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Background: FOXR2-activated central nervous system (CNS) neuroblastoma (CNS NB-FOXR2) is a recently identified subtype of brain tumor characterized by the elevated expression of the transcription factor FOXR2 mainly due to genomic rearrangements. However, the precise pathogenic mechanisms, including the cell type of origin, remain elusive., Methods: A gene expression analysis of patient tumors was performed to identify putative cell types of origin. Based on this prediction, a new human embryonic stem cell-based model was developed to validate the origin and to examine the molecular and cellular mechanisms underlying the formation of CNS NB-FOXR2., Results: Our data showed that CNS NB-FOXR2 tumors express a high level of lineage marker genes associated with the medial ganglionic eminence (MGE), a transient structure located in the developing ventral forebrain. Our model confirmed the cell-type-specific effect of FOXR2 on the proliferation and in vivo tumorigenicity. Additionally, we found that FOXR2 overexpression activated the MEK/ERK signaling pathway through a suppression of the endogenous RAS inhibitor DIRAS3. The MEK inhibitor trametinib suppressed the proliferation of FOXR2-expressing MGE progenitors more than nonexpressing cells., Conclusions: Our study collectively demonstrates that MGE progenitors are the cell of origin of CNS NB-FOXR2 and that FOXR2 activates the MEK/ERK signaling pathway, providing a potential therapeutic target., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Intranasal (i.n.) vaccination with adjuvant-free plasmid DNA encoding the leishmanial antigen LACK (LACK DNA) has shown to induce protective immunity against both cutaneous and visceral leishmaniasis in rodents. In the present work, we sought to evaluate the safety and effectiveness of d,l-glyceraldehyde cross-linked chitosan microparticles (CCM) as a LACK DNA non-intumescent mucoadhesive delivery system. CCM with 5 μm of diameter was prepared and adsorbed with a maximum of 2.4 % (w/w) of DNA with no volume alteration. Histological analysis of mouse nostrils instilled with LACK DNA / CCM showed microparticles to be not only mucoadherent but also mucopenetrant, inducing no local inflammation. Systemic safeness was confirmed by the observation that two nasal instillations one week apart did not alter the numbers of bronchoalveolar cells or blood eosinophils; did not alter ALT, AST and creatinine serum levels; and did not induce cutaneous hypersensitivity. When challenged in the footpad with Leishmania amazonensis , mice developed significantly lower parasite loads as compared with animals given naked LACK DNA or CCM alone. That was accompanied by increased stimulation of Th1-biased responses, as seen by the higher T-bet / GATA-3 ratio and IFN-γ levels. Together, these results demonstrate that CCM is a safe and effective mucopenetrating carrier that can increase the efficacy of i.n. LACK DNA vaccination against cutaneous leishmaniasis., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 Published by Elsevier Ltd.)

Leishmaniasis is a wide-spectrum disease caused by parasites from Leishmania genus. A well-modulated immune response that is established after the long-lasting clinical cure of leishmaniasis can represent a standard requirement for a vaccine. Previous studies demonstrated that Leishmania (Viannia) naiffi causes benign disease and its antigens induce well-modulated immune responses in vitro. In this work we aimed to identify the immunodominant proteins present in the soluble extract of L. naiffi (sLnAg) as candidates for composing a pan-specific anti-leishmaniasis vaccine. After immunoblotting using cured patients of cutaneous leishmaniasis sera and proteomics approaches, we identified a group of antigenic proteins from the sLnAg. In silico analyses allowed us to select mildly similar proteins to the host; in addition, we evaluated the binding potential and degree of promiscuity of the protein epitopes to HLA molecules and to B-cell receptors. We selected 24 immunodominant proteins from a sub-proteome with 328 proteins. Homology analysis allowed the identification of 13 proteins with the most orthologues among seven Leishmania species. This work demonstrated the potential of these proteins as promising vaccine targets capable of inducing humoral and cellular pan-specific immune responses in humans, which may in the future contribute to the control of leishmaniasis.

