Experimental Human Pneumococcal Carriage Augments IL-17A-dependent T-cell Defence of the Lung

Pneumococcal carriage is both immunising and a pre-requisite for mucosal and systemic disease. Murine models of pneumococcal colonisation show that IL-17A-secreting CD4⁺ T-cells (Th-17 cells) are essential for clearance of pneumococci from the nasopharynx. Pneumococcal-responding IL-17A-secreting CD4⁺ T-cells have not been described in the adult human lung and it is unknown whether they can be elicited by carriage and protect the lung from pneumococcal infection. We investigated the direct effect of experimental human pneumococcal nasal carriage (EHPC) on the frequency and phenotype of cognate CD4⁺ T-cells in broncho-alveolar lavage and blood using multi-parameter flow cytometry. We then examined whether they could augment ex vivo alveolar macrophage killing of pneumococci using an in vitro assay. We showed that human pneumococcal carriage leads to a 17.4-fold (p = 0.007) and 8-fold (p = 0.003) increase in the frequency of cognate IL-17A⁺ CD4⁺ T-cells in BAL and blood, respectively. The phenotype with the largest proportion were TNF⁺/IL-17A⁺ co-producing CD4⁺ memory T-cells (p<0.01); IFNγ⁺ CD4⁺ memory T-cells were not significantly increased following carriage. Pneumococci could stimulate large amounts of IL-17A protein from BAL cells in the absence of carriage but in the presence of cognate CD4⁺ memory T-cells, IL-17A protein levels were increased by a further 50%. Further to this we then show that alveolar macrophages, which express IL-17A receptors A and C, showed enhanced killing of opsonised pneumococci when stimulated with rhIL-17A (p = 0.013). Killing negatively correlated with RC (r = −0.9, p = 0.017) but not RA expression. We conclude that human pneumococcal carriage can increase the proportion of lung IL-17A-secreting CD4⁺ memory T-cells that may enhance innate cellular immunity against pathogenic challenge. These pathways may be utilised to enhance vaccine efficacy to protect the lung against pneumonia.     

Distinct and Atypical Intrinsic and Extrinsic Cell Death Pathways between Photoreceptor Cell Types upon Specific Ablation of Ranbp2 in Cone Photoreceptors

Non-autonomous cell-death is a cardinal feature of the disintegration of neural networks in neurodegenerative diseases, but the molecular bases of this process are poorly understood. The neural retina comprises a mosaic of rod and cone photoreceptors. Cone and rod photoreceptors degenerate upon rod-specific expression of heterogeneous mutations in functionally distinct genes, whereas cone-specific mutations are thought to cause only cone demise. Here we show that conditional ablation in cone photoreceptors of Ran-binding protein-2 (Ranbp2), a cell context-dependent pleiotropic protein linked to neuroprotection, familial necrotic encephalopathies, acute transverse myelitis and tumor-suppression, promotes early electrophysiological deficits, subcellular erosive destruction and non-apoptotic death of cones, whereas rod photoreceptors undergo cone-dependent non-autonomous apoptosis. Cone-specific Ranbp2 ablation causes the temporal activation of a cone-intrinsic molecular cascade highlighted by the early activation of metalloproteinase 11/stromelysin-3 and up-regulation of Crx and CoREST, followed by the down-modulation of cone-specific phototransduction genes, transient up-regulation of regulatory/survival genes and activation of caspase-7 without apoptosis. Conversely, PARP1⁺-apoptotic rods develop upon sequential activation of caspase-9 and caspase-3 and loss of membrane permeability. Rod photoreceptor demise ceases upon cone degeneration. These findings reveal novel roles of Ranbp2 in the modulation of intrinsic and extrinsic cell death mechanisms and pathways. They also unveil a novel spatiotemporal paradigm of progression of neurodegeneration upon cell-specific genetic damage whereby a cone to rod non-autonomous death pathway with intrinsically distinct cell-type death manifestations is triggered by cell-specific loss of Ranbp2. Finally, this study casts new light onto cell-death mechanisms that may be shared by human dystrophies with distinct retinal spatial signatures as well as with other etiologically distinct neurodegenerative disorders.     

