Patients infected with Leishmania braziliensis develop chronic lesions that often fail to respond to treatment with antiparasite drugs. To determine whether genes whose expression is highly variable in lesions between patients might influence disease outcome, we obtained biopsies of lesions from patients before treatment with pentavalent antimony and performed transcriptomic profiling on these clinical samples. We identified genes that were highly variably expressed between patients, and the variable expression of these genes correlated with treatment outcome. Among the most variable genes in all the patients were components of the cytolytic pathway, and the expression of these genes correlated with parasite load in the skin. We demonstrated that treatment failure was linked to the cytolytic pathway activated during infection. Using a host-pathogen marker profile of as few as three genes, we showed that eventual treatment outcome could be predicted before the start of treatment in two separate cohorts of patients with cutaneous leishmaniasis (n = 21 and n = 25). These findings raise the possibility of point-of-care diagnostic screening to identify patients at high risk of treatment failure and provide a rationale for a precision medicine approach to drug selection in cutaneous leishmaniasis. This work more broadly demonstrates the value of identifying genes of high variability in other diseases to better understand and predict diverse clinical outcomes.

Leishmania braziliensis are intracellular parasites that cause unique clinical forms of cutaneous leishmaniasis. Previous studies with other leishmania species demonstrated that reactive oxygen species (ROS) control promastigotes, the infective stage of the parasite, but not the amastigote form that exists in the mammalian host. Here we show that ROS inhibits growth of L. braziliensis amastigotes in resting monocytes, and that classical monocytes are primarily responsible for this control. ROS, but not nitric oxide, also contributed to killing of L. braziliensis by IFN-γ activated monocytes. Furthermore, by gene expression profiling of human lesions we found greater expression of genes associated with ROS, but not nitric oxide, compared to normal skin. This study shows that ROS are important for control of L. braziliensis both at the initial stages of infection, as well as at later time points, and highlights that monocyte subsets may play different roles during leishmaniasis.

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The effect of daidzein (D), 4-methylbenzylidene camphor (4-MBC) or estradiol-17b-benzoate (E 2 ) on muscle of osteoporotic rats during fracture healing was studied. After performing a metaphyseal tibia osteotomy in 96 osteoporotic 5-month-old female Sprague-Dawley rats, they received daily 50 mg D, 200 mg 4-MBC or 0 . 4 mg E 2 per kg body weight, or soy free (SF) diet up to 36 and 72 days. Mitochondrial activity, fiber area, and capillary density were analyzed in M. gastrocnemius. Osseous callus bridging of fracture was observed in half of the rats after 36 days. By day 72, fracture was healed in most of the animals. State 3 mitochondrial respiration significantly enhanced in E 2 , 4-MBC and D groups versus SF after 36 days (30, 32 and 32 vs 23 pmol O 2 /s per mg). It declined after 72 days, however, in E 2 group it was still at a higher level versus SF (25, 23 and 21 vs 20 pmol O 2 /s per mg). Size of fast oxidative glycolytic (FOG) and fast glycolytic (FG) fibers, capillary density did not differ significantly between the groups, however, at day 36 an increase in D and 4-MBC groups was detectable. FOG diameter was 64, 66, 68, and 58 mm and FG diameter was 88, 98, 95, and 89 mm in SF, D, 4-MBC, and E 2 groups. The ratio of capillaries to muscle fiber was 1 . 1, 1 . 4, 1 . 3, and 1 . 1 in SF, D, 4-MBC and E 2 groups by day 36. D and 4-MBC react similar to estrogen thereby improving oxidative cell metabolism in severe osteoporotic rats. The level of mitochondrial activity was higher, though no significant morphological differences could be shown.

Cutaneous leishmaniasis is a localized infection controlled by CD4+ T cells that produce IFN-γ within lesions. Phagocytic cells recruited to lesions, such as monocytes, are then exposed to IFN-γ which triggers their ability to kill the intracellular parasites. Consistent with this, transcriptional analysis of patient lesions identified an interferon stimulated gene (ISG) signature. To determine whether localized L. braziliensis infection triggers a systemic immune response that may influence the disease, we performed RNA sequencing (RNA-seq) on the blood of L. braziliensis-infected patients and healthy controls. Functional enrichment analysis identified an ISG signature as the dominant transcriptional response in the blood of patients. This ISG signature was associated with an increase in monocyte- and macrophage-specific marker genes in the blood and elevated serum levels IFN-γ. A cytotoxicity signature, which is a dominant feature in the lesions, was also observed in the blood and correlated with an increased abundance of cytolytic cells. Thus, two transcriptional signatures present in lesions were found systemically, although with a substantially reduced number of differentially expressed genes (DEGs). Finally, we found that the number of DEGs and ISGs in leishmaniasis was similar to tuberculosis–another localized infection–but significantly less than observed in malaria. In contrast, the cytolytic signature and increased cytolytic cell abundance was not found in tuberculosis or malaria. Our results indicate that systemic signatures can reflect what is occurring in leishmanial lesions. Furthermore, the presence of an ISG signature in blood monocytes and macrophages suggests a mechanism to limit systemic spread of the parasite, as well as enhance parasite control by pre-activating cells prior to lesion entry.

Estrogen regulates several biological processes in health and disease. Specifically, estrogen exerts antihypertrophic effects in the diseased heart. However, its role in the healthy heart remains elusive. Our initial aim was to identify the effects of 17β-estradiol (E2) on cardiac morphology and global gene expression in the healthy mouse heart. Two-month-old C57BL/6J mice were ovariectomized and treated with E2 or vehicle for 3 months. We report that E2 induced physiological hypertrophic growth in the healthy C57BL/6J mouse heart characterized by an increase in nuclear β-catenin. Hypothesizing that β-catenin mediates these effects of E2, we employed a model of cardiac β-catenin deletion. Our surprising finding is that E2 had the opposite effects in wild-type littermates, which were actually on the C57BL/6N background. Notably, E2 exerted no significant effect in hearts of mice with depleted β-catenin. We further demonstrate an E2-dependent increase in glycogen synthase kinase 3β (GSK3β) phosphorylation and endosomal markers in C57BL/6J but not C57BL/6N mice. Together, these findings indicate an E2-driven inhibition of GSK3β and consequent activation of β-catenin in C57BL/6J mice, whereas the opposite occurs in C57BL/6N mice. In conclusion, E2 exerts divergent effects on postnatal cardiac growth in mice with distinct genetic backgrounds modulating members of the GSK3β/β-catenin cascade.

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