Optimal fluid management is critical during mechanical ventilation to mitigate lung damage. Under normovolemia and protective ventilation, pulmonary tensile stress during pressure-support ventilation (PSV) results in comparable lung protection to compressive stress during pressure-controlled ventilation (PCV) in experimental acute lung injury (ALI). It is not yet known whether tensile stress can lead to comparable protection to compressive stress in ALI under a liberal fluid strategy (LF). A conservative fluid strategy (CF) was compared with LF during PSV and PCV on lungs and kidneys in an established model of ALI. Twenty-eight male Wistar rats received endotoxin intratracheally. After 24 h, they were treated with CF (minimum volume of Ringer's lactate to maintain normovolemia and mean arterial pressure ≥70 mmHg) or LF (~4 times higher than CF) combined with PSV or PCV (V T  = 6 ml/kg, PEEP = 3 cmH 2 O) for 1 h. Nonventilated animals (n = 4) were used for molecular biology analyses. CF-PSV compared with LF-PSV: (1) decreased the diffuse alveolar damage score (10 [7.8-12] vs. 25 [23-31.5], p = 0.006), mainly due to edema in axial and alveolar parenchyma; (2) increased birefringence for occludin and claudin-4 in lung tissue and expression of zonula-occludens-1 and metalloproteinase-9 in lung. LF compared with CF reduced neutrophil gelatinase-associated lipocalin and interleukin-6 expression in the kidneys in PSV and PCV. In conclusion, CF compared with LF combined with PSV yielded less lung epithelial cell damage in the current model of ALI. However, LF compared with CF resulted in less kidney injury markers, regardless of the ventilatory strategy., (© 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Introduction: Most studies of transcranial direct current stimulation (tDCS) for motor deficits in patients with stroke administered few sessions of tDCS and with low current amplitude., Methods: During 2015 to 2019, we randomized 60 inpatients with ischemic/hemorrhagic stroke and motor deficits to true or sham tDCS. Transcranial direct current stimulation was administered at 2- to 3-mA current strength, twice daily, 6 days a week, for 2 weeks; anode and cathode were placed over ipsilesional and contralesional motor cortices, respectively. All patients received individualized motor and cognitive rehabilitation. Motor outcomes were assessed 1 day before and 1 day after the tDCS course using the Fugl-Meyer Assessment, the Jebson-Taylor Hand Function Test, and the Barthel index (all coprimary outcomes). Mood and cognition were also assessed. Motor outcomes were compared between groups using age, baseline scores, and latency to treatment as covariates. The study was prospectively registered (CTRI/2017/01/007733)., Results: The mean age of the patients was 46.9 years. The sample was 73.3% male. Six patients did not complete the study. The covariates were significantly related to motor outcomes. Although all patients showed motor improvements, after adjusting for covariates, tDCS was not superior to sham treatment on any motor, mood, or cognitive outcome. Laterality of hemispheric lesion influenced spatial but not motor outcomes with tDCS. One true tDCS patient developed blistering under the anode and was withdrawn from the study; 3 more reported transient itching during sessions., Conclusions: An intensive course of tDCS, as delivered in this study, does not improve motor, mood, and cognitive outcomes in ischemic/hemorrhagic stroke in patients undergoing individualized rehabilitation. The study provides important leads for directions for future research., Competing Interests: The authors have no conflicts of interest to report., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Several infectious diseases are associated with hypothalamic-pituitary-adrenal (HPA) axis disorders by elevating circulating glucocorticoids (GCs), which are known to have an immunosuppressive potential. We conducted this study in golden hamsters, a suitable model for human visceral leishmaniasis (VL), to investigate the relationship of Leishmania (L.) infantum infection on cortisol production and VL severity., Methods: L. infantum-infected (n = 42) and uninfected hamsters (n = 30) were followed-up at 30, 120, and 180 days post-infection (dpi). Plasma cortisol was analyzed by radioimmunoassay and cytokines, inducible nitric oxide synthase (iNOS), and arginase by RT-qPCR., Results: All hamsters showed splenomegaly at 180 dpi. Increased parasite burden was associated with higher arginase expression and lower iNOS induction. Cortisol levels were elevated in infected animals in all-time points evaluated. Except for monocytes, all other leucocytes showed a strong negative correlation with cortisol, while transaminases were positively correlated. Immunological markers as interleukin (IL)-6, IL-1β, IL-10, and transforming growth-factor-β (TGF-β) were positively correlated to cortisol production, while interferon-γ (IFN-γ) presented a negative correlation. A network analysis showed cortisol as an important knot linking clinical status and immunological parameters., Conclusions: These results suggest that L. infantum increases the systemic levels of cortisol, which showed to be associated with hematological, biochemical, and immunological parameters associated to VL severity., Competing Interests: The authors have declared that no competing interests exist.

The golden hamster is a suitable model for studying cutaneous leishmaniasis (CL) due to Leishmania  ( Viannia )  braziliensis. Immunopathological mechanisms are well established in the L. (L.) major -mouse model, in which IL-4 instructs a Th2 response towards progressive infection. In the present study, we evaluated the natural history of L.   braziliensis infection from its first stages up to lesion establishment, with the aim of identifying immunological parameters associated with the disease outcome and parasitism fate. To this end, hamsters infected with 10 4 , 10 5 , or 10 6 promastigotes were monitored during the first hours (4h, 24h), early (15 days, 30 days) and late (50 days) post-infection (pi) phases. Cytokines, iNOS and arginase gene expression were quantified in the established lesions by reverse transcription-quantitative PCR. Compared to the 10 5 or 10 6 groups, 10 4 animals presented lower lesions sizes, less tissue damage, and lower IgG levels. Basal gene expression in normal skin was high for TGF-β, and intermediary for TNF, IL-6, and IL-4. At 4hpi, no cytokine induction was observed in the 10 4 group, while an upregulation of IL-6, IL-10, and IL-4 was observed in the 10 6 group. At 15dpi, lesion appearance was accompanied by an increased expression of all assessed cytokines, markedly in the 10 5 and 10 6 groups. Upregulation of all investigated cytokines was observed in the late phase, although less expressive in the 10 4 group. IFN-γ was the depending variable influencing tissue damage, while IL-6 was associated to parasite load. The network correlating gene expression and clinical and laboratorial parameters indicated inoculum-independent associations at 15 and 30dpi. A strong positive network correlation was observed in the 10 4 group, but not in the 10 5 or 10 6 groups. In conclusion, IL-4, IL-6, IL-10, and TGF-β are linked o L. braziliensis progression. However, a balanced cytokine network is the key for an immune response able to reduce the ongoing infection and reduce pathological damage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Paiva, Ribeiro-Romão, Resende-Vieira, Braga-Gomes, Oliveira, Saavedra, Silva-Couto, Albuquerque, Moreira, Pinto, Da-Cruz and Gomes-Silva.)

