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Thalia Pacheco-Fernandez,1,* Hannah Markle,1,* Chaitenya Verma,2 Ryan Huston,2,3 Sreenivas Gannavaram,1 Hira L Nakhasi,1 Abhay R Satoskar2,3 1Division of Emerging and Transfusion Transmitted Disease, Center for Biologics Evaluation and Research Food and Drug Administration, Silver Spring, MD, 20993, USA; 2Department of Pathology, Wexner Medical Center, The Ohio State University, Columbus, OH, 43201, USA; 3Department of Microbiology, Wexner Medical Center, The Ohio State University, Columbus, OH, 43201, USA*These authors contributed equally to this workCorrespondence: Abhay R Satoskar, Email abhay.satoskar@osumc.eduAbstract: Leishmaniasis is a neglected tropical disease endemic primarily to low- and middle-income countries, for which there has been inadequate development of affordable, safe, and efficacious therapies. Clinical manifestations of leishmaniasis range from self-healing skin lesions to lethal visceral infection with chances of relapse. Although treatments are available, secondary effects limit their use outside the clinic and negatively impact the quality of life of patients in endemic areas. Other non-medicinal treatments, such as thermotherapies, are limited to use in patients with cutaneous leishmaniasis but not with visceral infection. Recent studies shed light to mechanisms through which Leishmania can persist by hiding in cellular safe havens, even after chemotherapies. This review focuses on exploring the cellular niches that Leishmania parasites may be leveraging to persist within the host. Also, the cellular, metabolic, and molecular implications of Leishmania infection and how those could be targeted for therapeutic purposes are discussed. Other therapies, such as those developed against cancer or for manipulation of the ferroptosis pathway, are proposed as possible treatments against leishmaniasis due to their mechanisms of action. In particular, treatments that target hematopoietic stem cells and monocytes, which have recently been found to be necessary components to sustain the infection and provide a safe niche for the parasites are discussed in this review as potential field-deployable treatments against leishmaniasis.Keywords: leishmaniasis, field-deployable therapies, hematopoietic stem cells, monocytes, ferroptosis, repurposing drugs

Dinesh Mondal,1 Shinjiro Hamano,2 Md Golam Hasnain,1 Abhay R Satoskar3 1Centre for Nutrition and Food Security, Parasitology Laboratory, (icddr, b) Mohakhali, Dhaka, Bangladesh; 2Department of Parasitology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan; 3Departments of Pathology and Microbiology, Wexner Medical Center, The Ohio State University, Columbus, OH, USA Abstract: Post kala-azar dermal leishmaniasis (PKDL) is a skin complication resulting from infection with Leishmania donovani (LD) parasite. It mostly affects individuals who have previously suffered from visceral leishmaniasis (VL) caused by LD. In some cases, PKDL develops among people infected with LD, but do not show any symptoms of VL. Clinical presentation includes hypopigmented macules/papules/nodules or polymorphic lesions (combination of two or more lesions). Except for skin lesions, PKDL patients are generally healthy and usually do not seek medical care. These patients play an important role in interepidemic transmission of the infection and subsequent VL outbreak. Therefore, proper diagnosis and treatment of PKDL patients is important for the control of VL in endemic countries, especially in the Indian subcontinent where VL is anthroponotic. Here, we report the challenges in the estimation of PKDL burden, its diagnosis, and treatment, and suggest possible solutions based on recent literature, reports, published manuals, and web-based information. Keywords: post kala-azar dermal leishmaniasis, Bangladesh, challenge, treatment, diagnosis, prevention

Summary Almost all inbred mice are highly susceptible to parasites of the Leishmania mexicana complex that includes L. amazonensis and L. mexicana. Recent studies have reported that T cells from L. amazonensis-infected mice fail to respond to IL-12 due to impaired IL-12R expression. Here, we demonstrate that lymph node cells from L. mexicana-infected C57BL/6 and 129Sv/Ev mice respond efficiently to exogenous IL-12 in vitro and produce IFN-γ. Moreover, we also show that deletion of signal transducer and activator of transcription (STAT)4 gene in resistant STAT6–/– mice renders them susceptible to L. mexicana. These findings indicate that an inability to produce IL-12 rather than unresponsiveness to this cytokine is responsible for susceptibility to L. mexicana. Moreover, the data also demonstrate that the STAT4-mediated pathway is critical for the development of protective immunity against cutaneous leishmaniasis, regardless of the species of Leishmania and/or genetic background of the mice. [ABSTRACT FROM AUTHOR]

