New features on the survival of human-infective Trypanosoma rangeli in a murine model: Parasite accumulation is observed in lymphoid organs.

Trypanosoma rangeli is a non-pathogenic protozoan parasite that infects mammals, including humans, in Chagas disease-endemic areas of South and Central America. The parasite is transmitted to a mammalian host when an infected triatomine injects metacyclic trypomastigotes into the host's skin during a bloodmeal. Infected mammals behave as parasite reservoirs for several months and despite intensive research, some major aspects of T. rangeli-vertebrate interactions are still poorly understood. In particular, many questions still remain unanswered, e.g. parasite survival and development inside vertebrates, as no parasite multiplication sites have yet been identified. The present study used an insect bite transmission strategy to investigate whether the vector inoculation spot in the skin behave as a parasite-replication site. Histological data from the skin identified extracellular parasites in the dermis and hypodermis of infected mice in the first 24 hours post-infection, as well as the presence of inflammatory infiltrates in a period of up to 7 days. However, qPCR analyses demonstrated that T. rangeli is eliminated from the skin after 7 days of infection despite being still consistently found on circulating blood and secondary lymphoid tissues for up to 30 days post-infection. Interestingly, significant numbers of parasites were found in the spleen and mesenteric lymph nodes of infected mice during different periods of infection and steady basal numbers of flagellates are maintained in the host's bloodstream, which might behave as a transmission source to insect vectors. The presence of parasites in the spleen was confirmed by fluorescent photomicrography of free and cell-associated T. rangeli forms. Altogether our results suggest that this organ could possibly behave as a T. rangeli maintenance hotspot in vertebrates.

Human CD8+ T Cells Release Extracellular Traps Co-Localized With Cytotoxic Vesicles That Are Associated With Lesion Progression and Severity in Human Leishmaniasis.

Cell death plays a fundamental role in mounting protective and pathogenic immunity. Etosis is a cell death mechanism defined by the release of extracellular traps (ETs), which can foster inflammation and exert microbicidal activity. While etosis is often associated with innate cells, recent studies showed that B cells and CD4+ T cells can release ETs. Here we investigate whether CD8+ T cells can also release ETs, which might be related to cytotoxicity and tissue pathology. To these ends, we first employed an in vitro system stimulating human CD8+ T cells isolated from healthy volunteers with anti-CD3/anti-CD28. Using time-frame video, confocal and electron microscopy, we demonstrate that human CD8+ T cells release ETs upon stimulation (herein LETs - lymphocyte extracellular traps), which display unique morphology and functional characteristics. CD8+ T cell-derived LETs form long strands that co-localize with CD107a, a marker of vesicles containing cytotoxic granules. In addition, these structures connect the LET-releasing cell to other neighboring cells, often resulting in cell death. After demonstrating the release of LETs by human CD8+ T cells in vitro, we went on to study the occurrence of CD8-derived LETs in a human disease setting. Thus, we evaluated the occurrence of CD8-derived LETs in lesions from patients with human tegumentary leishmaniasis, where CD8+ T cells play a key role in mediating pathology. In addition, we evaluated the association of these structures with the intensity of the inflammatory infiltrate in early and late cutaneous, as well as in mucosal leishmaniasis lesions. We demonstrated that progression and severity of debilitating and mutilating forms of human tegumentary leishmaniasis are associated with the frequency of CD8+ T cells in etosis, as well as the occurrence of CD8-derived LETs carrying CD107a+ vesicles in the lesions. We propose that CD8+ T cell derived LETs may serve as a tool for delivering cytotoxic vesicles to distant target cells, providing insights into mechanisms of CD8+ T cell mediated pathology.

Renin angiotensin system molecules and nitric oxide local interactions in the adrenal gland of Trypanosoma cruzi infected rats.