Isolation, Characterization and Antifungal Activity of Proteinase Inhibitors from Capsicum chinense Jacq. Seeds

Capsicum species belong to the Solanaceae family and have great social, economic and agronomical significance. The present research presents data on the isolation and characterization of Capsicum chinense Jacq. peptides which were scrutinized in relation to their toxicity towards a diverse set of yeast species. The protein extract was separated with C18 reverse-phase chromatography in high performance liquid chromatography, resulting in three different peptide enriched fractions (PEFs) termed PEF1, PEF2 and PEF3. Tricine-SDS-PAGE of the PEF2 revealed peptides with molecular masses of approximately 5.0 and 8.5 kDa. These PEFs also exhibited strong antifungal activity against different yeasts. In the presence of the PEF2, Candida tropicalis exhibited morphological changes, including cellular agglomeration and formation of pseudohyphae. Determined N-terminal sequences of PEF2 and PEF3 were proven to be highly homologous to serine proteinase inhibitors, when analysed by comparative database sequence tools. For this reason were performed protease inhibitory activity assay. The PEFs displayed high inhibitory activity against trypsin and low inhibitory activity against chymotrypsin. PEF2 and PEF3 were considerably unsusceptible to a broad interval of pH and temperatures. Due to the myriad of application of Proteinase inhibitors (PIs) in fields ranging from plant protection against pathogens and pests to medicine such as in cancer and virus replication inhibition, the discovery of new PIs with new properties are of great interest.     

Joint and Independent Effects of Alcohol Drinking and Tobacco Smoking on Oral Cancer: A Large Case-Control Study

Alcohol drinking and tobacco smoking are assumed to have significant independent and joint effects on oral cancer (OC) development. This assumption is based on consistent reports from observational studies, which, however, overestimated the independent effects of smoking and drinking, because they did not account for the interaction effect in multivariable analyses. This case-control study sought to investigate the independent and the joint effects of smoking and drinking on OC in a homogeneous sample of adults. Case patients (N = 1,144) were affected by invasive oral/oropharyngeal squamous cell carcinoma confirmed histologically, diagnosed between 1998 and 2008 in four hospitals of São Paulo (Brazil). Control patients (N = 1,661) were not affected by drinking-, smoking-associated diseases, cancers, upper aero-digestive tract diseases. Cumulative tobacco and alcohol consumptions were assessed anamnestically. Patients were categorized into never/ever users and never/level-1/level-2 users, according to the median consumption level in controls. The effects of smoking and drinking on OC adjusted for age, gender, schooling level were assessed using logistic regression analysis; Model-1 did not account for the smoking-drinking interaction; Model-2 accounted for this interaction and included the resultant interaction terms. The models were compared using the likelihood ratio test. According to Model-1, the adjusted odds ratios (ORs) for smoking, drinking, smoking-drinking were 3.50 (95% confidence interval –95CI, 2.76–4.44), 3.60 (95CI, 2.86–4.53), 12.60 (95CI, 7.89–20.13), respectively. According to Model-2 these figures were 1.41 (95CI, 1.02–1.96), 0.78 (95CI, 0.48–1.27), 8.16 (95CI, 2.09–31.78). Analogous results were obtained using three levels of exposure to smoking and drinking. Model-2 showed statistically significant better goodness-of-fit statistics than Model-1. Drinking was not independently associated with OC, while the independent effect of smoking was lower than expected, suggesting that observational studies should be revised adequately accounting for the smoking-drinking interaction. OC control policies should focus on addictive behaviours rather than on single lifestyle risk factors.     

Human Hemorrhagic Pulmonary Leptospirosis: Pathological Findings and Pathophysiological Correlations

Background

Leptospirosis is a re-emerging zoonosis with protean clinical manifestations. Recently, the importance of pulmonary hemorrhage as a lethal complication of this disease has been recognized. In the present study, five human necropsies of leptospirosis (Weil‘s syndrome) with extensive pulmonary manifestations were analysed, and the antibodies expressed in blood vessels and cells involved in ion and water transport were used, seeking to better understand the pathophysiology of the lung injury associated with this disease.