Background: Carbohydrates on allergens are known to be important for allergenicity. However, most findings have been made with epitope analysis. In this study, we investigated the involvement of N-glycan on phospholipase A[sub 2] (PLA2), the major allergen of honeybee venom, in in vivo synthesis of specific IgE in mice. Methods: CBA/J and C57BL/6 mice were sensitized intranasally with either native or deglycosylated PLA2 in the absence of adjuvant. After repeated sensitization, serum Ab titers against PLA2 were determined. PLA2 was deglycosylated chemically with anhydrous trifluoromethanesulfonic acid (TFMS). Results: CBA/J mice showed PLA2-specific IgE production after repeated sensitization with native PLA2. They also produced PLA2-specific IgG1 predominantly, suggesting that Th2-type Ab production was induced. When we used deglycosylated PLA2 as a competitor in ELISA for detecting PLA2-specific IgE, deglycosylated PLA2 completely inhibited the binding between native PLA2 and IgE. Deglycosylated PLA2 had the same potential for inducing specific IgE synthesis as native PLA2, since sensitization with deglycosylated PLA2 also elicited IgE production in CBA/J mice. Conclusions: These results suggest that carbohydrate on PLA2 is less important than previously thought not only as a dominant IgE epitope but also in synthesis of PLA2-specific IgE in vivo. [ABSTRACT FROM AUTHOR]

Cutaneous leishmaniasis (CL) is characterized by extensive skin lesions, which are usually painless despite being associated with extensive inflammation. The molecular mechanisms responsible for this analgesia have not been identified. Through untargeted metabolomics, we found enriched anti-nociceptive metabolic pathways in L. mexicana -infected mice. Purines were elevated in infected macrophages and at the lesion site during chronic infection. These purines have anti-inflammatory and analgesic properties by acting through adenosine receptors, inhibiting TRPV1 channels, and promoting IL-10 production. We also found arachidonic acid (AA) metabolism enriched in the ear lesions compared to the non-infected controls. AA is a metabolite of anandamide (AEA) and 2-arachidonoylglycerol (2-AG). These endocannabinoids act on cannabinoid receptors 1 and 2 and TRPV1 channels to exert anti-inflammatory and analgesic effects. Our study provides evidence of metabolic pathways upregulated during L. mexicana infection that may mediate anti-nociceptive effects experienced by CL patients and identifies macrophages as a source of these metabolites., Competing Interests: This article reflects the views of the authors and should not be construed to represent FDA’s views or policies. A.R.S. and The Ohio State University hold a patent #WO2012145734A1 entitled: “Antileishmanial compositions and methods of use”., (© 2023 The Author(s).)

The leishmanin skin test was used for almost a century to detect exposure and immunity to Leishmania, the causative agent of leishmaniasis, a major neglected tropical disease. Due to a lack of antigen used for the intradermal injection, the leishmanin skin test is no longer available. As leishmaniasis control programs are advancing and new vaccines are entering clinical trials, it is essential to re-introduce the leishmanin skin test. Here we establish a Leishmania donovani strain and describe the production, under Good Laboratory Practice conditions, of leishmanin soluble antigen used to induce the leishmanin skin test in animal models of infection and vaccination. Using a mouse model of cutaneous leishmaniasis and a hamster model of visceral leishmaniasis, soluble antigen induces a leishmanin skin test response following infection and vaccination with live attenuated Leishmania major (LmCen -/- ). Both the CD4 + and CD8 + T-cells are necessary for the leishmanin skin test response. This study demonstrates the feasibility of large-scale production of leishmanin antigen addressing a major bottleneck for performing the leishmanin skin test in future surveillance and vaccine clinical trials., (© 2023. The Author(s).)

