Antibodies against Lutzomyia longipalpis saliva in the fox Cerdocyon thous and the sylvatic cycle of Leishmania chagasi

Sera of 11 wild Cerdocyon thous foxes from an endemic area for American visceral leishmaniasis were tested for the presence of antibodies against salivary gland homogenates (SGH) of Lutzomyia longipalpis. All foxes had higher levels of anti-Lu. longipalpis SGH antibodies than foxes from non-endemic areas, suggesting contact between foxes and the vector of visceral leishmaniasis. Sera of humans and dogs living in the same area were also tested for reactivity against Lu. longipalpis SGHs and had a lower proportion of reactivity than foxes. Antibodies against Leishmania chagasi were not detected in any of the foxes, but three foxes showed the presence of parasites in the bone marrow by direct examination, PCR or by infecting the vector. Both humans and dogs had higher levels of anti-Le. chagasi IgG antibodies than C. thous. The finding of an antibody response against saliva of Lu. longipalpis among C. thous together with the broad distribution of the vector in resting areas of infected foxes suggests that the natural foci of transmission of Le. chagasi exists independently of the transmission among dogs and humans.     

Targeting mitochondrial mRNA translation to tackle obesity‐induced insulin resistance: thumbs up for exercise

The control of renal vascular tone is important for the regulation of salt and water balance, blood pressure and the protection against damaging elevated glomerular pressure. The K⁺ conductance is a major factor in the regulation of the membrane potential (V_m) in vascular smooth muscle (VSMC) and endothelial cells (EC). The vascular tone is controlled by V_m via its effect on the opening probability of voltage‐operated Ca²⁺ channels (VOCC) in VSMC. When K⁺ conductance increases V_m becomes more negative and vasodilation follows, while deactivation of K⁺ channels leads to depolarization and vasoconstriction. K⁺ channels in EC indirectly participate in the control of vascular tone by endothelium‐derived vasodilation. Therefore, by regulating the tone of renal resistance vessels, K⁺ channels have a potential role in the control of fluid homoeostasis and blood pressure as well as in the protection of the renal parenchyma. The main classes of K⁺ channels (calcium activated (K_Ca), inward rectifier (K_ir), voltage activated (K_v) and ATP sensitive (K_ATP)) have been found in the renal vessels. In this review, we summarize results available in the literature and our own studies in the field. We compare the ambiguous in vitro and in vivo results. We discuss the role of single types of K⁺ channels and the integrated function of several classes. We also deal with the possible role of renal vascular K⁺ channels in the pathophysiology of hypertension, diabetes mellitus and sepsis.     

VEGF-induced neoangiogenesis is mediated by NAADP and two-pore channel-2–dependent Ca2+ signaling

