inflammatory cytokines TNF-α and IL-6 secretion and mRNA expression were downregulated by calcitriol (P < 0.05). No significant effects of calcitriol upon IFN-γ and IL-1β were observed. In addition, basal expression of TNF-α, IL-6 and IL-1β decreased as the cells formed syncytia. Our study supports an important autocrine/paracrine role of placental calcitriol in controlling adverse immunological responses at the feto–maternal interface, particularly in gestational pathologies associated with exacerbated inflammatory responses such as preeclampsia.     

Identification of a Wee1–Like Kinase Gene Essential for Procyclic Trypanosoma brucei Survival

Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases (CDKs). Activation of the cyclin B-cdc2 kinase complex is a pivotal step in mitotic initiation and the tyrosine kinase Wee1 is a key regulator of cell cycle sequence during G2/M transition and inhibits mitotic entry by phosphorylating the inhibitory tyrosine 15 on the cdc2 M-phase-inducing kinase. Wee1 degradation is essential for the exit from the G2 phase. In trypanosomatids, little is known about the genes that regulate cyclin B-cdc2 complexes at the G2/M transition of their cell cycle. Although canonical tyrosine kinases are absent in the genome of trypanosomatids, phosphorylation on protein tyrosine residues has been reported in Trypanosoma brucei. Here, we characterized a Wee1-like protein kinase gene from T. brucei. Expression of TbWee1 in a Schizosaccharomyces pombe strain null for Wee1 inhibited cell division and caused cell elongation. This demonstrates the lengthening of G2, which provided cells with extra time to grow before dividing. The Wee1-like protein kinase was expressed in the procyclic and bloodstream proliferative slender forms of T. brucei and the role of Wee1 in cell cycle progression was analyzed by generating RNA interference cell lines. In the procyclic form of T. brucei, the knock-down of TbWee1 expression by RNAi led to inhibition of parasite growth. Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines. Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase. We also showed that TbWee1 is an essential gene necessary for proper cell cycle progression and parasite growth in T. brucei. Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M.     

Immunologic evaluation and validation of methods using synthetic peptides derived from Mycobacterium tuberculosis for the diagnosis of tuberculosis infection

The goal of this study was to demonstrate the usefulness of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB). This assay used 20 amino acid-long, non-overlapped synthetic peptides that spanned the complete Mycobacterium tuberculosis ESAT-6 and Ag85A sequences. The validation cohort consisted of 1,102 individuals who were grouped into the following five diagnostic groups: 455 patients with PTB, 60 patients with EPTB, 40 individuals with non-EPTB, 33 individuals with leprosy and 514 healthy controls. For the PTB group, two ESAT-6 peptides (12033 and 12034) had the highest sensitivity levels of 96.9% and 96.2%, respectively, and an Ag85A-peptide (29878) was the most specific (97.4%) in the PTB groups. For the EPTB group, two Ag85A peptides (11005 and 11006) were observed to have a sensitivity of 98.3% and an Ag85A-peptide (29878) was also the most specific (96.4%). When combinations of peptides were used, such as 12033 and 12034 or 11005 and 11006, 99.5% and 100% sensitivities in the PTB and EPTB groups were observed, respectively. In conclusion, for a cohort that consists entirely of individuals from Venezuela, a multi-antigen immunoassay using highly sensitive ESAT-6 and Ag85A peptides alone and in combination could be used to more rapidly diagnose PTB and EPTB infection.     

Melatonin attenuates the effects of sub-acute administration of lead on kidneys in rats without altering the lead-induced reduction in nitric oxide

Exposure to lead induces oxidative stress and renal damage. Although most forms of oxidative stress are characterized by simultaneous elevation of nitrogen and oxidative species, lead-induced oxidative stress is unusual in that it is associated with a reduction in nitric oxide (NO) levels in the kidney. The role of NO in kidney injury is controversial; some studies suggest that it is associated with renal injury, whereas others show that it exerts protective effects. Concentration-dependent effects have also been proposed, linking low levels with vasodilatation and high levels with toxicity. The aim of this study was to evaluate the effects of melatonin co-exposure on the lead-induced reduction in renal NO levels. We found that sub-acute intraperitoneal administration of 10 mg/kg/day of lead for 15 days induced toxic levels of lead in the blood and caused renal toxicity (pathological and functional). Under our experimental conditions, lead induced an increase in lipid peroxidation and a decrease in NO. Melatonin co-treatment decreased lead-induced oxidative stress (peroxidation level) and toxic effects on kidneys without altering the lead-induced reduction in renal NO. These results suggest that, in our experimental model, the reduction in renal NO levels by lead exposure is not the only responsible factor for lead-induced kidney damage.     

