During visceral leishmaniasis (VL), T helper 1 (Th1)-based inflammation is induced to control intracellular parasites. Inflammation-based pathology has been shown to be dampened by interleukin 10 and eventual Programmed Death1 (PD1)-mediated T cell exhaustion. Cell type(s) responsible for the initiation of T cell-produced IL-10 during VL are unknown. CD19+, CD5−, CD1d−, IgDhi regulatory B cells from healthy controls produced IL-10 in absence of infection or stimulation in contrast to IgDlo/neg B cells. IgDhi B cells may have a de novo vs. induced regulatory program. IgDhi B cells increased three-fold in population size as VL progressed. B cells from VL dogs were necessary and sufficient to suppress T helper 1 (Th1) cell effector function. IgDhi B cells induced T cell and IgDlo B cell IL-10 production. Blockage of B cell-specific PD-L1 restored Th1 responses. IgDhi regulatory B cells represent a novel regulatory B cell which may precipitate T cell exhaustion during VL.

Members of the Leishmania genus are the causative agents of the life-threatening disease leishmaniasis. New drugs are being sought due to increasing resistance and adverse side effects with current treatments. The knowledge that dUTPase is an essential enzyme and that the all ␣-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric ␤-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding. These findings will aid in the development of dUTPase inhibitors as potential new lead antitrypanosomal compounds.Various members of the genus Leishmania cause leishmaniasis, which threatens ϳ350 million people worldwide and gives rise to about two million clinical cases each year, of which ϳ25% are of the fatal visceral form (1). The disease is largely endemic to developing countries, and current treatments are expensive and can result in undesirable side effects for the patient (1). This, combined with the increasing drug resistance that is developing in the Leishmania species, means that new and novel anti-parasitic drug targets are urgently required.Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) 2 represents such a target. dUTPases catalyze the hydrolysis of dUTP to dUMP and pyrophosphate (2). This provides the starting material for the synthesis of dTMP by thymidylate synthase and in addition maintains the ratio of dTTP:dUTP in the cell at a high enough level to prevent excessive misincorporation of dUMP into the genome during DNA replication (3). dUTPase activity is essential as was shown by gene knock-outs in Escherichia coli and Saccharomyces cerevisiae. Inhibition of dUTPase activity results in futile cycles of DNA repair that ultimately cause the fragmentation of DNA and cell death (4, 5). dUTPase activity also appears to be essential in L. major (6) and the related parasite Trypanosoma brucei, where an RNAi approach was used to knock down dUTPase expression, resulting in decreased cell proliferation and growth in both procyclic and bloodstream forms of the organism (7). dUTPases have been characterized extensively both biochemically and structurally in many species. Most organisms, including humans, have trimeric dUTPases with structures largely consisting of ␤-pleated sheet (8). Three active sites are formed at the trimer interfaces by...

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Infection with Leishmania causes diseases with variable presentation. The most severe form is visceral leishmaniasis (VL), caused by either L. donovani or L. infantum. Despite efforts to eliminate VL, to date, molecular detection in resource-poor settings have lacked the accuracy and rapidity that would enable widespread field use and the need for accurate, sensitive assays to detect asymptomatic Leishmania infection has become apparent. The domestic dog serves as the primary reservoir host of L. infantum. Study of this reservoir population provides an opportunity to evaluate the sensitivity and specificity of diagnostics for well-defined, symptomatic, canine visceral leishmaniasis (CVL) and asymptomatic L. infantum infection. Blood samples from an L. infantum-endemic population of US hunting dogs were evaluated with Dual-Path Platform (DPP®) CVL compared to those obtained via direct detection methods (culture- and Leishmania-specific quantitative polymerase chain reaction, qPCR) and immunofluorescence anti-Leishmania antibody test (IFAT). Statistically significant correlations were found between DPP® CVL development time and clinical status, culture status, circulating DNA levels, and IFAT titer. DPP® CVL results correlated with both clinical severity of disease and serological evidence of asymptomatic L. infantum infection. By precisely documenting the minimum time required for the development of a clear positive result in DPP® CVL, this test could be used in a rapid, semi-quantitative manner for the evaluation of asymptomatic and symptomatic CVL. Our results also indicate that a similar test could be used to improve our understanding of human VL.

Visceral Leishmaniasis is a deadly disease caused by Leishmania infantum , endemic in more than 98 countries across the globe. Although the most common means of transmission is via a sand fly vector, there is growing evidence that vertical transmission may be critical for maintaining L . infantum infection within the reservoir, canine, population. Vertical transmission is also an important cause of infant morbidity and mortality particularly in sub-Saharan Africa. While vertical transmission of visceralizing species of Leishmania has been reported around the globe, risk factors associated with this unique means of Leishmania transmission have not been identified therefore interventions regarding this means of transmission have been virtually non-existent. Furthermore, the basic reproductive number, (R 0 ), or number of new L . infantum infections that one infected mother or dam can cause has not been established for vertical transmission, also hampering the ability to assess the impact of this means of transmission within reservoir of human hosts. Canine Leishmaniosis (CanL) is enzootic within a U.S. hunting dog population. CanL is transmitted within this population via transplacental transmission with no reported vector transmission, despite many repeated attempts to find infected sand flies associated with these dogs and kennels. This population with predominantly, if not solely, vertical transmission of L . infantum was used to evaluate the critical risk factors for vertical transmission of Leishmania and establish the R 0 of vertical L . infantum infection. Evaluation of 124 animals born to eighteen dams diagnostically positive for infection with L . infantum showed that there was a 13.84x greater chance of being positive for L . infantum within their lifetime if the mother was also positive within her lifetime (RR: 13.84, 95% CI: 3.54–54.20, p-value: <0.0001). The basic reproductive number for vertically transmitted L . infantum within this cohort was 4.12. These results underscore that there is a high risk of L . infantum infection to transmit from mother to offspring. Targeted public health interventions and control efforts that address vertical transmission of L . infantum are necessary in endemic countries to eliminate visceral leishmaniasis.