Although phagocytic cells are documented targets of Leishmania parasites, it is unclear whether other cell types can be infected. Here, we use unbiased single-cell RNA sequencing (scRNA-seq) to simultaneously analyze host cell and Leishmania donovani transcriptomes to identify and annotate parasitized cells in spleen and bone marrow in chronically infected mice. Our dual-scRNA-seq methodology allows the detection of heterogeneous parasitized populations. In the spleen, monocytes and macrophages are the dominant parasitized cells, while megakaryocytes, basophils, and natural killer (NK) cells are found to be unexpectedly infected. In the bone marrow, the hematopoietic stem cells (HSCs) expressing phagocytic receptors FcγR and CD93 are the main parasitized cells. Additionally, we also detect parasitized cycling basal cells, eosinophils, and macrophages in chronically infected mice. Flow cytometric analysis confirms the presence of parasitized HSCs. Our unbiased dual-scRNA-seq method identifies rare, parasitized cells, potentially implicated in pathogenesis, persistence, and protective immunity, using a non-targeted approach., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Leishmaniasis is a tropical disease prevalent in 90 countries. Presently, there is no approved vaccine for human use. We developed a live attenuated L. mexicana Cen -/- (LmexCen -/- ) strain as a vaccine candidate that showed excellent efficacy, characterized by reduced Th2 and enhanced Th1 responses in C57BL/6 and BALB/c mice, respectively, compared to wild-type L. mexicana (Lmex WT ) infection. Toward understanding the immune mechanisms of protection, we applied untargeted mass spectrometric analysis to LmexCen -/- and Lmex WT infections. Data showed enrichment of the pentose phosphate pathway (PPP) in ears immunized with LmexCen -/- versus naive and Lmex WT infection. PPP promotes M1 polarization in macrophages, suggesting a switch to a pro-inflammatory phenotype following LmexCen -/- inoculation. Accordingly, PPP inhibition in macrophages infected with LmexCen -/- reduced the production of nitric oxide and interleukin (IL)-1β, hallmarks of classical activation. Overall, our study revealed the immune regulatory mechanisms that may be critical for the induction of protective immunity., Competing Interests: The FDA is currently a co-owner of two US patents that claim attenuated Leishmania species with the centrin gene deletion (US7,887,812 and US 8,877,213). This article reflects the views of the authors and should not be construed to represent FDA’s views or policies., (© 2023 The Author(s).)

Leishmaniasis is a parasitic disease that is prevalent in 90 countries, and yet no licensed human vaccine exists against it. Toward control of leishmaniasis, we have developed Leishmania major centrin gene deletion mutant strains ( LmCen -/- ) as a live attenuated vaccine, which induces a strong IFN-γ-mediated protection to the host. However, the immune mechanisms of such protection remain to be understood. Metabolomic reprogramming of the host cells following Leishmania infection has been shown to play a critical role in pathogenicity and shaping the immune response following infection. Here, we applied untargeted mass spectrometric analysis to study the metabolic changes induced by infection with LmCen -/- and compared those with virulent L. major parasite infection to identify the immune mechanism of protection. Our data show that immunization with LmCen -/- parasites, in contrast to virulent L. major infection promotes a pro-inflammatory response by utilizing tryptophan to produce melatonin and downregulate anti-inflammatory kynurenine-AhR and FICZ-AhR signaling., Competing Interests: The FDA is currently a co-owner of two US patents that claim attenuated Leishmania species with the centrin gene deletion (US7,887,812 and US8,877,213). This article reflects the views of the authors and should not be construed to represent FDA’s views or policies. The remaining authors declare no competing interests.