Leishmania (Viannia) braziliensis is one of the main etiological agents of tegumentary leishmaniasis in Latin America. The establishment of a successful infection in host cells requires several key events including phagocytosis, phagolysosomal maturation impairment, and parasite replication. Autophagy is accountable for the physiological turnover of cellular organelles, degradation of macromolecular structures, and pathogen elimination. In many cases, autophagy control leads to a successful infection, both impairing pathogen elimination or providing nutrients. Here, we have investigated the relationship between autophagy and L. braziliensis infection. We observed that BECLIN1 expression was upregulated early on infection in both in vitro macrophage cultures and biopsies of cutaneous lesions from L. braziliensis infected patients. On the other hand, LC3B expression was downregulated in cutaneous lesions biopsies. A transient pattern of LC3+ cells was observed along L. braziliensis infection, but the number of LC3 puncta did not vary. Additionally, autophagy induction, with rapamycin treatment or through starvation, reduced infection. As expected, rapamycin increased the percentage of LC3+ cells and the number of puncta, but the presence of parasite restricted this effect, indicating LC3-associated autophagy impairment by L. braziliensis. Finally, silencing LC3B but not BECLIN1 promoted infection, confirming BECLIN1 independent and LC3B-related control by the parasite. Taken together, these data indicate macrophage autophagic machinery manipulation by L. braziliensis, resulting in successful establishment and survival into the host cell., (Copyright © 2021. Published by Elsevier B.V.)

Background: Pressure-support ventilation may worsen lung damage due to increased dynamic transpulmonary driving pressure. The authors hypothesized that, at the same tidal volume (VT) and dynamic transpulmonary driving pressure, pressure-support and pressure-controlled ventilation would yield comparable lung damage in mild lung injury., Methods: Male Wistar rats received endotoxin intratracheally and, after 24 h, were ventilated in pressure-support mode. Rats were then randomized to 2 h of pressure-controlled ventilation with VT, dynamic transpulmonary driving pressure, dynamic transpulmonary driving pressure, and inspiratory time similar to those of pressure-support ventilation. The primary outcome was the difference in dynamic transpulmonary driving pressure between pressure-support and pressure-controlled ventilation at similar VT; secondary outcomes were lung and diaphragm damage., Results: At VT = 6 ml/kg, dynamic transpulmonary driving pressure was higher in pressure-support than pressure-controlled ventilation (12.0 ± 2.2 vs. 8.0 ± 1.8 cm H2O), whereas static transpulmonary driving pressure did not differ (6.7 ± 0.6 vs. 7.0 ± 0.3 cm H2O). Diffuse alveolar damage score and gene expression of markers associated with lung inflammation (interleukin-6), alveolar-stretch (amphiregulin), epithelial cell damage (club cell protein 16), and fibrogenesis (metalloproteinase-9 and type III procollagen), as well as diaphragm inflammation (tumor necrosis factor-α) and proteolysis (muscle RING-finger-1) were comparable between groups. At similar dynamic transpulmonary driving pressure, as well as dynamic transpulmonary driving pressure and inspiratory time, pressure-controlled ventilation increased VT, static transpulmonary driving pressure, diffuse alveolar damage score, and gene expression of markers of lung inflammation, alveolar stretch, fibrogenesis, diaphragm inflammation, and proteolysis compared to pressure-support ventilation., Conclusions: In the mild lung injury model use herein, at the same VT, pressure-support compared to pressure-controlled ventilation did not affect biologic markers. However, pressure-support ventilation was associated with a major difference between static and dynamic transpulmonary driving pressure; when the same dynamic transpulmonary driving pressure and inspiratory time were used for pressure-controlled ventilation, greater lung and diaphragm injury occurred compared to pressure-support ventilation.