Chagas disease (CD) is a tropical zoonosis caused by the protozoan Trypanosoma cruzi. Severe autonomic dysfunction like reduced cardiac catecholamine-containing or acetylcholinesterase-positive innervation have been reported in CD. Renin-angiotensin system (RAS) seems to participate in the regulation of adrenal catecholamine secretion by adrenal medullary chromaffin cells, which might be dependent of nitric oxide (NO) pathways. To investigate the levels of RAS components in the adrenal gland during the acute infection with Y strain T. cruzi and in response to acute administration of an inhibitor of the enzyme NO synthase, L-NAME. Male Holtzman rats were inoculated intraperitoneally with Y strain T. cruzi and received L-NAME or tap water from one day before the infection until 13 or 17 days post-inoculation (dpi). The concentration of RAS molecules in the adrenal tissue was evaluated by ELISA immunoassay. Angiotensin converting enzyme 1 (ACE1) levels were significantly lower at 17 dpi when compared to 13 dpi. No significant differences were found compared with baseline, and no changes were detected in adrenal tissue levels of angiotensin converting enzyme 2 (ACE2), angiotensin II, or angiotensin-(1-7). Moreover, the treatment with L-NAME did not influence the levels of RAS components in adrenal tissue during the course of T. cruzi infection. We provided the first evidence that levels of RAS molecules change in the adrenal gland during acute phase of T. cruzi infection. Future studies are necessary to fully address the role of NO in RAS-associated adrenal gland function in CD.

Interactions between local renin angiotensin system and nitric oxide in the brain of Trypanosoma cruzi infected rats.

Chagas' disease (CD) is a zoonosis caused by the protozoan Trypanosoma cruzi. Besides being an important cause of cardiomyopathy, central nervous system (CNS) manifestations have also been reported in CD. Renin-Angiotensin System (RAS) plays a pathophysiological role in several brain disorders such as cerebrovascular and neurodegenerative diseases. A link between RAS and nitric oxide (NO) pathways has been described in CNS. For instance, Angiotensin-(1-7) increases NO expression in the brain, which may, in turn, help to control parasite load in response to T. cruzi infection. Herein, we investigated the levels of RAS components in the brain cortex in acute T. cruzi infection and the effect of L-NAME administration, an inhibitor of the enzyme NO synthase, in CNS infection and in RAS molecules. Male Holtzman rats were inoculated intraperitoneally with T. cruzi Y strain and received L-NAME or tap water from one day before the infection until 13 days post infection (dpi). Parasitemia was evaluated on alternate days from day 3 post-infection until day 13 in both T. cruzi infected groups. Histopathological analysis of the brain cortex was also performed. Brain cortex was collected from non-infected (controls) and infected rats at 13 dpi for RAS components assessment. Infected rats receiving L-NAME presented higher parasitemia, brain parasitism and inflammation compared with non-treated infected animals. The administration of L-NAME significantly decreased the levels of Angiotensin I Converting Enzyme 2 (ACE2). In conclusion, we provided preliminary evidence of the interaction between RAS and NO during the acute phase of T. cruzi infection.

Effect of blockade of nitric oxide in heart tissue levels of Renin Angiotensin System components in acute experimental Chagas disease.

Chagas disease (CD) is an important cause of cardiomyopathy in South America. The pathophysiology of CD is still a matter of debate. Renin Angiotensin System (RAS) components are clearly involved in cardiovascular diseases. RAS molecules interact with nitric oxide (NO) pathway in blood vessel and heart tissue. Thus, the aim of this study is to investigate possible changes in RAS molecules during the infection with Y strain T. cruzi and in response to acute administration of an inhibitor of the enzyme NO synthase, l-NAME. Male Holtzman rats were inoculated intraperitoneally with Y strain T. cruzi and received l-NAME or tap water from one day before the infection until 13 or 17 days post infection (dpi). Angiotensin converting enzyme 1 (ACE1) levels were significantly higher at day 17 when compared to baseline in atrium, whereas, in ventricle, ACE2 levels were significantly higher in 13 dpi when compared to baseline. In response to l-NAME treatment, atrium tissue levels of ACE1 were significantly reduced in treated animals at day 17, while Angiotensin-(1-7) concentration in atrium significantly increased in this group at the same time-point. No changes were detected in RAS components in the ventricle. ACE2 levels in Soleus muscle were significantly reduced in treated animals at day 13. In conclusion, changes in RAS molecules were detected during acute phase of T. cruzi infection and the inhibition of NO synthesis clearly interfered with expression of ACE1 and Angiotensin-(1-7) in the atrium.

Mast cell-nerve interaction in the colon of Trypanosoma cruzi-infected individuals with chagasic megacolon.