Leishmaniasis is considered as one of the 20 neglected tropical diseases. Current methods of leishmanial diagnosis depend on conventional laboratory-based techniques, which are time-consuming, costly and require special equipment and trained personnel. In this context, we aimed to provide an immuno field effect transistors (ImmunoFET) biosensor that matches the conventional standards for point-of-care (POC) monitoring and detection of Leishmania (L.) donovani/Leishmania major. Crude antigens prepared by repeated freeze thawing of L. donovani/L. major stationary phase promastigotes were used for ELISA and ImmunoFETs. Lesishmania-specific antigens were serially diluted in 1× PBS from a concentration of 10 6 -10 2 parasites/mL. A specific polyclonal antibody-based sandwich ELISA was established for the detection of Leishmania antigens. An immunoFET technology-based POC novel assay was constructed for the detection of Leishmania antigens. Interactions between antigen-antibody at the gate surface generate an electrical signal that can be measured by semiconductor field-effect principles. Sensitivity was considered and measured as the change in current divided by the initial current. The final L. donovani/L. major crude antigen protein concentrations were measured as 1.50 mg/mL. Sandwich ELISA against the Leishmania 40S ribosomal protein detected Leishmania antigens could detect as few as 100 L. donovani/L. major parasites. An immunoFET biosensor was constructed based on the optimization of aluminium gallium nitride/gallium nitride (AlGaN/GaN) surface oxidation methods. The device surface was composed by an AlGaN/GaN wafer with a 23 nm AlGaN barrier layer, a 2 μm GaN layer on the silicon carbide (SiC) substrate for Leishmania binding, and coated with a specific antibody against the Leishmania 40S ribosomal protein, which was successfully detected at concentrations from 10 6 to 10 2 parasites/mL in 1× PBS. At the concentration of 10 4 parasites, the immunoFETs device sensitivities were 13% and 0.052% in the sub-threshold regime and the saturation regime, respectively. Leishmania parasites were successfully detected by the ImmunoFET biosensor at a diluted concentration as low as 150 ng/mL. In this study, the developed ImmunoFET biosensor performed well. ImmunoFET biosensors can be used as an alternative diagnostic method to ELISA. Increasing the sensitivity and optimization of immuno-FET biosensors might allow earlier and faster detection of leishmaniasis., (© 2023 John Wiley & Sons Ltd.)

Human infection with the protozoan parasite Trypanosoma cruzi causes Chagas disease for which there are no prophylactic vaccines. Cyclophilin 19 is a secreted cis-trans peptidyl isomerase expressed in all life stages of Trypanosoma cruzi. This protein in the insect stage leads to the inactivation of insect anti-parasitic peptides and parasite transformation whereas in the intracellular amastigotes it participates in generating ROS promoting the growth of parasites. We have generated a parasite mutant with depleted expression of Cyp19 by removal of 2 of 3 genes encoding this protein using double allelic homologous recombination. The mutant parasite line failed to replicate when inoculated into host cells in vitro or in mice indicating that Cyp19 is critical for infectivity. The mutant parasite line also fails to replicate in or cause clinical disease in immuno-deficient mice further validating their lack of virulence. Repeated inoculation of mutant parasites into immuno-competent mice elicits parasite-specific trypanolytic antibodies and a Th-1 biased immune response and challenge of mutant immunized mice with virulent wild-type parasites is 100% effective at preventing death from acute disease. These results suggest that parasite Cyp19 may be candidate for small molecule drug targeting and that the mutant parasite line may warrant further immunization studies for prevention of Chagas disease., (© 2023. The Author(s).)

Leishmaniasis is one of the top neglected tropical diseases with significant morbidity and mortality in low and middle-income countries (LMIC). However, this disease is also spreading in the developed world. Currently, there is a lack of effective strategies to control this disease. Vaccination can be an effective measure to control leishmaniasis and has the potential to achieve disease elimination. Recently, we have generated centrin gene-deleted new world L. mexicana (LmexCen -/- ) parasites using CRISPR/Cas9 and showed that they protect mice against a homologous L. mexicana infection that causes cutaneous disease. In this study, we tested whether LmexCen -/- parasites can also protect against visceral leishmaniasis caused by L. donovani in a hamster model. We showed that immunization with LmexCen -/- parasites is safe and does not cause lesions. Furthermore, such immunization conferred protection against visceral leishmaniasis caused by a needle-initiated L. donovani challenge, as indicated by a significant reduction in the parasite burdens in the spleen and liver as well as reduced mortality. Similar control of parasite burden was also observed against a sand fly mediated L. donovani challenge. Importantly, immunization with LmexCen -/- down-regulated the disease promoting cytokines IL-10 and IL-4 and increased pro-inflammatory cytokine IFN-γ resulting in higher IFN-γ/IL-10 and IFN-γ/IL4 ratios compared to non-immunized animals. LmexCen -/- immunization also resulted in long-lasting protection against L. donovani infection. Taken together, our study demonstrates that immunization with LmexCen -/- parasites is safe and efficacious against the Old World visceral leishmaniasis., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Introduction: Despite their effectiveness and indispensability, many drugs are poorly solvated in aqueous solutions. Over recent decades, the need for targeted drug delivery has led to the development of pharmaceutical formulations with enhanced lipid solubility to improve their delivery properties. Therefore, a dependable approach for administering lipid-soluble drugs needs to be developed., Areas Covered: The advent of 3D printing or additive manufacturing (AM) has revolutionized the development of medical devices, which can effectively enable the delivery of lipophilic drugs to the targeted tissues. This review focuses on the use of microneedles and iontophoresis for transdermal drug delivery. Microneedle arrays, inkjet printing, and fused deposition modeling have emerged as valuable approaches for delivering several classes of drugs. In addition, iontophoresis has been successfully employed for the effective delivery of macromolecular drugs., Expert Opinion: Microneedle arrays, inkjet printing, and fused deposition are potentially useful for many drug delivery applications; however, the clinical and commercial adoption rates of these technologies are relatively low. Additional efforts is needed to enable the pharmaceutical community to fully realize the benefits of these technologies.