Background: The authors hypothesized that low tidal volume (VT) would minimize ventilator-induced lung injury regardless of the degree of mechanical power. The authors investigated the impact of power, obtained by different combinations of VT and respiratory rate (RR), on ventilator-induced lung injury in experimental mild acute respiratory distress syndrome (ARDS)., Methods: Forty Wistar rats received Escherichia coli lipopolysaccharide intratracheally. After 24 h, 32 rats were randomly assigned to be mechanically ventilated (2 h) with a combination of different VT (6 ml/kg and 11 ml/kg) and RR that resulted in low and high power. Power was calculated as energy (ΔP,L/E,L) × RR (ΔP,L = transpulmonary driving pressure; E,L = lung elastance), and was threefold higher in high than in low power groups. Eight rats were not mechanically ventilated and used for molecular biology analysis., Results: Diffuse alveolar damage score, which represents the severity of edema, atelectasis, and overdistension, was increased in high VT compared to low VT, in both low (low VT: 11 [9 to 14], high VT: 18 [15 to 20]) and high (low VT: 19 [16 to 25], high VT: 29 [27 to 30]) power groups. At high VT, interleukin-6 and amphiregulin expressions were higher in high-power than in low-power groups. At high power, amphiregulin and club cell protein 16 expressions were higher in high VT than in low VT. Mechanical energy and power correlated well with diffuse alveolar damage score and interleukin-6, amphiregulin, and club cell protein 16 expression., Conclusions: In experimental mild ARDS, even at low VT, high mechanical power promoted ventilator-induced lung injury. To minimize ventilator-induced lung injury, low VT should be combined with low power.

Introduction: Electroconvulsive therapy (ECT) is associated with memory deficits on neuropsychological assessment. The association of ECT with nonmemory cognitive deficits has been poorly studied., Methods: We present a 40-year-old woman who showed a bizarre form of spatial cognition impairment on a subtest of the Tactual Performance Test (TPT) after recovering from depression with 6 alternate day, thrice-weekly, inpatient ECT treatments. This woman was part of a naturalistic, nonblind study that examined nonmemory cognitive deficits in antidepressant-treated depressed patients who did and did not receive ECT., Results: The impairment was in the form of bizarrely drawn reproductions of differently shaped wooden blocks that had been presented to the patient when she was blindfolded. The impairment was still evident when she was retested (3 hours later) under substantially simplified conditions but was much attenuated approximately 2.5 weeks later., Conclusions: On the surface, it seems that ECT had induced severe impairment in spatial cognition and that the impairment showed the familiar pattern of attenuation with the passage of time. However, another recovered patient in the study, who did not receive ECT, also showed substantial spatial deficits on the same subtest of the TPT, and the attenuation of the deficits across time in the ECT-treated patient was probably a result of repeated exposure to the task. We suggest that not all patients who seem to experience spectacular cognitive impairment after ECT have deficits that are attributable to ECT.

Competing Interests: There are no conflicts of interest.

Competing Interests: There are no conflicts of interest.

Background: Cutaneous leishmaniasis (CL) is a neglected disease with a broad spectrum of clinical manifestations, ranging from small cutaneous nodules to severe mucosal tissue destruction. Leishmania (Viannia) braziliensis is the main species attributed to CL in the Americas. However, studies of experimental infection are limited in the murine model due to the self-resolutive pattern of the disease. Previously, our group demonstrated that the hamster model reproduces many of the clinical and histopathological features observed in humans. Herein, we standardized a RT-qPCR gene expression assay to evaluate a panel of immunological markers and a qPCR assay in order to quantify with high sensitivity and reproducibility the parasite load in skin lesions., Methods: Hamsters were intradermally infected in the footpad with 10(5) promastigotes of L. (V.) braziliensis and 110 days post-infection skin lesions and popliteal lymph nodes were removed for RNA and DNA extraction, both from the same tissue fragment. Gene expression of IFN-ɣ, IL-10, TGF-β TNF, IL-4, IL-6, iNOS and arginase were measured using non-infected animal tissue as a calibrator. Parasite load was quantified from DNA extracted from lesions by qPCR targeting Leishmania kDNA and normalized by hamster GAPDH, using a SYBR Green-based absolute quantification methodology., Results: A relative quantification RT-qPCR assay was standardized for the evaluation of mRNA levels from skin and lymph node samples of golden hamsters, with PCR efficiencies ranging from 92.3 to 116.4 %. In uninfected animals, higher basal mRNA levels in lymph nodes were observed for IFN-ɣ, TGF-β, TNF and IL-4 (111.4 ± 92.2; 5.6 ± 1.2; 5.3 ± 1.7; and 60.3 ± 26.8, respectively) in comparison to skin. In golden hamsters infected with L. (V.) braziliensis, an increase in the expression of all immunological markers evaluated was observed, ranging from 2.7 ± 0.2 for TGF-β to 1018.5 ± 809.0 for iNOS in skin lesions, and 2.4 ± 1.6 for TGF-β to 600.2 ± 666.4 for iNOS in popliteal lymph nodes. Interestingly, significantly higher levels of IFN-ɣ, TNF and IL-10 mRNA were observed in skin in comparison to lymph nodes, while a lower significant level of arginase mRNA was observed in skin. In parallel, parasite loads were quantified by qPCR from the skin lesions of infected animals, ranging from 27.0 to 6647.0, with a median of 553.4 (416.7-1504.0) parasites/mg skin equivalents, whereas lesion size varied from 0.3 to 3.1 mm. Despite the tendency of larger lesions to present higher parasite load, the correlation observed was not statistically significant., Conclusions: In this study, we describe for the first time a sensitive, reproducible and cheaper molecular assay to quantify from the same tissue fragment the gene expression of immunological markers and the parasite load in skin lesions, observing a mixed profile of immune response in the hamster model infected by L. (V.) braziliensis.

