Simone G. Ramos

5-Lipoxygenase Deficiency Impairs Innate and Adaptive Immune Responses during Fungal Infection

5-lipoxygenase-derived products have been implicated in both the inhibition and promotion of chronic infection. Here, we sought to investigate the roles of endogenous 5-lipoxygenase products and exogenous leukotrienes during Histoplasma capsulatum infection in vivo and in vitro. 5-LO deficiency led to increased lung CFU, decreased nitric oxide production and a deficient primary immune response during active fungal infection. Moreover, H. capsulatum-infected 5-LO−/− mice showed an intense influx of neutrophils and an impaired ability to generate and recruit effector T cells to the lung. The fungal susceptibility of 5-LO−/− mice correlated with a lower rate of macrophage ingestion of IgG-H. capsulatum relative to WT macrophages. Conversely, exogenous LTB4 and LTC4 restored macrophage phagocytosis in 5-LO deficient mice. Our results demonstrate that leukotrienes are required to control chronic fungal infection by amplifying both the innate and adaptive immune response during histoplasmosis.

Protection against tuberculosis by a single intranasal administration of DNA-hsp65 vaccine complexed with cationic liposomes

BackgroundThe greatest challenges in vaccine development include optimization of DNA vaccines for use in humans, creation of effective single-dose vaccines, development of delivery systems that do not involve live viruses, and the identification of effective new adjuvants. Herein, we describe a novel, simple technique for efficiently vaccinating mice against tuberculosis (TB). Our technique consists of a single-dose, genetic vaccine formulation of DNA-hsp65 complexed with cationic liposomes and administered intranasally.ResultsWe developed a novel and non-toxic formulation of cationic liposomes, in which the DNA-hsp65 vaccine was entrapped (ENTR-hsp65) or complexed (COMP-hsp65), and used to immunize mice by intramuscular or intranasal routes. Although both liposome formulations induced a typical Th1 pattern of immune response, the intramuscular route of delivery did not reduce the number of bacilli. However, a single intranasal immunization with COMP-hsp65, carrying as few as 25 μg of plasmid DNA, leads to a remarkable reduction of the amount of bacilli in lungs. These effects were accompanied by increasing levels of IFN-γ and lung parenchyma preservation, results similar to those found in mice vaccinated intramuscularly four times with naked DNA-hsp65 (total of 400 μg).ConclusionOur objective was to overcome the significant obstacles currently facing DNA vaccine development. Our results in the mouse TB model showed that a single intranasal dose of COMP-hsp65 elicited a cellular immune response that was as strong as that induced by four intramuscular doses of naked-DNA. This formulation allowed a 16-fold reduction in the amount of DNA administered. Moreover, we demonstrated that this vaccine is safe, biocompatible, stable, and easily manufactured at a low cost. We believe that this strategy can be applied to human vaccines to TB in a single dose or in prime-boost protocols, leading to a tremendous impact on the control of this infectious disease.

Sudden Infant Death Syndrome (SIDS): a study of cardiac conduction system.

The theory that Sudden Infant Death Syndrome (SIDS) may be related to lethal cardiac arrhythmias or heart block due to structural abnormalities of the conduction system is attractive and still of particular interest. We analyze 69 autopsied cases of SIDS (46 males and 23 females, infants ranging in age from 3 to 365 days) and 24 age-matched cases of explained death (ED) as controls (16 males and 8 females), infants who died from extracardiac cause (cerebral and respiratory). SIDS and ED groups were divided into three subgroups according to the age: (A) from 3 to 60 days; (B) from 61 to 120 days; (C) from 121 to 365 days. Histological observations were focused on the cardiac conduction system (CCS) which was examined on serial sections with the technique devised by one of the present authors (L. Rossi). The following findings were observed: resorptive degeneration (97.10% of SIDS, 75% of ED), His bundle dispersion (33. 33% of SIDS, 16.66% of ED), Mahaim fibers (21.73% of SIDS, 8.3% of ED), cartilaginous meta-hyperplasia (5.79% of SIDS, 4.16% of ED), persistent fetal dispersion (24.63% of SIDS, 16.66% of ED), intramural right bundle (20.29% of SIDS, 25% of ED), left sided His bundle (20.29% of SIDS and 8.3% of ED), hemorrhage of the atrio-ventricular junction (15.94% of SIDS), septation of the bifurcation (13.04% of SIDS), atrio-ventricular node (AVN) dispersion (7.24% of SIDS), sino-atrial node hypoplasia (5.79% of SIDS), Zahn node (1.45% of SIDS), His bundle hypoplasia (1.45% of SIDS), intramural left bundle (1.45% of SIDS), AVN dualism (2.89% of SIDS), and His bundle dualism (2.89% of SIDS, 4.16% of ED). Only the presence of resorptive degeneration was significantly higher in SIDS than in ED cases (p = 0.004). Regarding the subgroups, the only significant difference was the higher presence of intramural right bundle in SIDS-A than in SIDS-B (p = 0.01). Despite the non-specificity of most of these findings, we believe that these changes, associated with particular conditions and/or neurovegetative stimuli, could cause potentially malignant arrhythmias. These data suggest the need for an accurate approach and examination of the cardiac conduction system in all cases of sudden death in infancy

