Pam3CSK4 adjuvant given intranasally boosts anti-Leishmania immunogenicity but not protective immune responses conferred by LaAg vaccine against visceral leishmaniasis

The use of adjuvants in vaccine formulations is a well-established practice to improve immunogenicity and protective immunity against diseases. Previously, we have demonstrated the feasibility of intranasal vaccination with the antigen of killed Leishmania amazonensis promastigotes (LaAg) against experimental leishmaniasis. In this work, we sought to optimize the immunogenic effect and protective immunity against murine visceral leishmaniasis conferred by intranasal delivery of LaAg in combination with a synthetic TLR1/TLR2 agonist (Pam3CSK4). Intranasal vaccination with LaAg/PAM did not show toxicity or adverse effects, induced the increase of delayed-type hypersensitivity response and the production of inflammatory cytokines after parasite antigen recall. However, mice vaccinated with LaAg/PAM and challenged with Leishmania infantum presented significant reduction of parasite burden in both liver and spleen, similar to those vaccinated with LaAg. Although LaAg/PAM intranasal vaccination had induced higher frequencies of specific CD4⁺ and CD8⁺ T cells and increased levels of IgG2a antibody isotype in serum, both LaAg and LaAg/PAM groups presented similar levels of IL-4 and IFN-y and decreased production of IL-10 when compared to controls. Our results provide the first evidence of the feasibility of intranasal immunization with antigens of killed Leishmania in association with a TLR agonist, which may be explored for developing an effective and alternative strategy for vaccination against visceral leishmaniasis.     

Diverse FSH and testosterone signaling pathways in the Sertoli cell.

FSH and testosterone exert different regulatory effects on the seminiferous epithelium; they act through multiple and complex signaling routes to direct the development of the germ cells into mature spermatozoa. In addition to their well-known pathways of action, both hormones have recently been recognized to have new signaling routes that are linked to the Ca(2+) ion, including, among others, the regulation of cell proliferation by FSH and the regulation of cell migration by testosterone.     

Baroreflex dysfunction in rats submitted to protein restriction

Earlier studies from the authors' laboratory showed that malnourishment induces alterations in the cardiovascular homeostasis increasing the basal mean arterial pressure and heart rate. In this study, the authors evaluated whether the sympathetic and parasympathetic efferent activities contribute to changes in the cardiovascular homeostasis through altered modulation of the arterial baroreflex of malnourished rats. After weaning, male Fischer rats were given 15% (Normal Protein--NP) or 6% (Low Protein--LP) protein diet for 35 d. The baroreflex gain and latency were evaluated before and after selective autonomic blockades in control and malnourished rats. It was observed that malnourishment affected the baroreflex gain in response to activation and deactivation of the arterial baroreflex. Moreover, malnourished rats showed increased baroreflex latency as compared to that of control rats. Regarding the autonomic efferent activity directed to the heart, the data showed increased sympathetic and decreased parasympathetic efferent activities in malnourished rats, and such alterations could be related to the observed changes in the arterial baroreflex gain as well as in the basal mean arterial pressure and heart rate.     

Non-classic androgen actions in Sertoli cell membrane in whole seminiferous tubules: effects of nandrolone decanoate and catechin

Studies show a mechanism of action of testosterone, nandrolone and catechin as agonists of the membrane androgen receptor. The aim of this work is to investigate the non-classical effect of androgens and catechin in Sertoli cells from immature rats. The membrane potential of Sertoli cells in whole seminiferous tubules was recorded using a standard single microelectrode technique. It was performed a topical application of testosterone (1 μM), nandrolone (0.1, 0.5 and 1 μM) and the flavonoid catechin (0.1, 0.5 and 1 μM) alone and also after infusion with flutamide (1 μM), diazoxide (100 μM) or U73122 (1 μM). The immature testes were incubated for 5 min in KRb with (45)Ca(2+), with or without nandrolone (1 μM). The results were given as mean±SEM. The data were analyzed using ANOVA for repeated measures with Bonferroni post-test. Testosterone produces a depolarization in the membrane potential at 120 s after application. Catechin (1 μM) and nandrolone (1 μM) have shown a similar response to testosterone: depolarization at 120 s after the application. The same response of catechin and nandrolone was observed at different doses. The effects of testosterone, catechin and nandrolone were not affected after perfusion with flutamide. Perfusion with diazoxide and U73122 nullified the effect of nandrolone (1 μM) and catechin (1 μM). Nandrolone and testosterone increased (45)Ca(2+) uptake with or without flutamide within 5min. These results indicate that nandrolone and catechin act through a receptor on the plasmatic membrane, as well as testosterone, showing a non-classical pathway in Sertoli cells from immature rat testes.     