Background: The World Bank and Lancet Commission in 2015 have prioritized surgery in Low-Income Countries (LIC) and Lower-Middle Income Countries (LMICs). This is consistent with the shift in the global burden of disease from communicable to noncommunicable diseases over the past 20 years. Essential surgery must be performed safely, with adequate anesthesia monitoring and intervention. Unfortunately, a huge barrier to providing safe surgery includes the paucity of an anesthesia workforce. In this study, we qualitatively evaluated the anesthesia capacity of Mozambique, a LIC in Africa with limited access to anesthesia and safe surgical care. Country-based solutions are suggested that can expand to other LIC and LMICs., Methods: A comprehensive review of the Mozambique anesthesia system was conducted through interviews with personnel in the Ministry of Health (MOH), a school of medicine, a public central referral hospital, a general first referral hospital, a private care hospital, and leaders in the physician anesthesia community. Personnel databases were acquired from the MOH and Maputo Central Hospital., Results: Quantitative results reveal minimal anesthesia capacity (290 anesthesia providers for a population of >25 million or 0.01:10,000). The majority of physician anesthesiologists practice in urban settings, and many work in the private sector. There is minimal capacity for growth given only 1 Mozambique anesthesia residency with inadequate resources. The most commonly perceived barriers to safe anesthesia in this critical shortage are lack of teachers, lack of medical student interest in and exposure to anesthesia, need for more schools, low allocation to anesthesia from the list of available specialist prospects by MOH, and low public payments to anesthesiologists. Qualitative results show assets of a good health system design, a supportive environment for learning in the residency, improvement in anesthetic care in past decades, and a desire for more educational opportunities and teachers., Conclusions: Mozambique has a strong health system design but few resources for surgery and safe anesthesia. At present, similar to other LICs, human resources, access to essential medicines, and safety monitoring limit safe anesthesia in Mozambique.

Background: Depression is among the commonest of psychiatric disorders, and inflammatory mechanisms have been suggested to play a role in its pathophysiology. Interferons are a superfamily of proinflammatory cytokines that play a role in host defence mechanisms. Interferons are used in the treatment of a variety of autoimmune (e.g. multiple sclerosis), viral (e.g. chronic hepatitis B and C), and malignant (e.g. malignant melanoma, hairy cell leukemia) disorders; depression, however, is a notable and clinically troublesome adverse effect., Objective: This article seeks to present a simple explanation and update for the reader about what interferons are, how interferons are classified, the clinical conditions in which interferons are used, the occurrence of depression as a clinical adverse effect of interferon therapy, possible mechanisms that explain interferon-related depression, the treatment of interferon-related depression, and the prevention of interferon-related depression., Methods: A qualitative literature review is presented., Results and Conclusions: Irrespective of the indication for IFN therapy, IFNs are associated with a 30- 70% risk of treatment-emergent depression. This risk could be due to the IFN, or to an interaction between the IFN and the indication for which it was prescribed. Various neurohormonal, neurochemical, neurohistological, and other mechanisms have been put forth to explain IFN-related depression. Prophylactic treatment with antidepressants reduces the risk of IFN-related depression; antidepressants also effectively treat the condition. Recent alternatives to IFNs have shown to decrease the risk of treatment-emergent depression.

Microcystin-LR (MC-LR) is a harmful cyanotoxin able to induce adverse outcomes in the respiratory system. We aimed to examine the lungs and nasal epithelium of mice following a sub-chronic exposure to MC-LR. Swiss mice were intranasally instilled with 10 μL of distilled water (CTRL, n = 10) or 6.7 ng/kg of MC-LR diluted in 10 μL of distilled water (TOX, n = 8) during 30 consecutive days. Respiratory mechanics was measured in vivo and histology measurements (morphology and inflammation) were assessed in lungs and nasal epithelium samples 24 h after the last intranasal instillation. Despite the lack of changes in the nasal epithelium, TOX mice displayed an increased amount of PMN cells in the lungs (× 10(-3)/μm(2)), higher lung static elastance (cmH2O/mL), resistive and viscoelastic/inhomogeneous pressures (cmH2O) (7.87 ± 3.78, 33.96 ± 2.64, 1.03 ± 0.12, 1.01 ± 0.08, respectively) than CTRL (5.37 ± 4.02, 26.65 ± 1.24, 0.78 ± 0.06, 0.72 ± 0.05, respectively). Overall, our findings suggest that the nasal epithelium appears more resistant than lungs in this model of MC-LR intoxication., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Previous results have shown that oral and intranasal administration of particulate Leishmania (Leishmania) amazonensis antigens (LaAg) partially protects mice against L. amazonensis infection. However, vaccination studies on species of the subgenus Viannia, the main causative agent of cutaneous and mucosal leishmaniasis in the Americas, have been hampered by the lack of easy-to-handle bio-models that accurately mimic the human disease. Recently, we demonstrated that the golden hamster is an appropriate model for studying the immunopathogenesis of cutaneous leishmaniasis caused by L. (Viannia) braziliensis. Using the golden hamster model, our current study investigated whether the protective effect of intranasal immunisation with LaAg can be extended to L. braziliensis infection., Methodology/principal Findings: Golden hamsters vaccinated with either two intranasal (IN) doses of LaAg (10 µg) or two intramuscular doses of LaAg (20 µg) were challenged 2 weeks post-vaccination with L. braziliensis. The results showed that IN immunisation with LaAg significantly reduced lesion growth and parasitic load as well as serum IgG and IgG2 levels. At the experimental endpoint on day 114 post-infection, IN-immunised hamsters that were considered protected expressed IFN-γ and IL10 mRNA levels that returned to uninfected skin levels. In contrast to the nasal route, intramuscular (IM) immunisation failed to provide protection., Conclusions/significance: These results demonstrate for the first time that the nasal route of immunisation can induce cross protection against L. braziliensis infection.