Chagas disease is an infection caused by the parasite Trypanosoma cruzi that affects millions of people worldwide and is endemic in Latin America. Megacolon is the most frequent complication of the digestive chronic form and happens due to lesions of the enteric nervous system. The neuronal lesions seem to initiate in the acute phase and persist during the chronic phase, albeit the mechanisms involved in this process are still debated. Among the cells of the immune system possibly involved in this pathological process is the mast cell (MC) due to its well-known role in the bi-directional communication between the immune and nervous systems. Using ultrastructural analysis, we found an increased number of degranulated MCs in close proximity to nerve fibers in infected patients when compared with uninfected controls. We also immunostained MCs for the two pro-inflammatory molecules tryptase and chymase, the first being also important in neuronal death. The number of MCs immunostained for tryptase or chymase was increased in patients with megacolon, whereas increased tryptase staining was additionally observed in patients without megacolon. Moreover, we detected the expression of the tryptase receptor PAR2 in neurons of the enteric nervous system, which correlated to the tryptase staining results. Altogether, the data presented herein point to the participation of MCs on the denervation process that occurs in the development of T. cruzi-induced megacolon.

Physicochemical Characterization and Skin Permeation of Cationic Transfersomes Containing the Synthetic Peptide PnPP-19.

BACKGROUND: PnPP-19 is a 19-amino-acid synthetic peptide previously described as a novel drug for the treatment of erectile dysfunction. OBJECTIVE: The aim of this work was to evaluate the physicochemical properties of cationic transfersomes containing PnPP-19 and the skin permeation of free PnPP-19 and PnPP-19-loaded transfersomes. METHODS: Three different liposomal preparation methods were evaluated. Cationic transfersomes contained egg phosphatidyl choline: stearylamine (9:1 w/w) and Tween 20 (84.6:15.4 lipid:Tween, w/w). Lipid concentration varied from 20 to 40 mM. We evaluated the entrapment percentage, mean diameter, zeta potential and stability at 4 °C of the formulations. The skin permeation assays were performed with abdominal human skin using Franz diffusion cell with 3 cm2 diffusion area at 32 °C and a fluorescent derivative of the peptide, containing 5-TAMRA, bound to PnPP-19 C-terminal region, where an extra lysine was inserted. RESULTS: Our results showed variable entrapment efficiencies, from 6% to 30%, depending on the preparation method and the lipid concentration used. The reverse phase evaporation method using a total lipid concentration equal to 40 mM led to the best entrapment percentage (30.2 + 4.5%). Free PnPP-19 was able to permeate skin at a rate of 10.8 ng/cm2/h. However, PnPP-19 was specifically hydrolyzed by skin proteases, generating a fragment of 15 amino acid residues. Encapsulated PnPP-19 permeated the skin at a rate of 19.8 ng/cm2/h. CONCLUSION: The encapsulation of PnPP-19 in cationic transfersomes protected the peptide from degradation, favoring its topical administration.

A single session of moderate intensity walking increases brain-derived neurotrophic factor (BDNF) in the chronic post-stroke patients.

Background Aerobic exercise, even for short durations, may promote an increase in serum concentrations of brain-derived neurotrophic factor (BDNF). However, it is necessary to determine the optimal exercise types and intensities to increase BDNF levels. Objectives This aim of this study was investigate the effects of mild and moderate intensity acute aerobic exercise on serum BDNF levels in patients in the chronic post-stroke phase. Methods The participants answered a socio-demographic questionnaire, cognitive assessment (Mini Mental State Examination), assessment of depressive symptoms (Hamilton Depression Scale), fatigue (Fatigue Severity Scale) and functional capacity (6-minute walk test). Blood samples were collected before and after each session. The measurement of the concentration of BDNF was performed using the enzyme-linked immunosorbent assay . Patients were asked to walk for 30-min in the target training zone (mild intensity, 50-63% of maximum heart rate, and moderate intensity, 64-76% of maximum heart rate), once each week for 2 consecutive weeks. Results Our results indicate that 30 min of acute aerobic exercise at a moderate intensity, but not at a mild intensity, increases serum BDNF levels in the chronic post-stroke phase. Conclusions This study suggests a potential mechanism for the beneficial effects of exercise as a component of recovery from stroke, and provides the basis for future studies that will elucidate the specific parameters for clinical applications.

Cardiac autonomic denervation and expression of neurotrophins (NGF and BDNF) and their receptors during experimental Chagas disease.