Nutrient Uptake and Organic Acid Anion Metabolism in Lupins and Peas Supplied with Nitrate

Unlike many plants reported in the literature, lupins do notexcrete OH^- in amounts equivalent to the net excess of inorganicanion uptake over inorganic cation uptake. To investigate themechanisms involved in the maintenance of charge balance, nutrientuptake and organic anion accumulation of lupins and peas suppliedwith a range of NO^-₃ concentrations, were compared. Lupins absorbed less NO^-₃ than peas on a dry weight basis, whichlargely accounted for the smaller excess of anion uptake overcation uptake in lupins than in peas at the same NO^-₃ supply.When anion uptake exceeded cation uptake, peas excreted an equivalentcharge of OH^-, whereas lupins excreted much smaller amountsof OH^- than the excess of anion over cation uptake. It was calculatedthat lupins excreted significant amounts of organic anions whenanion uptake exceeded cation uptake, whereas organic anion excretionfrom peas was negligible, regardless of their NO^-₃ supply andcation-anion balance. In this study, organic anion excretion was measured from lupinroots grown in near-sterile conditions while supplied with NO^-₃at 0, 500 and 2000 µM. Although complete sterility wasnot achieved, there was close agreement between the organicanion excreted and the excess anion over cation uptake.Copyright1994, 1999 Academic Press Lupinus angustifolius L., Pisum sativum L., organic acid, nutrient uptake     

Testosterone rapidly stimulates insulin release from isolated pancreatic islets through a non-genomic dependent mechanism.

The action of testosterone on the 45Ca2+ uptake and insulin secretion was studied in short-term experiments using isolated pancreatic islets of Langerhans. Testosterone (1 microM) stimulated 45Ca2+ uptake within 60 seconds of incubation on similar proportion than tolbutamide. Also, the hormone rapidly increased insulin release (34%; 180 seconds) on the presence of non-stimulatory concentrations of glucose (3 mM). Impermeant testosterone-BSA significantly stimulated the secretion of insulin to a lower percentage (10%). The action of the hormone is specific--neither 17beta-E2 nor progesterone stimulated insulin secretion in the presence of 3 mM glucose. The action of testosterone on insulin secretion was dose-dependent, and at rat plasma physiological concentrations (25 nM), stimulus was 17% (p < 0.05). In conclusion, in isolated pancreatic islets experiments, physiological concentration of testosterone rapidly stimulate insulin secretion and 45Ca2+ uptake through a membrane bound mechanism.     

Functional characterization of the Aspergillus fumigatusPHO80 homologue

Phosphate is an ion that is essential for fungal growth. The systems for inorganic phosphate (P_i) acquisition in eukaryotic cells (PHO) have been characterized as a low-affinity (that assures a supply of P_i at normal or high external P_i concentrations) and a high-affinity (activated in response to P_i starvation). Here, as an initial step to understand the PHO pathway in Aspergillus fumigatus, we characterized the PHO80 homologue, PhoB^PHO80. We show that the ΔphoB^PHO80 mutant has a polar growth defect (i.e., a delayed germ tube emergence) and, by phenotypic and phosphate uptake analyses, establish a link between PhoB^PHO80, calcineurin and calcium metabolism. Microarray hybridizations carried out with RNA obtained from wild-type and ΔphoB^PHO80 mutant cells identify Afu4g03610 (phoD^PHO84), Afu7g06350 (phoE^PHO89), Afu4g06020 (phoC^PHO81), and Afu2g09040 (vacuolar transporter Vtc4) as more expressed both in the ΔphoB^PHO80 mutant background and under phosphate-limiting conditions of 0.1 mM P_i. Epifluorescence microscopy revealed accumulation of poly-phosphate in ΔphoB^PHO80 vacuoles, which was independent of extracellular phosphate concentration. Surprisingly, a phoD^PHO84 deletion mutant is indistinguishable phenotypically from the corresponding wild-type strain. mRNA analyses suggest that protein kinase A absence supports the expression of PHO genes in A. fumigatus. Furthermore, ΔphoB^PHO80 and ΔphoD^PHO84 mutant are fully virulent in a murine low dose model for invasive aspergillosis.     