The golden hamster (Mesocricetus auratus) is a susceptible model to Leishmania (Viannia) spp.; however, available studies employ different infection protocols, which account for clinical and pathological presentation differences. Herein, L. (V.) braziliensis preparations were standardized to contain 10(4), 10(5), or 10(6) parasites to determine an optimal inoculum that ensured cutaneous lesions without causing a disseminated infection in hamsters. Lesion development was followed for 105 days by size measurements, and skin, draining lymph node, spleen, and sera were investigated to check parasite load, spleen visceralization, cytokine expression, histopathological changes, and anti-Leishmania IgG levels. The lesion emergence time was inversely proportional to the parasite concentration in the inocula. Animals infected by 10(4) parasites presented nodular lesions, while those infected with 10(6) parasites often exhibited ulcerated lesions. The differences in the final lesion sizes were observed between 10(4) and 10(5) inocula or 10(4) and 10(6) inocula. High IFNG expression, anti-Leishmania IgG levels, and parasite load occurred independently of the inoculum used. A mild inflammatory skin involvement was observed in animals infected with 10(4) parasites, while extensive tissue damage and parasite spleen visceralization occurred with 10(5) and 10(6) parasites. These results indicate that inocula with different concentrations of parasites generate differences in the time of lesion emergence, clinical presentation, and systemic commitment, despite high and similar IFNG expression and parasite load. This suggests that a modulation in the immune response to different parasite numbers occurs in an early phase of the infection, which could dictate the establishment and magnitude of the chronic phase of the disease., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The interest in developing new sunscreens is increasing due to the harmful effects of UV radiation on the skin, such as erythema, accelerated skin ageing (photoageing) and the induction of skin cancer. However, many molecular sunscreens penetrate into the skin causing photoallergies, phototoxic reactions and skin irritation. Thus, the aim of this work was the preparation and characterization of polymeric and solid lipid nanoparticles to act carriers of benzophenone-3 (BZ3), aiming to improve the safety of sunscreen products by increasing the sun protection factor (SPF), decreasing BZ3 skin penetration and decreasing BZ3 concentration in sunscreen formulation. BZ3 was encapsulated in poly(epsilon-caprolactone) (PCL) nanoparticles by the nanoprecipitation method and in solid lipid nanoparticles (SLN) by the hot high pressure homogenization method. The particles were stable for 40 days. The BZ3 encapsulated in PCL nanoparticles was released faster than BZ3 encapsulated in SLN. The sun protection factor increased when BZ3 was encapsulated in both nanostructures. However, BZ3 encapsulated in PCL nanoparticles decreased its skin permeation more than SLN-BZ3. Furthermore, BZ3 encapsulated in SLN did not exhibit cytotoxic or phototoxic effects in human keratinocytes (HaCaT cells) and BABL/c 3T3 fibroblasts, whereas PCL nanoparticles with BZ3 showed phototoxic potential in HaCaT cells. Nevertheless, BZ3 free and encapsulated in PCL nanoparticles or in SLN did not show allergic reactions in mice. Our results suggest that these nanostructures are interesting carriers for sunscreen.

The objective of this work was to verify if hydrophilic gels containing benzophenone-3 loaded nanocapsules (HG-NCBZ3) could improve the sunscreen in vitro effectiveness against UVA radiation and its photostability compared to a conventional hydrogel containing the free sunscreen (HG-BZ3). In parallel, the immune response of the nanostructured system was evaluated by mouse ear swelling test and the local lymph node assay. The nanocapsules were prepared by interfacial deposition of poly(epsilon-caprolactone) and characterized in terms of particle size, polydispersity index, zeta potential, drug content and encapsulation efficiency. HG-NCBZ3 UV scans showed higher absorption intensity values than HG-NCplacebo, prepared using unloaded nanocapsules. Films of the gels were irradiated with UVA light and the BZ3 recovery was evaluated by HPLC. BZ3 recovery decreased from 100% to 29% for HG-BZ3 and to 56% for HG-NCBZ3 after 13 h. After wavelength scans within 13 h, the relative areas under the curves (AUC) decreased from 1.00 to 0.62 for HG-BZ3 and remained constant for HG-NCBZ3. Sensitization assay showed that stimulation indexes lower than 3 for all the hydrogel samples. Formulations did not induce increases higher than 10% in ear swelling, indicating that the hydrogels did not cause cutaneous sensitization in mice. The nanoencapsulation improved both the photostability and the effectiveness of BZ3 compared to the non-encapsulated sunscreen and the topical application of free and nanoencapsulated BZ3 did not produce significant allergy response in mice.