Captopril reduces collagen and mast cell and eosinophil accumulation in pig serum-induced rat liver fibrosis.

The effect of captopril on the development of hepatic septal fibrosis in a specific experimental model produced by repeated injections of whole pig serum into the peritoneal cavity of rats was studied. The results afforded four basic conclusions. First, the experimental model used seems to be a pure form of septal fibrosis, which depends on active tissue fibroplasia, without hepatocyte necrosis. The fibrotic septa, located between limiting plates of adjacent classic hepatic lobules, and delimiting the classic liver lobule, consisted of collagen fibers infiltrated by eosinophils, mast cells, fat‐storing cells (Ito cells), transitional cells and interstitial fibro‐blasts. Second, the angiotensin‐converting enzyme inhibitor captopril attenuated the hepatic fibrosis induced by pig serum administration, as proven by a decrease In hepatic hydroxyproline concentration and histological examination of the liver. Third, this attenuation of hepatic fibrosis might be related, at least in part, to diminished mast cell and eosinophil accumulation in the hepatic tissue. Finally, these data may indicate a novel action of angiotensin‐converting enzyme inhibitor in general, and for captopril in particular, as drugs potentially capable of reducing eosinophils in fibrotic processes.

Ultrasonic tissue characterization of vulnerable carotid plaque: correlation between videodensitometric method and histological examination

BackgroundTo establish the correlation between quantitative analysis based on B-mode ultrasound images of vulnerable carotid plaque and histological examination of the surgically removed plaque, on the basis of a videodensitometric digital texture characterization.MethodsTwenty-five patients (18 males, mean age 67 ± 6.9 years) admitted for carotid endarterectomy for extracranial high-grade internal carotid artery stenosis (≥ 70% luminal narrowing) underwent to quantitative ultrasonic tissue characterization of carotid plaque before surgery. A computer software (Carotid Plaque Analysis Software) was developed to perform the videodensitometric analysis. The patients were divided into 2 groups according to symptomatology (group I, 15 symptomatic patients; and group II, 10 patients asymptomatic). Tissue specimens were analysed for lipid, fibromuscular tissue and calcium.ResultsThe first order statistic parameter mean gray level was able to distinguish the groups I and II (p = 0.04). The second order parameter energy also was able to distinguish the groups (p = 0,02). A histological correlation showed a tendency of mean gray level to have progressively greater values from specimens with < 50% to >75% of fibrosis.ConclusionVideodensitometric computer analysis of scan images may be used to identify vulnerable and potentially unstable lipid-rich carotid plaques, which are less echogenic in density than stable or asymptomatic, more densely fibrotic plaques.

Hyaluronidase recruits mesenchymal-like cells to the lung and ameliorates fibrosis

Hyaluronidases (HYALs) comprise a group of enzymes that degrade hyaluronic acid (HA). In this report, we reveal that a single intranasal inoculation of HYAL induces an increase in mononuclear cells within the bronchoalveolar space demonstrating a mesenchymal-like phenotype, expressing stem cell antigen-1 (SCA-1), CD44 and CD73 but not CD34, CD45, CD3, CD4, CD8 or CD19. This influx of mesenchymal stem cell (MSC)-like cells was dependent on leukotriene production within the lung parenchyma. These findings prompted experiments demonstrating that HYAL treatment potently blocked bleomycin-induced lung injury and fibrosis while decreasing transforming growth factor (TGF)-β production and collagen deposition. These data suggest that HYAL is a novel and promising tool to use autologous MSC-like cells in the treatment of pulmonary fibrosis.

