BackgroundProtozoan parasites from the genus Leishmania cause broad clinical manifestations known as leishmaniases, which affect millions of people worldwide. Cutaneous leishmaniasis (CL), caused by L. major, is one the most common forms of the disease in the Old World. There is no preventive or therapeutic human vaccine available for L. major CL, and existing drug treatments are expensive, have toxic side effects, and resistant parasite strains have been reported. Hence, further therapeutic interventions against the disease are necessary. Terminal, non-reducing, and linear α-galactopyranosyl (α-Gal) epitopes are abundantly found on the plasma membrane glycolipids of L. major known as glycoinositolphospholipids. The absence of these α-Gal epitopes in human cells makes these glycans highly immunogenic and thus potential targets for vaccine development against CL.Methodology/Principal findingsHere, we evaluated three neoglycoproteins (NGPs), containing synthetic α-Gal epitopes covalently attached to bovine serum albumin (BSA), as vaccine candidates against L. major, using α1,3-galactosyltransferase-knockout (α1,3GalT-KO) mice. These transgenic mice, similarly to humans, do not express nonreducing, linear α-Gal epitopes in their cells and are, therefore, capable of producing high levels of anti-α-Gal antibodies. We observed that Galα(1,6)Galβ-BSA (NGP5B), but not Galα(1,4)Galβ-BSA (NGP12B) or Galα(1,3)Galα-BSA (NGP17B), was able to significantly reduce the size of footpad lesions by 96% in comparison to control groups. Furthermore, we observed a robust humoral and cellular immune response with production of high levels of protective lytic anti-α-Gal antibodies and induction of Th1 cytokines.Conclusions/SignificanceWe propose that NGP5B is an attractive candidate for the study of potential synthetic α-Gal-neoglycoprotein-based vaccines against L. major infection.

The causative agent of Chagas disease, Trypanosoma cruzi, is transmitted by triatomine vectors. The insect is endemic in the Americas, including the United States, where epidemiological studies are limited, particularly in the Southwestern region. Here, we have determined the prevalence of T. cruzi in triatomines, and feral cats and dogs, and wild animals, the infecting parasite genotypes and the mammalian host bloodmeal sources of the triatomines at four different geographical sites in the U.S.-Mexico border, including El Paso County, Texas, and nearby cities in New Mexico. Using qualitative polymerase chain reaction to detect T. cruzi infections, we found 66.4% (n = 225) of triatomines, 45.3% (n = 95) of feral dogs, 39.2% (n = 24) of feral cats, and 71.4% (n = 7) of wild animals positive for T. cruzi. Over 95% of T. cruzi genotypes or discrete typing units (DTUs) identified were TcI and some TcIV. Furthermore, Triatoma rubida was the triatomine species most frequently (98.2%) collected in all samples analyzed. These findings suggest a high prevalence of T. cruzi infections among triatomines, and feral and wild animals in the studied sites. Therefore, our results underscore the urgent need for implementation of a systematic epidemiological surveillance program for T. cruzi infections in insect vectors, and feral and wild animals, and Chagas disease in the human population in the southwestern region of the United States.

Leishmania major is the causative agent of cutaneous leishmaniasis (CL). No human vaccine is available for CL, and current drug regimens present several drawbacks, such as emerging resistance, severe toxicity, medium effectiveness, and/or high cost. Thus, the need for better treatment options against CL is a priority. In the present study, we validate the enzyme methionine aminopeptidase 1 of L. major (MetAP1Lm), a metalloprotease that catalyzes the removal of N-terminal methionine from peptides and proteins, as a chemotherapeutic target against CL infection. The in vitro antileishmanial activities of eight novel MetAP1 inhibitors (OJT001 to OJT008) were investigated. Three compounds, OJT006, OJT007, and OJT008, demonstrated potent antiproliferative effects in macrophages infected with L. major amastigotes and promastigotes at submicromolar concentrations, with no cytotoxicity against host cells. Importantly, the leishmanicidal effect in transgenic L. major promastigotes overexpressing MetAP1Lm was diminished by almost 10-fold in comparison to the effect in wild-type promastigotes. Furthermore, the in vivo activities of OJT006, OJT007, and OJT008 were investigated in L. major-infected BALB/c mice. In comparison to the footpad parasite load in the control group, OJT008 decreased the footpad parasite load significantly, by 86%, and exhibited no toxicity in treated mice. We propose MetAP1 inhibitor OJT008 as a potential chemotherapeutic candidate against CL infection caused by L. major infection.

To examine the molecular basis for the decreased pituitary growth hormone (GH) and thyrotropin (TSH) content during restricted feeding, fasting and diabetes, we measured steady-state levels of mRNA for TSH-alpha, TSH-beta and GH in the pituitary from normal rats either fed ad libitum (C), limited to 75%, 50% and 25% (FR75, FR50, FR25, respectively) of ad libitum intake, or deprived of food for 2 and 4 days (F2 and F4, respectively), and also in streptozotocin-diabetic (D) and D insulin-treated animals. The results from these experimental groups were compared with those in thyroidectomized (Tx) rats. Pituitary mRNA was quantified by Northern blot hybridization with cDNA probes specific for rat TSH-alpha, TSH-beta and GH. Although changes in the pituitary GH mRNA during restricted feeding, fasting and diabetes were similar qualitatively to those induced by hypothyroidism, GH mRNA levels in Tx rats (> 10% of C values) were less than in the other experimental groups (p < 0.001). Pituitaries from FR50, FR25 and D rats also contained less GH mRNA than F2 and F4 animals (p < 0.05). Thyroidectomy resulted in a marked increase in both TSH-beta and TSH-alpha mRNAs, the changes in TSH-beta mRNA being greater than those in TSH-alpha mRNA. In contrast, FR50, FR25, F2, F4 and D rats exhibited a decrease in pituitary TSH-beta mRNA (60%, 50%, 35%, 36% and 33%, respectively, of C values; p < 0.01-0.05) and in TSH-alpha mRNA levels (81%, 64%, 46%, 43% and 36%, respectively, of normal values; p < 0.02-0.05), TSH-beta mRNA showing the greater changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Leishmania major (L. major) is a protozoan parasite that causes cutaneous leishmaniasis. About 12 million people are currently infected with an annual incidence of 1.3 million cases. The purpose of this study was to synthesize a small library of novel thiophene derivatives, and evaluate its parasitic activity, and potential mechanism of action (MOA). We developed a structure–activity relationship (SAR) study of the thiophene molecule 5A. Overall, eight thiophene derivatives of 5A were synthesized and purified by silica gel column chromatography. Of these eight analogs, the molecule 5D showed the highest in vitro activity against Leishmania major promastigotes (EC50 0.09 ± 0.02 µM), with an inhibition of the proliferation of intracellular amastigotes higher than 75% at only 0.63 µM and an excellent selective index. Moreover, the effect of 5D on L. major promastigotes was associated with generation of reactive oxygen species (ROS), and in silico docking studies suggested that 5D may play a role in inhibiting trypanothione reductase. In summary, the combined SAR study and the in vitro evaluation of 5A derivatives allowed the identification of the novel molecule 5D, which exhibited potent in vitro anti-leishmanial activity resulting in ROS production leading to cell death with no significant cytotoxicity towards mammalian cells.