Twenty-two synthetic analogues of neolignans comprising beta-ketoethers and beta-ketosulfides were obtained from condensation reactions among beta-bromoketones and phenols or thiophenols, respectively, in basic solutions, and assayed in vitro for activity against intracellular Leishmania amazonensis and Leishmania donovani amastigotes, the causative agents of cutaneous and visceral leishmaniasis. The highest selective activity was found for compounds with sulfur bridges, whereas beta-ketosulphoxides and beta-ketosulphones had significantly less growth inhibitory activity. Compounds 2-[(4-chlorophenyl)thio]propan-1-one and 1-(3,4-dimethoxy)-2-[(4-methylphenyl)thio]propan-1-one were the most potent, inhibiting the growth parasite species by over 90% at microgram/mL, but only compound 1-(3,4-dimethoxy)-2-[(4-methylphenyl)thio]propan-1-one was selectively toxic to the parasites.

We previously showed the opposing effect of systemic and mucosal vaccination with whole Leishmania amazonensis antigen (LaAg). Here, the role played by lipophosphoglycan (LPG) as the key disease-promoting component of intramuscular (i.m.) LaAg and its usefulness as a defined intranasal vaccine was investigated in murine cutaneous leishmaniasis. BALB/c mice were twice vaccinated by the i.m. route with 25mug of intact LaAg or with LaAg that was pretreated with anti-LPG 3A1-La monoclonal antibody, prior to infection with L. amazonensis. LPG neutralization rendered the otherwise disease-promoting LaAg antigen protective, as observed by the smaller lesion sizes and reduced parasite burden. The increased resistance was accompanied by a markedly lower antigen-driven TGF-beta and IL-10 responses in the lesion-draining lymph nodes, concomitant with significantly higher IFN-gamma production. To test for intranasal efficacy, 10 microg of affinity-purified LPG and its parental LaAg were twice instilled in the nostrils prior to L. amazonensis infection. In both cases, similarly slower lesion growth and lower parasite burden were found that was associated with increased IFN-gamma and IL-10 responses in the lesion-draining lymph nodes. These results support a role for LPG in the dual route-related effect of LaAg and shows its strong potential as a defined needle-free and adjuvant-free vaccine for cutaneous leishmaniasis.

We previously showed that intranasal (i.n.) vaccination with pCIneo plasmid encoding the leishmanial LACK gene (pCIneo-LACK) induces long-lasting protective immunity against cutaneous leishmaniasis in mice. In this work, we proposed to investigate whether the efficacy of i.n. pCIneo-LACK is extensive to visceral leishmaniasis. BALB/c mice received two i.n. doses of 30 microg pCIneo-LACK prior to intravenous (i.v.) infection with Leishmania chagasi. Vaccinated mice developed significantly lower parasite burden in the liver and spleen than control mice receiving empty pCIneo or saline. The spleen cells of vaccinated mice produced significantly increased IFN-gamma and IL-4 concomitant with decreased IL-10 production during infection. Serum levels of specific IgG were elevated whereas TNF-alpha were decreased as compared with controls. These results show that the practical needle-free i.n. pCIneo-LACK vaccine displays potential broad-spectrum activity against leishmaniasis.

In this study we determined whether exposing mice to hyperbaric oxygen (HBO) would alter various disease parameters of a susceptible mouse strain infected with Leishmania amazonensis. BALB/c mice exposed to HBO (100% O2 at a pressure of 2.5 ATA, 1h before parasite inoculation and subsequently for 20 days) showed significant delay in lesion development and reduction in lesion parasite burdens compared with HBO-unexposed mice. Circulating levels of interferon gamma (IFN-gamma) and tumor necrosis factor (TNF-alpha) were significantly elevated in HBO-exposed as compared to HBO-unexposed mice. Concanavalin A-stimulated lymph nodes cultures from HBO-exposed mice released significantly more IFN-gamma and less interleukin 10 (IL-10) than cultures from HBO-unexposed mice, consistent with a skewed Th1 response. These results demonstrate, for the first time, that HBO can play a pathogen control role during leishmaniasis. Further studies are needed to elucidate whether hyperoxia alone or increased atmospheric pressure alone can exert a similar effect.