Principal Findings

Prominent vascular damage was present in the lung microcirculation, with decreased CD34 and preserved aquaporin 1 expression. At the periphery and even inside the extensive areas of edema and intraalveolar hemorrhage, enlarged, apparently hypertrophic type I pneumocytes (PI) were detected and interpreted as a non-specific attempt of clearence of the intraalveolar fluid, in which ionic transport, particularly of sodium, plays a predominant role, as suggested by the apparently increased ENaC and aquaporin 5 expression. Connexin 43 was present in most pneumocytes, and in the cytoplasm of the more preserved endothelial cells. The number of type II pneumocytes (PII) was slightly decreased when compared to normal lungs and those of patients with septicemia from other causes, a fact that may contribute to the progressively low PI count, resulting in deficient restoration after damage to the alveolar epithelial integrity and, consequently, a poor outcome of the pulmonary edema and hemorrhage.

Conclusions

Pathogenesis of lung injury in human leptospirosis was discussed, and the possibility of primary non-inflammatory vascular damage was considered, so far of undefinite etiopathogenesis, as the initial pathological manifestation of the disease.     

Chemokine Receptors CCR6 and CXCR3 Are Necessary for CD4+ T Cell Mediated Ocular Surface Disease in Experimental Dry Eye Disease

CD4⁺ T cells are essential to pathogenesis of ocular surface disease in dry eye. Two subtypes of CD4⁺ T cells, Th1 and Th17 cells, function concurrently in dry eye to mediate disease. This occurs in spite of the cross-regulation of IFN-γ and IL-17A, the prototypical cytokines Th1 and Th17 cells, respectively. Essential to an effective immune response are chemokines that direct and summon lymphocytes to specific tissues. T cell trafficking has been extensively studied in other models, but this is the first study to examine the role of chemokine receptors in ocular immune responses. Here, we demonstrate that the chemokine receptors, CCR6 and CXCR3, which are expressed on Th17 and Th1 cells, respectively, are required for the pathogenesis of dry eye disease, as CCR6KO and CXCR3KO mice do not develop disease under desiccating stress. CD4⁺ T cells from CCR6KO and CXCR3KO mice exposed to desiccating stress (DS) do not migrate to the ocular surface, but remain in the superficial cervical lymph nodes. In agreement with this, CD4⁺ T cells from CCR6 and CXCR3 deficient donors exposed to DS, when adoptively transferred to T cell deficient recipients manifest minimal signs of dry eye disease, including significantly less T cell infiltration, goblet cell loss, and expression of inflammatory cytokine and matrix metalloproteinase expression compared to wild-type donors. These findings highlight the important interaction of chemokine receptors on T cells and chemokine ligand expression on epithelial cells of the cornea and conjunctiva in dry eye pathogenesis and reveal potential new therapeutic targets for dry eye disease.     

Mesenchymal Stem Cells (MSC) Prevented the Progression of Renovascular Hypertension,Improved Renal Function and Architecture

Renovascular hypertension induced by 2 Kidney-1 Clip (2K-1C) is a renin-angiotensin-system (RAS)-dependent model, leading to renal vascular rarefaction and renal failure. RAS inhibitors are not able to reduce arterial pressure (AP) and/or preserve the renal function, and thus, alternative therapies are needed. Three weeks after left renal artery occlusion, fluorescently tagged mesenchymal stem cells (MSC) (2×10⁵ cells/animal) were injected weekly into the tail vein in 2K-1C hypertensive rats. Flow cytometry showed labeled MSC in the cortex and medulla of the clipped kidney. MSC prevented a further increase in the AP, significantly reduced proteinuria and decreased sympathetic hyperactivity in 2K-1C rats. Renal function parameters were unchanged, except for an increase in urinary volume observed in 2K-1C rats, which was not corrected by MSC. The treatment improved the morphology and decreased the fibrotic areas in the clipped kidney and also significantly reduced renal vascular rarefaction typical of 2K-1C model. Expression levels of IL-1β, TNF-α angiotensinogen, ACE, and Ang II receptor AT₁ were elevated, whereas AT₂ levels were decreased in the medulla of the clipped kidney. MSC normalized these expression levels. In conclusion, MSC therapy in the 2K-1C model (i) prevented the progressive increase of AP, (ii) improved renal morphology and microvascular rarefaction, (iii) reduced fibrosis, proteinuria and inflammatory cytokines, (iv) suppressed the intrarenal RAS, iv) decreased sympathetic hyperactivity in anesthetized animals and v) MSC were detected at the CNS suggesting that the cells crossed the blood-brain barrier. This therapy may be a promising strategy to treat renovascular hypertension and its renal consequences in the near future.     