Effect of insulin on exocrine pancreatic secretion in healthy and diabetic anaesthetised rats

This investigation characterised the effects of exogenous insulin on exocrine pancreatic secretion in anaesthetised healthy and diabetic rats. Animals were rendered diabetic by a single injection of streptozotocin (STZ, 60 mg kg(-1) I.P.). Age-matched controls were injected citrate buffer. Rats were tested for hyperglycaemia 4 days after STZ injection and 7-8 weeks later when they were used for the experiments. Following anaesthesia (1 g kg(-1) urethane I.P.), laparotomy was performed and the pancreatic duct cannulated for collection of pure pancreatic juice. Basal pancreatic juice flow rate in diabetic rats was significantly (p < 0.001) increased whereas protein and amylase outputs were significantly (p < 0.001) decreased compared to control rats. Insulin (1 IU, I.P.) produced in healthy rats significant increases in pancreatic flow rate, amylase secretion and protein output compared to basal (p < 0.05). Insulin action also included a reduction in blood glucose (152.7 +/- 16.9 mg dl(-1), n = 6, prior to insulin and 42.0 +/- 8.4 mg dl(-1), n = 4, 100 min later). In fact, flow rate and glycaemia showed a strong negative correlation (p < 0.01, Pearson). Pretreatment with atropine (0.2 mg kg(-1), I.V.) abolished the effects of insulin on secretory parameters despite a similar reduction in glycaemia; in this series of experiments the correlation between flow rate and blood glucose was lost. In diabetic rats, insulin (4 IU, I.P.) did not modify exocrine pancreatic secretion. There was a fall in blood glucose (467.6 +/- 14.0 mg dl(-1), n = 10, prior to insulin and 386.6 +/- 43.6 mg dl(-1), n = 7, 120 min later). Rats, however, did not become hypoglycaemic. Similar results were observed in diabetic atropinized rats. The results of this study indicate that the effects of insulin on exocrine pancreatic secretion in anaesthetised healthy rats are mediated by hypoglycaemia-evoked vagal cholinergic activation.     

Differences in nocturnal basal and rhythmic prolactin secretion in untreated compared to treated HIV-infected men are associated with CD4+ T-lymphocytes

The existence of decreased hypothalamic dopaminergic tone in HIV-infected men has been suggested. In a cross-sectional study, we determined 12 h nocturnal basal and pulsatile prolactin (PRL) release levels (by blood sampling every 10 min) and their correlation with CD4+ T cells in seven volunteer HIV-negative, healthy men (group 1), and 21 normoprolactinemic, euthyroid, HIV-infected men divided into 3 groups (each group = 7): (i) group 2, asymptomatic HIV-infected stage A1 men, untreated; (ii) group 3, AIDS stage C3 without active opportunistic infections, untreated; and (iii) group 4, previously stage C3 after at least 6 months of successful highly active antiretroviral therapy. Serum PRL was measured by radioimmunoanalysis and the results were analysed by waveform-independent deconvolution analysis. CD4+ T lymphocytes were measured by flow cytometry and viral load by a nucleic acid sequence-based amplification assay. No differences were detected in the first two groups. In the third group, however, 100% of prolactin secretion was found to be pulsatile with a shorter secretory burst duration (P = 0.04), and a greater circulating half-life and pulse amplitude (P < or = 0.04). Group 4 had the greatest basal prolactin secretion (P < or = 0.04), and a shorter secretory burst duration (P = 0.04 vs group 2), circulating half-life (P = 0.01 vs group 3) and intersecretory burst interval (P = 0.06 vs group 1). PRL approximate entropy was similar among all groups. Linear correlations existed between CD4+ T cell counts and PRL secretory burst half duration (r = 0.62, P = 0.002) and amplitude (r = -0.63, P = 0.001), and in circulating serum half-life (r = - 0.61, P = 0.002) in HIV-infected groups. Viral load showed no correlations. It is suggested that differential changes in nocturnal prolactin secretion among HIV-infected men occurred while maintaining the normal coordinate feedback and/or feedforward control within the lactotropic axis. These changes may represent an adaptative mechanism to sustain, by different means, the maximal physiologic PRL production to stimulate the highest cellular immune response and/or reconstitution in attempting to survive.