Leishmania amazonensis and Leishmania braziliensis are the main causal agents of anergic diffuse cutaneous leishmaniasis and hyperergic mucosal leishmaniasis in man, respectively. In this work we demonstrate that intramuscular vaccination of BALB/c mice with whole antigens of L. amazonensis (LaAg) but not L. braziliensis (LbAg) results in increased susceptibility to cutaneous leishmaniasis. LaAg vaccination resulted in an increased capacity of the draining lymph nodes to produce IL-10 and TGF-beta during antigen recall responses. In vitro cultivation with LaAg but not LbAg induced increased apoptosis of CD8+ T cells. Following infection with L. amazonensis, LaAg-vaccinated mice produced significantly more TGF-beta and a higher serum IgG1/IgG2a antibody ratio compared with LbAg-vaccinated and non-vaccinated animals. The association of TGF-beta with enhanced susceptibility to infection was confirmed in mice co-vaccinated with LaAg and neutralizing anti-TGF-beta antibodies. Upon parasite challenge, these animals developed much smaller lesion sizes and parasite burdens, comparable with non-vaccinated controls. The disease-promoting effect of LaAg vaccination is not a general event, as in contrast to BALB/c, the disease outcome in C57Bl/6 mice was unaltered. Together, these findings indicate that species-specific components of L. amazonensis activate overt TGF-beta production that predisposes more susceptible individuals to aggravated disease following vaccination.

Leishmania amazonensis is the main agent of diffuse cutaneous leishmaniasis, a disease associated with anergic immune responses. In this study we show that the crude antigen of Leishmania amazonensis (LaAg) but not L. braziliensis promastigotes (LbAg) contains substances that suppress mitogenic and spontaneous proliferative responses of T cells. The suppressive substances in LaAg are thermoresistant (100 degrees C/1h) and partially dependent on protease activity. T cell anergy was not due to a decreased production of growth factors as it was not reverted by addition of exogenous IL-2, IL-4, IFN-gamma or IL-12. LaAg did not inhibit anti-CD3-induced T cell activation, suggesting that anergy was due to a defect in antigen presentation. It was also not due to cell necrosis, but was accompanied by expressive DNA fragmentation in lymph node cells, indicative of apoptosis. Although pre-incubation of macrophages with LaAg prevented their capacity to present antigens, this effect was not due to apoptosis of the former. These results suggest that the T cell anergy found in diffuse leishmaniasis may be the result of parasite antigen-driven apoptosis of those cells following defective antigen presentation.

We have previously demonstrated that oral delivery of a disease-promoting particulated antigen of Leishmania amazonensis (LaAg) partially protects mice against cutaneous leishmaniasis. In the present work, we sought to optimize a mucosal vaccine by using the intranasal route for delivery of different antigen preparations, including (i) LaAg, (ii) soluble recombinant p36/LACK leishmanial antigen (LACK), and (iii) plasmid DNA encoding LACK (LACK DNA). BALB/c mice that received two intranasal doses of 10 microg of LaAg and were challenged 1 week postvaccination with L. amazonensis developed delayed but effective control of lesion growth. A diminished parasite burden was accompanied by enhancement of both gamma interferon (IFN-gamma) and interleukin-10 levels in the lesion-draining lymph nodes. The vaccine efficacy improved with time. At 4 months postvaccination, when a strong parasite-specific TH1-type response was present in vivo, the infection was controlled for at least 5 months after challenge. In contrast to the particulated LaAg, soluble LACK (10 microg/dose) had no effect. Interestingly, LACK DNA (30 microg/dose), but not empty DNA, promoted rapid and durable protective immunity. Parasite growth was effectively controlled, and at 5 months after challenge LACK-reactive cells in both the mucosal and lesion-draining lymph nodes produced high levels of IFN-gamma. These results demonstrate for the first time the feasibility of using the intranasal route for long-lived memory vaccination against cutaneous leishmaniasis with adjuvant-free crude antigens or DNA.

The induction of oral tolerance against disease-inducing antigens has emerged as a feasible strategy to prevent immunopathologies. In this study, we investigated the effect of oral immunization with whole antigens of Leishmania amazonensis promastigotes (LaAg) on murine cutaneous leishmaniasis. We found that two oral doses with 100 microg LaAg rendered BALB/c and C57BL/6 mice more resistant against subsequent infection with L. amazonensis. The oral vaccine also partially protected BALB/c mice against Leishmania major infection. Unlike the oral route, hepatic immunization was without effect, indicating a requirement for antigen passage through the gut mucosa. Oral LaAg significantly impaired the capacity of infected BALB/c mice to mount a disease-associated hypersensitivity response, compatible with peripheral tolerization. Both IFN-gamma and IL-10, but not IL-4 were greatly elevated in the mesenteric lymph nodes whereas only IFN-gamma was increased in the peripheral lymph nodes, compatible with a TH1 cytokine response. Gamma delta TCR+ T cells may be an important component in antigenic sensitization of the gut mucosa since their depletion during oral immunization reverted protection. These results demonstrate for the first time the feasibility of using the oral route of immunization to induce protection against cutaneous leishmaniasis using a crude parasite antigen.