Preconditioning of Microglia by α-Synuclein Strongly Affects the Response Induced by Toll-like Receptor (TLR) Stimulation

In recent years, it has become accepted that α-synuclein (αSyn) has a key role in the microglia-mediated neuroinflammation, which accompanies the development of Parkinson’s disease and other related disorders, such as Dementia with Lewy Bodies and Alzheimer’s disease. Nevertheless, the cellular and molecular mechanisms underlying its pathological actions, especially in the sporadic forms of the diseases, are not completely understood. Intriguingly, several epidemiological and animal model studies have revealed a link between certain microbial infections and the onset or progression of sporadic forms of these neurodegenerative disorders. In this work, we have characterized the effect of toll-like receptor (TLR) stimulation on primary murine microglial cultures and analysed the impact of priming cells with extracellular wild-type (Wt) αSyn on the subsequent TLR stimulation of cells with a set of TLR ligands. By assaying key interleukins and chemokines we report that specific stimuli, in particular Pam3Csk4 (Pam3) and single-stranded RNA40 (ssRNA), can differentially affect the TLR2/1- and TLR7-mediated responses of microglia when pre-conditioned with αSyn by augmenting IL-6, MCP-1/CCL2 or IP-10/CXCL10 secretion levels. Furthermore, we report a skewing of αSyn-primed microglia stimulated with ssRNA (TLR7) or Pam3 (TLR2/1) towards intermediate but at the same time differential, M1/M2 phenotypes. Finally, we show that the levels and intracellular location of activated caspase-3 protein change significantly in αSyn-primed microglia after stimulation with these particular TLR agonists. Overall, we report a remarkable impact of non-aggregated αSyn pre-sensitization of microglia on TLR-mediated immunity, a phenomenon that could contribute to triggering the onset of sporadic α-synuclein-related neuropathologies.     

Small tropical forest trees have a greater capacity to adjust carbon metabolism to long-term drought than large canopy trees

The response of small understory trees to long-term drought is vital in determining the future composition, carbon stocks and dynamics of tropical forests. Long-term drought is, however, also likely to expose understory trees to increased light availability driven by drought-induced mortality. Relatively little is known about the potential for understory trees to adjust their physiology to both decreasing water and increasing light availability. We analysed data on maximum photosynthetic capacity (J_max, V_cmax), leaf respiration (R_leaf), leaf mass per area (LMA), leaf thickness and leaf nitrogen and phosphorus concentrations from 66 small trees across 12 common genera at the world's longest running tropical rainfall exclusion experiment and compared responses to those from 61 surviving canopy trees. Small trees increased J_max, V_cmax, R_leaf and LMA (71, 29, 32, 15% respectively) in response to the drought treatment, but leaf thickness and leaf nutrient concentrations did not change. Small trees were significantly more responsive than large canopy trees to the drought treatment, suggesting greater phenotypic plasticity and resilience to prolonged drought, although differences among taxa were observed. Our results highlight that small tropical trees have greater capacity to respond to ecosystem level changes and have the potential to regenerate resilient forests following future droughts.     

Polymorphism and genetic diversity of Isospora parnaitatiaiensis Silva,Rodrigues, Lopes,Berto, Luz,Ferreira & Lopes, 2015 (Eimeriidae) from antbirds (Thamnophilidae) in Brazil

Systematic Parasitology - Isospora parnaitatiaiensis Silva, Rodrigues, Lopes, Berto, Luz, Ferreira &amp; Lopes, 2015 was identified from a new host, the plain antvireo Dysithamnus mentalis...