Vascular endothelial growth factor (VEGF) and its receptors VEGFR1/VEGFR2 play major roles in controlling angiogenesis, including vascularization of solid tumors. Here we describe a specific Ca²⁺ signaling pathway linked to the VEGFR2 receptor subtype, controlling the critical angiogenic responses of endothelial cells (ECs) to VEGF. Key steps of this pathway are the involvement of the potent Ca²⁺ mobilizing messenger, nicotinic acid adenine-dinucleotide phosphate (NAADP), and the specific engagement of the two-pore channel TPC2 subtype on acidic intracellular Ca²⁺ stores, resulting in Ca²⁺ release and angiogenic responses. Targeting this intracellular pathway pharmacologically using the NAADP antagonist Ned-19 or genetically using Tpcn2^−/− mice was found to inhibit angiogenic responses to VEGF in vitro and in vivo. In human umbilical vein endothelial cells (HUVECs) Ned-19 abolished VEGF-induced Ca²⁺ release, impairing phosphorylation of ERK1/2, Akt, eNOS, JNK, cell proliferation, cell migration, and capillary-like tube formation. Interestingly, Tpcn2 shRNA treatment abolished VEGF-induced Ca²⁺ release and capillary-like tube formation. Importantly, in vivo VEGF-induced vessel formation in matrigel plugs in mice was abolished by Ned-19 and, most notably, failed to occur in Tpcn2^−/− mice, but was unaffected in Tpcn1^−/− animals. These results demonstrate that a VEGFR2/NAADP/TPC2/Ca²⁺ signaling pathway is critical for VEGF-induced angiogenesis in vitro and in vivo. Given that VEGF can elicit both pro- and antiangiogenic responses depending upon the balance of signal transduction pathways activated, targeting specific VEGFR2 downstream signaling pathways could modify this balance, potentially leading to more finely tailored therapeutic strategies.In the adult the formation of new capillaries is an uncommon occurrence mostly restricted to pathological rather than physiological conditions, the majority of blood vessels remaining quiescent once organ growth is accomplished (1). Physiological neoangiogenesis is generally restricted to body sites undergoing regeneration or restructuring (e.g., tissue lesion repair and corpus luteum formation), whereas pathological neoangiogenesis takes place in different diseases ranging from macular degeneration to atherosclerosis, and is vital for the highly noxious development of solid tumors, thus representing a promising target for therapeutic strategies (2). Vascular endothelial growth factors (VEGF), and in particular the family member VEGF-A, are major regulators of angiogenesis and regulate ECs, mainly through the stimulation of VEGF receptor-2 (VEGFR2), a receptor tyrosine kinase, to induce cell proliferation, migration, and sprouting in the early stages of angiogenesis (3, 4). Antiangiogenic agents that target VEGF signaling have become an important component of therapies in multiple cancers, but their use is limited by acquisition of resistance to their therapeutic effects (5, 6). When overall VEGF receptor (VEGFR) signaling is experimentally impaired by the use of blocking antibodies or of specific tyrosine kinase inhibitors, alternative cellular and tissue strategies nullify the success of such interventions (5, 7, 8). Resistance to anti-VEGF therapies may occur through a variety of mechanisms, including evocation of alternative compensatory factors, selection of hypoxia-resistant tumor cells, action of proangiogenic circulating cells, and increased circulating nontumor proangiogenic factors. Moreover, cross-interactions (both cellular and humoral) between ECs and other environmental cues have to be taken into account for the ultimate aim of tailoring therapeutic interventions according to the specific pattern of the angiogenic microenvironment and EC conditions (5–7). The search for novel key downstream effectors is therefore of potential significance in the perspective of angiogenesis control in cancer progression.Autophosphorylation of VEGFR2 upon binding VEGF results in the activation of downstream signaling cascades through complex and manifold molecular interactions that transmit signals leading to angiogenic responses. Stimulation of different EC types via VEGFR2 results in increases in intracellular free calcium concentrations [Ca²⁺]_i (9, 10) and the crucial role of this signaling element in the regulation of EC functions and angiogenesis is recognized (11, 12), and thought to be largely mediated by the phospholipase Cγ (PLCγ)/inositol 1,4,5 trisphosphate (IP₃) signaling pathway (10). It has been reported that IP₃ releases Ca²⁺ from intracellular stores in ECs, increasing [Ca²⁺]_i, and is augmented by store-operated Ca²⁺ influx (13). This signaling primes the endothelium for angiogenesis through the activation of downstream effectors such as endothelial nitric oxide synthase (eNOS), protein kinases C (PKC), and mitogen-activated protein kinases (MAPKs). Indeed, it has been reported that the interplay between IP₃-dependent Ca²⁺ mobilization and store-operated Ca²⁺ entry produces Ca²⁺ signals whose inhibition impairs the angiogenic effect of VEGF (14, 15). Given the complexity of both VEGF and Ca²⁺ signaling, and the crucial finding that VEGF evokes pro- and antiangiogenic responses, it is clear that the specificity of VEGF-evoked Ca²⁺ signatures deserves further investigation.Differences in Ca²⁺ signatures, which are key to determining specific Ca²⁺-dependent cellular responses, rely upon often complex spatiotemporal variations in [Ca²⁺]_i (16). A major determinant of these are based on functionally distinct intracellular Ca²⁺-mobilizing messengers, namely IP₃ and cyclic adenosine diphosphoribose (cADPR), which mobilize Ca²⁺ from the endoplasmic reticulum (ER) stores, and nicotinic acid adenine dinucleotide phosphate (NAADP), which triggers Ca²⁺ release from acidic organelles, such as lysosomes and endosomes (17, 18). NAADP likely targets a channel distinct from IP₃ and ryanodine receptors (RyRs), known as two-pore channels (TPCs) (19–25), and the resulting localized NAADP-evoked Ca²⁺ signals may in some cases be globalized via IP₃ and RyRs through Ca²⁺-induced Ca²⁺ release (26, 27). However, in a few cell types, direct activation of RyRs and Ca²⁺ influx channels by NAADP have also been proposed as alternative mechanisms (28, 29). It has been demonstrated that NAADP-sensitive Ca²⁺ stores are present in the endothelium, and that NAADP is capable of regulating vascular smooth muscle contractility and blood pressure by EC-dependent mechanisms (30). In addition, we have previously demonstrated that NAADP is a specific and essential intracellular mediator of ECs histamine H₁ receptors, evoking [Ca²⁺]_i release and secretion of von Willebrand factor, which requires the functional expression of TPCs (31).In the present work, we identify a novel pathway for VEGFR2 signal transduction whereby receptor activation leads to NAADP and TPC2-dependent Ca²⁺ release from acidic Ca²⁺ stores, which in turn controls angiogenic response in vitro and in vivo. These findings demonstrate, to our knowledge for the first time, the direct relationship between NAADP-mediated Ca²⁺ release and the signaling mechanisms underlying ECs angiogenesis mediated by VEGF.     