Although cardiac dysautonomia is a distinctive feature of Chagas disease, its clinical and functional significance is still being speculated. Neurotrophic factors are potentially involved; however, studies of their effect in this infection are rare. Ultrastructural abnormalities in autonomic varicosities, levels of both nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF), as well as the expression of their receptors, were analysed in the heart of a rat model of Trypanosoma infection. Predominantly, at the early stage of the infection, cardiac autonomic varicosities displayed several signs of degeneration parallel to the elevation of cardiac levels of NGF, as well as expression of the receptors TrkA and p75NTR. For BDNF and TrkB, the changes were less conspicuous. Data obtained here can contribute to further clarify the factors related to the autonomic nervous system's adaptive changes that could determine the evolution of different clinical forms of Chagas disease; mainly, the cardiac form.

Demyelination/remyelination and expression of interleukin-1ß, substance P, nerve growth factor, and glial-derived neurotrophic factor during trigeminal neuropathic pain in rats.

The etiology of trigeminal neuropathic pain is not clear, but there is evidence that demyelination, expression of cytokines, neuropeptides, and neurotrophic factors are crucial contributors. In order to elucidate mechanisms underlying trigeminal neuropathic pain, we evaluated the time course of morphological changes in myelinated and unmyelinated trigeminal nerve fibers, expression of cytokine IL-1ß, neuropeptide substance P (SP), nerve growth factor (NGF), and glial derived neurotrophic factor (GDNF) in peripheral and ganglion tissues, using a rat model of trigeminal neuropathic pain. Chronic constriction injury (CCI) of the infraorbital nerve (IoN), or a sham surgery, was performed. Mechanical allodynia was evaluated from day 3 to day 15 post-surgery. Trigeminal nerves were divided into 2 sections - distal to CCI and ganglion - for morphological analyses, immunohistochemistry (IL-1ß, SP), and protein quantification by ELISA (NGF, GDNF). At early postoperative time points, decreased mechanical responses were observed, which were associated with demyelination, glial cell proliferation, increased immunoexpression of IL-1 ß and SP, and impaired GDNF production. In the late postoperative period, mechanical allodynia was present with partial recovery of myelination, glial cell proliferation, and increased immunoreactivity of IL-1ß and SP. Our data show that demyelination/remyelination processes are related to the development of pain behavior. IL-1ß may have effects both in ganglia and nerves, while SP may be an important mediator at the nerve endings. Additionally, low levels of GDNF may produce impaired signaling, which may be involved in generation of pain.

Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes.

Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole.

Mast cells in the colon of Trypanosoma cruzi-infected patients: are they involved in the recruitment, survival and/or activation of eosinophils?

Megacolon is frequently observed in patients who develop the digestive form of Chagas disease. It is characterized by dilation of the rectum-sigmoid portion and thickening of the colon wall. Microscopically, the affected organ presents denervation, which has been considered as consequence of an inflammatory process that begins at the acute phase and persists in the chronic phase of infection. Inflammatory infiltrates are composed of lymphocytes, macrophages, natural killer cells, mast cells, and eosinophils. In this study, we hypothesized that mast cells producing tryptase could influence the migration and the activation of eosinophils at the site, thereby contributing to the immunopathology of the chronic phase. We seek evidence of interactions between mast cells and eosinophils through (1) evaluation of eosinophils, regarding the expression of PAR2, a tryptase receptor; (2) correlation analysis between densities of mast cells and eosinophils; and (3) ultrastructural studies. The electron microscopy studies revealed signs of activation of mast cells and eosinophils, as well as physical interaction between these cells. Immunohistochemistry and correlation analyses point to the participation of tryptase immunoreactive mast cells in the migration and/or survival of eosinophils at the affected organ.

Neuroimmunopathology of Trypanosoma cruzi-induced megaoesophagus: Is there a role for mast cell proteases?

Tryptase and chymase are mast cell (MC)-specific proteases, which influence in the activation of inflammatory cells. In this study, we quantified tryptase- or chymase-expressing MCs in the oesophaguses of Chagas patients, and searched for a correlation between those data with area of nerve fibres that expressed either PGP9.5 (pan-marker) or vasoactive intestinal polypeptide (VIP), which is a neuromediator that has anti-inflammatory activity. Samples from the oesophaguses of 14 individuals Trypanosoma cruzi-infected and from six uninfected individuals were analysed by immunohistochemistry. It was demonstrated that the number of tryptase-IR MCs in infected individuals increased when compared with controls, regardless of whether the individuals had megaoesophagus, whereas the number of chymase-IR MCs increased only in infected individuals without megaoesophagus. Negative correlations were observed between tryptase-IR MCs and the density of nerve fibres that expressed VIP or PGP 9.5-IR. The participation of chymase and tryptase in this type of immunopathology is discussed.

