Regulated expression of the Leishmania major surface virulence factor lipophosphoglycan using conditionally destabilized fusion proteins

Surface glycoconjugates play important roles in the infectious cycle of Leishmania major, including the abundant lipophosphoglycan (LPG) implicated in parasite survival in the sand fly vector and the initial stages of establishment in the mammalian host macrophage. We describe a system for inducible expression of LPG, applying a novel protein-based system that allows controlled degradation of a key LPG biosynthetic enzyme, UDP-galactopyranose mutase (UGM). This methodology relies on a mutated FK506-binding protein (FKBP) destabilizing domain (dd) fused to the protein of interest; in the absence of rapamycin analogs, such as Shld1, the dd domain is destabilized, leading to proteasomal degradation, whereas drug treatment confers stabilization. Tests in L. major using dd fusions to a panel of reporters and cellular proteins confirmed its functionality, with a high degree of regulation and low background, and we established the kinetics of protein activation and/or loss. Two inexpensive and widely available ligands, FK506 and rapamycin, functioned similarly to Shld1, without effect on Leishmania growth or differentiation. We generated parasites lacking UGM through deletion of the GLF gene and substitution with a ddGLF fusion construct, either as chromosomal knockins or through episomal complementation; these showed little or no LPG expression in the absence of inducer, whereas in its presence, high levels of LPG were attained rapidly. Complement lysis tests confirmed the correct integrity of the Leishmania LPG coat. These data suggest that the dd approach has great promise in the study of LPG and other pathways relevant to parasite survival and virulence.     

Falha de integridade dos elétrodos de desfibrilhação Riata e Riata ST: um problema atual

Riata and Riata ST silicone defibrillation leads are prone to externalization of conductors due to inside-out abrasion in the high-voltage system, causing structural damage which may be accompanied by electrical failure. These situations are easily detected by fluoroscopy or radiology and by inspection of intracardiac electrograms and/or measurement of impedance. However, older pulse generators do not automatically perform all the measurements needed to assess the integrity of the high-voltage electrical system, nor do they have patient notifier alerts in case of dysfunction.The authors describe the case of a patient in whom structural damage was detected on fluoroscopy during pulse generator replacement. They discuss the best strategy in these patients, considering current knowledge of this dysfunction.     

Multifunctional CD4⁺ T cells in patients with American cutaneous leishmaniasis

Leishmaniasis is a group of important parasitic diseases affecting millions worldwide. To understand more clearly the quality of T helper type 1 (Th1) response stimulated after Leishmania infection, we applied a multiparametric flow cytometry protocol to evaluate multifunctional T cells induced by crude antigen extracts obtained from promastigotes of Leishmania braziliensis (LbAg) and Leishmania amazonensis (LaAg) in peripheral blood mononuclear cells from healed cutaneous leishmaniasis patients. Although no significant difference was detected in the percentage of total interferon (IFN)-γ-producing CD4(+) T cells induced by both antigens, multiparametric flow cytometry analysis revealed clear differences in the quality of Th1 responses. LbAg induced an important proportion of multifunctional CD4(+) T cells (28% of the total Th1 response evaluated), whereas LaAg induced predominantly single-positive cells (68%), and 57% of those were IFN-γ single-positives. Multifunctional CD4(+) T cells showed the highest mean fluorescence intensity (MFI) for the three Th1 cytokines assessed and MFIs for IFN-γ and interleukin-2 from those cells stimulated with LbAg were significantly higher than those obtained after LaAg stimulation. These major differences observed in the generation of multifunctional CD4(+) T cells suggest that the quality of the Th1 response induced by L. amazonensis antigens can be involved in the mechanisms responsible for the high susceptibility observed in L. amazonensis-infected individuals. Ultimately, our results call attention to the importance of studying a Th1 response regarding its quality, not just its magnitude, and indicate that this kind of evaluation might help understanding of the complex and diverse immunopathogenesis of American tegumentary leishmaniasis.     

Wild, synanthropic and domestic hosts of Leishmania in an endemic area of cutaneous leishmaniasis in Minas Gerais State, Brazil

Domestic, synanthropic and wild hosts of Leishmania spp. parasites were studied in an area endemic for American tegumentary leishmaniasis (ATL), specifically in northern Minas Gerais State, Brazil. Domestic dogs and small forest mammals are reservoir hosts for L. (Leishmania) infantum. However, the role that these animals play in the transmission cycle of the Leishmania spp. that cause cutaneous leishmaniasis is not well known. This study evaluated 72 rodents, 25 marsupials and 98 domestic dogs found in two villages of the Xakriabá Indigenous Territory, an area of intense ATL transmission. A total of 23 dogs (23.47%) were shown to be positive according to at least one test; 8 dogs (8.16%) tested positive in a single serological test and 15 dogs (15.31%) tested positive by IFAT and ELISA. Eleven dogs were euthanised to allow for molecular diagnosis, of which nine (81.8%) tested positive by PCR for Leishmania in at least one tissue. Seven animals were infected only with L. (L.) infantum, whilst two displayed a mixed infection of L. (L.) infantum and L. (V.) braziliensis. Isoenzymatic characterisation identified L. (L.) infantum parasites isolated from the bone marrow of two dogs. Of the 97 small mammals captured, 24 tested positive for Leishmania by PCR. The results showed that L. (V.) braziliensis, L. (L.) infantum and L. (V.) guyanensis are circulating among wild and synanthropic mammals present in the Xakriabá Reserve, highlighting the epidemiological diversity of ATL in this region.     

RESOLUTION OF CUTANEOUS LEISHMANIASIS AFTER ACUTE ECZEMA DUE TO INTRALESIONAL MEGLUMINE ANTIMONIATE

We report a case of a 42 year-old female, who came to a leishmaniasisreference center in Rio de Janeiro, Brazil, presenting a cutaneous leishmaniasislesion in the right forearm. Treatment with low-dose intramuscular meglumineantimoniate (MA) (5 mg Sb⁵⁺/kg/day) was initiated, with improvement after28 days, although with the development of generalized eczema. After 87 days, thelesion worsened. Patient refused treatment with amphotericin B. MA was theninfiltrated in the lesion, in two sessions, resulting in local eczema, with bullaeformation; however, twenty days after, both the ulcer and eczema receded.Intralesional administration of MA should be used carefully when previous cutaneoushypersensitivity is detected.     

Nail abrasion

Certain diseases of the nail complex cause hyperkeratosis or alterations of the shape of the nail plate. These conditions may be painful, may decrease the penetration of topical medicaments and may be ugly. The nail plate abrasion, performed with dermabrader device or sandpaper, has application in patients suffering from onychomycosis, psoriasis, subnail infections and haematomas. The technique facilitates the collection of scales for mycological examination, decreases treatment time (of topical monotherapy) for onychomycosis and provides greater comfort for the patient by reducing nail plate thickness. It can also be useful for the partial removal of the nail plate in cases of haematomas and subnail infections. Nail abrasion is an effective and inexpensive method, easily applied in either nail pathologies with hyperkeratosis of the nail plate or in those requiring partial removal of the plate.     

Direction of the mental canal in human mandibles

The direction of the mental canal was calculated in the horizontal and vertical planes with a view to aiding anaesthetic techniques. Based on our data, it is advisable to give the following approximate inclinations to the needle when penetrating the mental canal in anaesthetic procedures: 55 degrees postero-anteriorly in relation to a horizontal plane and 40 degrees latero-medially in relation to a horizontal line tangent to the body of the mandible at the level of the mental foramen.