Leishmaniasis, caused by an infection of the Leishmania protozoa, is a neglected tropical disease and a major health problem in tropical and subtropical regions of the world, with approximately 350 million people worldwide at risk and 2 million new cases occurring annually. Current treatments for leishmaniasis are not highly efficacious and are associated with high costs, especially in low- and middle-income endemic countries, and high toxicity. Due to a surge in the incidence of leishmaniases worldwide, the development of new strategies such as a prophylactic vaccine has become a high priority. However, the ability of Leishmania to undermine immune recognition has limited our efforts to design safe and efficacious vaccines against leishmaniasis. Numerous antileishmanial vaccine preparations based on DNA, subunit, and heat-killed parasites with or without adjuvants have been tried in several animal models but very few have progressed beyond the experimental stage. However, it is known that people who recover from Leishmania infection can be protected lifelong against future infection, suggesting that a successful vaccine requires a controlled infection to develop immunologic memory and subsequent long-term immunity. Live attenuated Leishmania parasites that are non-pathogenic and provide a complete range of antigens similarly to their wild-type counterparts could evoke such memory and, thus, would be effective vaccine candidates. Our laboratory has developed several live attenuated Leishmania vaccines by targeted centrin gene disruptions either by homologous recombination or, more recently, by using genome editing technologies involving CRISPR-Cas9. In this review, we focused on the sequential history of centrin gene-deleted Leishmania vaccine development, along with the characterization of its safety and efficacy. Further, we discussed other major considerations regarding the transition of dermotropic live attenuated centrin gene-deleted parasites from the laboratory to human clinical trials.

Leishmaniasis is a vector-borne parasitic disease transmitted through the bite of a sand fly with no available vaccine for humans. Recently, we have developed a live attenuated Leishmania major centrin gene-deleted parasite strain ( LmCen -/- ) that induced protection against homologous and heterologous challenges. We demonstrated that the protection is mediated by IFN (Interferon) γ-secreting CD4 + T-effector cells and multifunctional T cells, which is analogous to leishmanization. In addition, in a leishmanization model, skin tissue-resident memory T (TRM) cells were also shown to be crucial for host protection. In this study, we evaluated the generation and function of skin TRM cells following immunization with LmCen -/- parasites and compared those with leishmanization. We show that immunization with LmCen -/- generated skin CD4+ TRM cells and is supported by the induction of cytokines and chemokines essential for their production and survival similar to leishmanization. Following challenge with wild-type L. major , TRM cells specific to L. major were rapidly recruited and proliferated at the site of infection in the immunized mice. Furthermore, upon challenge, CD4 + TRM cells induce higher levels of IFNγ and Granzyme B in the immunized and leishmanized mice than in non-immunized mice. Taken together, our studies demonstrate that the genetically modified live attenuated LmCen -/- vaccine generates functional CD4 + skin TRM cells, similar to leishmanization, that may play a crucial role in host protection along with effector T cells as shown in our previous study., Competing Interests: The FDA is currently a co-owner of two US patents that claim attenuated Leishmania species with the Centrin gene deletion (US7,887,812 and US 8,877,213). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ismail, Karmakar, Bhattacharya, Sepahpour, Takeda, Hamano, Matlashewski, Satoskar, Gannavaram, Dey and Nakhasi.)