Chronic sympathectomy of the caudal artery delays cutaneous heat loss during passive heating.

The present study aimed to investigate the chronic effects of caudal artery sympathectomy on thermoregulatory adjustments induced by passive heating. Male Wistar rats were subjected to two surgical procedures: caudal artery denervation (CAD) or sham surgery (Sham-CAD) and intraperitoneal implantation of a temperature sensor. On the day of the experiments, the animals were exposed to an ambient temperature of 36°C for 60min or allowed to rest under thermoneutral conditions (26°C). During the experiments, the tail skin temperature (T(skin)) and the core body temperature (T(core)) were measured. Under thermoneutral conditions, although sympathetic denervation did not change the average values of T(core) and T(skin), CAD rats exhibited decreased T(skin) variability compared with Sham-CAD rats (0.020±0.005°C vs. 0.031±0.005°C; P=0.024). During heat exposure, no differences were observed in the T(core) between the groups. In contrast, although peak T(skin) values were not affected by chronic sympathectomy of the caudal artery, CAD animals showed a delayed increase in T(skin); the time until the stabilization of T(skin) was three-fold longer in CAD rats than in Sham-CAD rats (15.3±2.5min vs. 4.9±0.6min; P=0.001). In conclusion, chronic sympathectomy of the caudal artery delays cutaneous heat loss during passive heating and decreases T(skin) variability under thermoneutral conditions. Taken together, our results indicate that the sympathetic innervation of cutaneous vessels is essential for the precise regulation of tail heat loss.

Cardiac NGF and GDNF expression during Trypanosoma cruzi infection in rats.

In rats, autonomic nerve endings are damaged during Trypanosoma cruzi-induced myocarditis. Gradual recovery occurs after the acute phase. The present work shows the cardiac levels of glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF), and their cellular sources during T. cruzi infection in rats. Atrial and ventricular NGF levels (ELISA) increased significantly at day 20 post inoculation, the time-point of maximal sympathetic denervation. ELISA failed to show significant increase of cardiac GDNF levels. However immunohistochemistry showed a significant increase of anti-GDNF gold particles over atrial granules at day 20. Light microscopy showed stronger NGF immunostaining in atrial cardiomyocytes and several blood capillaries. In situ hybridization showed NGF and GDNF mRNAs in atrial and ventricular myocytes of both infected and uninfected animals. Endothelial cells exhibited NGF mRNA and protein only in infected rats. No evidence of neurotrophic factor expression by the infiltrating mononuclear cells was found. This is the first report on neurotrophic factor expression during T. cruzi infection. Our findings indicate an important role for NGF in the regenerative phenomena subsequent to a myocarditis able to damage sympathetic nerve endings, with preservation of preterminals and nerve trunks. GDNF could have a minor or a more transient participation.

Morfologia e histoquímica de glicogênio e mucossubstâncias na próstata e glândulas bulbo-uretrais de Marmosa cinerea Temminck, 1824 (Marsupialia - Didelphidae) / Morphology, glycogen and mucosubstances histochemistry of the prostate and bulbo-urethral glands of the Marmosa cinerea Temminck, 1824 (Marsupialia - Didelphidae)

Sao descritos os aspectos histológicos e a distribuiçao de glicogênio e mucossubstâncias na próstata e nos três pares de glândulas bulbo-uretrais (Cowper) de Marmosa cinerea, marsupial Didelphidae com ampla distribuiçao geográfica no Brasil. Os três segmentos prostáticos, cranial, médio e caudal, apresentam túbulos secretores disseminados na mucosa uretral e sao divididos em zonas externa, média e interna, de acordo com as características morfológicas e tintoriais do epitélio secretor. No segmento cranial, o epitélio secreta glicogênio e mucossubstâncias neutras. No segmento médio sao produzidas mucossubstâncias neutras e sialomucinas, sendo sugerido um mecanismo apócrino de secreçao neste segmento. O epitélio do segmento caudal produz glicogênio, mucossubstâncias neutras e proteínas. O par lateral de bulbo-uretrais é formado por túbulos secretores longos e ramificados, que produzem mucossubstâncias neutras. O par intermédio constitui-se de túbulo-ácinos revestidos por células com porçao apical expandida e que secretam mucossubstâncias neutras, sialomucinas e sulfomucinas. Os túbulo-ácinos do par medial sao tortuosos e suas células produzem mucossubstâncias neutras.