Responding to misinformation and criticisms regarding United States cat predation estimates

Misinformation (or denialism), the disingenuous assertion of information contradicting overwhelming scientific consensus, increasingly poses a challenge for invasion biology. The issue of free-ranging domestic cats (Felis catus) provides an example of this misinformation: overwhelming consensus shows that cats are invasive species that impact wildlife and human health yet free-ranging cat advocates propagate misinformation about such impacts to support policies keeping cats on the landscape. These advocates also attempt to discredit peer-reviewed scientific research on cat impacts, as exemplified by the response to a high-profile paper estimating cats annually kill billions of U.S. birds and mammals (Loss et al. in Nat Commun 4:1396, 2013). Although favorably received by scientific and invasive species management communities, an effort was launched to discredit this paper by criticizing its methods, including a report commissioned by a feral cat advocacy group and a post by a feral cat blogger. These same organizations and individuals have made similar criticisms at scientific conferences and policy roundtables. Given the realized effects of this campaign in influencing invasive species policy, we here respond to these criticisms and show they are characterized by numerous errors and misrepresentations. We conclude that the criticisms are part of the broader campaign to fabricate doubt about outdoor cat impacts and stymie policies favoring removal of cats from the landscape. Because misinformation surrounding cats is emblematic of the broader issue of misinformation and denialism, this response will not only facilitate evidence-based policy for managing cats but also stimulate research and discussion into causes and impacts of misinformation in invasion biology.     

Isoproterenol opens K+(ATP) channels via a beta2-adrenoceptor-linked mechanism in Sertoli cells from immature rats.

In the present study, we investigated the mechanism by which isoproterenol hyperpolarises membrane potential (MP) in Sertoli cells from seminiferous tubules of 15-day-old rat testes. Modification of MP and resistance (R0) was analysed using conventional intracellular glass microelectrodes. Isoproterenol (2 x 10(-6) M) induced an immediate and significant hyperpolarisation in the Sertoli-cell membrane. The beta2-AR antagonist, butoxamine (1 x 10(-6) M), nullified isoproterenol action. The effect of the beta1 antagonist, metoprolol (1 x 10(-6) M), was light and non-significant. Sulphonylurea glibenclamide inhibition of the K+(ATP) channels suppressed isoproterenol action, and testosterone, while depolarising Sertoli-cell MP closing the K+(ATP) channels through the PLC/PIP2 pathway, reduced beta-AR agonist-induced hyperpolarisation. Also, polycations LaCl3 and spermine reversed isoproterenol's hyperpolarisation effect, probably depolarising the membrane potential through ionic interaction neutralising the action of isoproterenol on K+(ATP) channels. Adenylate cyclase agonist forskolin (0.1 microM) rapidly hyperpolarised Sertoli-cell MP, mimicking the isoproterenol effect. These effects indicate that isoproterenol's action on K+(ATP) channel probably involves the known signalling cascade beta-AR/Gs/AC/cAMP/PKA. These results suggest that the isoproterenol-induced hyperpolarisation is mediated by the opening of K+(ATP) channels in Sertoli cells. This beta-adrenergic hyperpolarisation might play a physiological role in the modulation of MP.     

Search for Viroids and Other Small RNAs Associated with Forest Damage

We examined whether viroids may be involved as the causative agents of forest damages which have been observed in Germany during the last decade. A crude RNA extract was prepared from healthy and diseased copper beech, spruce, and pine trees and was analysed by two-dimensional gel electrophoresis which resolves RNA bands in the molecular weight range from 25,000 to 10⁶. Viroids are known to consist of single stranded circular RNA and are clearly differentiable in the electrophoresis used here from cellular RNA. Viroids could not be found in any of the samples investigated. In all samples from diseased trees, however, other additional RNA bands were discovered which were more similar to cellular RNA than to viroids in their electrophoretic properties. It is uncertain whether these diseased-associated RNAs are due to an infection or originate from a modified nucleic acid metabolism.