Leishmaniasis is a neglected protozoan disease affecting over 12 million people globally with no approved vaccines for human use. New World cutaneous leishmaniasis (CL) caused by L. mexicana is characterized by the development of chronic non-healing skin lesions. Using the CRISPR/Cas9 technique, we have generated live attenuated centrin knockout L. mexicana (LmexCen -/- ) parasites. Centrin is a cytoskeletal protein important for cellular division in eukaryotes and, in Leishmania, is required only for intracellular amastigote replication. We have investigated the safety and immunogenicity characteristics of LmexCen -/- parasites by evaluating their survival and the cytokine production in bone-marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) in vitro. Our data shows that LmexCen -/- amastigotes present a growth defect, which results in significantly lower parasitic burdens and increased protective cytokine production in infected BMDMs and BMDCs, compared to the wild type (WT) parasites. We have also determined the safety and efficacy of LmexCen -/- in vivo using experimental murine models of L. mexicana. We demonstrate that LmexCen -/- parasites are safe and do not cause lesions in susceptible mouse models. Immunization with LmexCen -/- is also efficacious against challenge with WT L. mexicana parasites in genetically different BALB/c and C57BL/6 mouse models. Vaccinated mice did not develop cutaneous lesions, displayed protective immunity, and showed significantly lower parasitic burdens at the infection site and draining lymph nodes compared to the control group. Overall, we demonstrate that LmexCen -/- parasites are safe and efficacious against New World cutaneous leishmaniasis in pre-clinical models., (© 2022. The Author(s).)

Our earlier in vitro and in vivo studies have revealed that the phytosterol, pentalinonsterol (cholest-4,20,24-trien-3-one) (PEN), isolated from the roots of Pentalinon andrieuxii, possesss immunomodulatory properties in macrophages and dendritic cells. Leishmaniasis, caused by the infection of Leishmania spp. (a protozoan parasite), is emerging as the second-leading cause of mortality among the tropical diseases and there is an unmet need for a pharmacological intervention of leishmaniasis. Given the beneficial immunomodulatory actions and lipophilic properties of PEN, the objective of this study was to elucidate the mechanism(s) of action of the immunomodulatory action(s) of PEN in macrophages through the modulation of phospholipase A 2 (PLA 2 ) activity that might be crucial in the antileishmanial action of PEN. Therefore, in this study, we investigated whether PEN would modulate the activity of PLA 2 in RAW 264.7 macrophages and mouse bone marrow-derived primary macrophages (BMDMs) in vitro and further determined how the upstream PLA 2 activation would regulate the downstream cytokine release in the macrophages. Our current results demonstrated that (i) PEN induced PLA 2 activation (arachidonic acid release) in a dose- and time-dependent manner that was regulated upstream by the mitogen-activated protein kinases (MAPKs); (ii) the PEN-induced activation of PLA 2 was attenuated by the cPLA 2 -specific pharmacological inhibitors; and (iii) the cPLA 2 -specific pharmacological inhibitors attenuated the release of inflammatory cytokines from the macrophages. For the first time, our current study demonstrated that PEN exhibited its immunomodulatory actions through the activation of cPLA 2 in the macrophages, which potentially could be used in the development of a pharmacological intervention against leishmaniasis., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The basic aim of the study was to develop and evaluate the triple drug loaded cationic nano-vesicles (cNVs), where miltefosine was used as a replacement of surfactant (apart from its anti-leishmanial role), in addition to meglumine antimoniate (MAM) and imiquimod (Imq), as a combination therapy for the topical treatment of cutaneous leishmaniasis (CL). The optimized formulation was nano-sized (86.2 ± 2.7 nm) with high entrapment efficiency (63.8 ± 2.1% (MAM) and 81.4 ± 2.3% (Imq)). In-vivo skin irritation assay showed reduced irritation potential and a decrease in the cytotoxicity of cNVs as compared to conventional NVs (having sodium deoxycholate as a surfactant). A synergistic interaction between drugs was observed against intracellular amastigotes, whereas the in-vivo antileishmanial study presented a significant reduction in the parasitic burden. The results suggested the potential of surfactant free, triple drug loaded cNVs as an efficient vehicle for the safe topical treatment of CL., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Introduction: Leishmaniasis is a major public health problem and the second most lethal parasitic disease in the world due to the lack of effective treatments and vaccines. Even when not lethal, leishmaniasis significantly affects individuals and communities through life-long disabilities, psycho-sociological trauma, poverty, and gender disparity in treatment., Areas Covered: This review discusses the most relevant and recent research available on Pubmed and GoogleScholar highlighting leishmaniasis' global impact, pathogenesis, treatment options, and lack of effective control strategies. An effective vaccine is necessary to prevent morbidity and mortality, lower health care costs, and reduce the economic burden of leishmaniasis for endemic low- and middle-income countries. Since there are several forms of leishmaniasis, a pan- Leishmania vaccine without geographical restrictions is needed. This review also focuses on recent advances and common challenges in developing prophylactic strategies against leishmaniasis., Expert Opinion: Despite advances in pre-clinical vaccine research, approval of a human leishmaniasis vaccine still faces major challenges - including manufacturing of candidate vaccines under Good Manufacturing Practices, developing well-designed clinical trials suitable in endemic countries, and defined correlates of protection. In addition, there is a need to explore Challenge Human Infection Model to avoid large trials because of fluctuating incidence and prevalence of leishmanasis.