Nasal vaccination with attenuated Salmonella expressing VapA: TLR2 activation is not essential for protection against R. equi infection

Virulent strains of Rhodococcus equi have a large plasmid of 80–90 kb, which encodes several virulence-associated proteins (Vap), including VapA, a lipoprotein highly associated with disease. We have previously demonstrated that oral immunisation with attenuated Salmonella enterica Typhimurium strain expressing the antigen VapA (STM VapA+) induces specific and long-term humoral and cellular immunity against R. equi. It was shown that VapA activates Toll-like receptor 2 (TLR2) on macrophages by establishing an interaction that ultimately favours immunity against R. equi infection. The purpose of this study was to evaluate the immune response triggered by nasal immunisation with STM VapA+ and to determine whether TLR2 supports the vaccine effect. We developed an optimised protocol for a single nasal immunisation that conferred protection against R. equi infection in mice, which was manifested by efficient R. equi clearance in challenged animals. Nasal vaccination with STM VapA+ has also induced protection in Tlr2^−/− mice and mice with non-functional TLR4. Moreover, spleen cells of vaccinated mice augmented T-bet expression, as well as the production of IL-12, IFN-γ, nitric oxide and hydrogen peroxide. Notably, the population of CD4⁺ T cells with memory phenotype significantly increased in the spleens of vaccinated mice challenged 1 or 5 months after immunisation. In these animals, the spleen bacterial burden was also reduced. When similar experimental procedures were performed in TLR2 knockout mice, an increase in CD4⁺ T cells with memory phenotype was not observed. Consequently, we conclude that nasal vaccination with attenuated Salmonella expressing the R. equi virulence factor VapA confers long-lasting protection against experimental rhodoccocosis and that TLR2 engagement was not crucial to induce this protection but may be required for a long-term immune response.     

Molecular characterization of arthropod-borne hematozoans in wild mammals from Brazil,Venezuela and Spain

A survey of Babesia, Theileria and Hepatozoon was conducted in wild mammals, including the capybara (Hydrochaeris hydrochaeris; n = 14) from Brazil, the jaguar (Panthera onca; n = 2) and crab-eating raccoon (Procyon cancrivorus; n = 4) from Venezuela, and the red deer (Cervus elaphus; n = 70), red squirrel (Sciurus vulgaris; n = 5) and Eurasian pine marten (Martes martes; n = 3) from Spain. Diagnostic procedures included both microscopy and molecular methods (PCR and sequencing of the 18S rRNA gene). Microscopic examination of blood smears revealed no hematozoan infections — unlike the molecular analyses. Nine Brazilian capybaras were found to be infected with Hepatozoon canis (prevalence 64%), two of which were coinfected with a previously unknown babesid (prevalence 14%) loosely related to Theileria equi (90% 18S rRNA gene similarity according to BLAS® analysis). One jaguar and one crab-eating raccoon from Venezuela were infected by H. canis. Four of the red deer were infected with theilerids (5.7% prevalence), two with Theileria sp. and two with T. annulata. One red squirrel and three pine martens were infected with Hepatozoon sp. The isolate form the red squirrel was phylogenetically related to Hepatozoon sp. reported in Spanish bank voles, whereas those infecting the pine martens were related to Hepatozoon felis reported in Spanish cats. In conclusion, the molecular findings show that some non-canid mammals are carriers of H. canis in South America, while red deer may carry T. annulata in Europe. Small mammals in Europe appear to be unlikely hosts of H. canis and H. felis.     

Inbreeding effect on morbidity: I. Three analyses (one with cousin and sib controls) of two surveys among Brazilian whites and Negroes

Data from two surveys performed in Brazil and involving 5,719 persons have been subdivided into three subsamples (one with cousin and sib controls). The totals led to estimates of B (abnormal equivalents per gamete) around 0.5, of A (the damage in a noninbred population) around 0.01-0.02, and of B/A from 14 to 77. No statistically significant difference was found between whites and nonwhites (mulattoes and Negroes). Inbreeding seems to disclose a predominantly mutational load.     

Cleft lip rhinoplasty: the role of bone and cartilage grafts

Correction of the cleft lip nasal deformity involves repositioning of the lower lateral cartilage on the cleft side to raise the dome, lengthening the columella and bringing it toward the midline, and correcting any asymmetries of the nasal floor. Additional structural support in the form of bone or cartilage grafts is often required in order to achieve the desired projection and angularity. Our experience with these grafts in a large number of patients over the past 20 years has shown them to produce good, predictable results that are long lasting, with minimum donor site morbidity. Long-term follow-up indicates that these grafts maintain their volume and original features, resulting in satisfaction among our patients.     

Deep orbital reconstruction for correction of post-traumatic enophthalmos

Post-traumatic enophthalmos is a complex orbital deformity resulting from an injury disrupting orbital bone and ligament support, allowing displacement and a change in shape of the orbital soft-tissue contents. Clinically, this is manifested by inferior and posterior displacement of the globe, pseudoptosis, and deepening of the supratarsal fold. Soft-tissue changes such as canthal malposition and shortening of the horizontal dimension of the palpebral fissure are present when unreduced nasoethmoidal orbital fractures exist. The essential principles of surgical correction include full dissection of the bony orbital soft tissues including the posterior orbit with restoration of bony orbital volume by the judicious insertion of bone grafts to correct the vertical as well as the anteroposterior position of the ocular globe. The combination of techniques of craniofacial exposure, osteotomy, and bone grafting allow the condition of post-traumatic enophthalmos to be greatly improved with minimal complications.