Helminth Coinfection Does Not Affect Therapeutic Effect of a DNA Vaccine in Mice Harboring Tuberculosis

Background Helminthiasis and tuberculosis (TB) coincide geographically and there is much interest in exploring how concurrent worm infections might alter immune responses against bacilli and might necessitate altered therapeutic approaches. A DNA vaccine that codifies heat shock protein Hsp65 from M. leprae (DNAhsp65) has been used in therapy during experimental tuberculosis. This study focused on the impact of the co-existence of worms and TB on the therapeutic effects of DNAhsp65. Methodology/Principal Findings Mice were infected with Toxocara canis or with Schistosoma mansoni, followed by coinfection with M. tuberculosis and treatment with DNAhsp65. While T. canis infection did not increase vulnerability to pulmonary TB, S. mansoni enhanced susceptibility to TB as shown by higher numbers of bacteria in the lungs and spleen, which was associated with an increase in Th2 and regulatory cytokines. However, in coinfected mice, the therapeutic effect of DNAhsp65 was not abrogated, as indicated by colony forming units and analysis of histopathological changes. In vitro studies indicated that Hsp65-specific IFN-γ production was correlated with vaccine-induced protection in coinfected mice. Moreover, in S. mansoni-coinfected mice, DNA treatment inhibited in vivo TGF-β and IL-10 production, which could be associated with long-term protection. Conclusions/Significance We have demonstrated that the therapeutic effects of DNAhsp65 in experimental TB infection are persistent in the presence of an unrelated Th2 immune response induced by helminth infections.

Turbulent flow/low wall shear stress and stretch differentially affect aorta remodeling in rats.

Objective The present investigation was carried out to evaluate the relationship between local hemodynamic forces and intimal and medial remodeling in the proximal and distal segments of the arterial walls of rats in relation to severe infradiaphragmatic stenosis of the aorta. Methods Young male rats were divided randomly into an operated group, animals submitted to surgical abdominal aorta stenosis, and a sham-operated group, a control group of animals submitted to sham operation to simulate abdominal aorta stenosis. Results and conclusions Constricted aortas showed two distinct adaptive remodeling responses to hemodynamic stimuli induced by infradiaphragmatic coarctation. The first is remodeling in the hypertensive prestenotic segment with increased circumferential wall tension (CWT), associated with normal tensile stress, laminar flow/normal wall shear stress characterized by enlarged heterogeneous endothelial cells, elongated in the direction of the blood flow, diffusely distributed neointimal plaques, appearing as discrete bulging towards the vascular lumen and medial thickening. Our findings suggest that increased CWT caused by hypertension play a pivotal role in the remodeling of the prestenotic segment through biomechanical effects on oxidative stress and increased expression of transforming growth factor beta. The second is remodeling in the normotensive poststenotic segment with turbulent flow/low wall shear stress and normal CWT and tensile stress characterized by groups of endothelial cells with phenotypic alterations and focally distributed neointimal plaques, similar but many of them larger than those found in the prestenotic segments. Further studies are needed to determine how the mechanical forces of turbulent flow/low shear stress are detected and transduced into chemical signaling by the cells of the artery walls and then converted into pathophysiologically relevant phenotypic changes.

TLR2-dependent mast cell activation contributes to the control of Mycobacterium tuberculosis infection

Mast Cells (MCs) express toll-like receptor 2 (TLR2), a receptor known to be triggered by several major mycobacterial ligands and involved in resistance against Mycobacterium tuberculosis (MTB) infection. This study investigated whether adoptive transfer of TLR2 positive MCs (TLR2(+/+)) corrects the increased susceptibility of TLR2(-/-) mice to MTB infection. TLR2(-/-) mice displayed increased mycobacterial burden, diminished myeloid cell recruitment and proinflammatory cytokine production accompanied by defective granuloma formation. The reconstitution of these mice with TLR2(+/+) MCs, but not TLR2(-/-), confers better control of the infection, promotes the normalization of myeloid cell recruitment associated with reestablishment of the granuloma formation. In addition, adoptive transfer of TLR2(+/+) MC to TLR2(-/-) mice resulted in regulation of the pulmonary levels of IL-beta, IL-6, TNF-alpha, enhanced Th1 response and activated CD8(+) T cell homing to the lungs. Our results suggest that activation of MCs via TLR2 is required to compensate the defect in protective immunity and inability of TLR2(-/-) mice to control MTB infection.

Gut microbiota translocation to the pancreatic lymph nodes triggers NOD2 activation and contributes to T1D onset

Streptozotocin causes T1D by inducing the translocation of intestinal bacteria into pancreatic lymph nodes and driving the development of pathogenic Th1 and Th17 cells through NOD2 receptor.