Leishmaniasis is endemic to the tropical and subtropical regions of the world and is transmitted by the bite of an infected sand fly. The multifaceted interactions between Leishmania , the host innate immune cells, and the adaptive immunity determine the severity of pathogenesis and disease development. Leishmania parasites establish a chronic infection by subversion and attenuation of the microbicidal functions of phagocytic innate immune cells such as neutrophils, macrophages and dendritic cells (DCs). Other innate cells such as inflammatory monocytes, mast cells and NK cells, also contribute to resistance and/or susceptibility to Leishmania infection. In addition to the cytokine/chemokine signals from the innate immune cells, recent studies identified the subtle shifts in the metabolic pathways of the innate cells that activate distinct immune signal cascades. The nexus between metabolic pathways, epigenetic reprogramming and the immune signaling cascades that drive the divergent innate immune responses, remains to be fully understood in Leishmania pathogenesis. Further, development of safe and efficacious vaccines against Leishmaniasis requires a broader understanding of the early interactions between the parasites and innate immune cells. In this review we focus on the current understanding of the specific role of innate immune cells, the metabolomic and epigenetic reprogramming and immune regulation that occurs during visceral leishmaniasis, and the strategies used by the parasite to evade and modulate host immunity. We highlight how such pathways could be exploited in the development of safe and efficacious Leishmania vaccines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Volpedo, Pacheco-Fernandez, Bhattacharya, Oljuskin, Dey, Gannavaram, Satoskar and Nakhasi.)

Signal Transducer and Activator of Transcription (STAT) 1 signaling is critical for IFN-γ-mediated immune responses and resistance to protozoan and viral infections. However, its role in immunoregulation during helminth parasitic infections is not fully understood. Here, we used STAT1 -/- mice to investigate the role of this transcription factor during a helminth infection caused by the cestode Taenia crassiceps and show that STAT1 is a central molecule favoring susceptibility to this infection. STAT1 -/- mice displayed lower parasite burdens at 8 weeks post-infection compared to STAT1 +/+ mice. STAT1 mediated the recruitment of inflammatory monocytes and the development of alternatively activated macrophages (M2) at the site of infection. The absence of STAT1 prevented the recruitment of CD11b + Ly6C hi Ly6G - monocytic cells and therefore their suppressive activity. This failure was associated with the defective expression of CCR2 on CD11b + Ly6C hi Ly6G - cells. Importantly, CD11b + Ly6C hi Ly6G - cells highly expressed PDL-1 and suppressed T-cell proliferation elicited by anti-CD3 stimulation. PDL-1 + cells were mostly absent in STAT1 -/- mice. Furthermore, only STAT1 +/+ mice developed M2 macrophages at 8 weeks post-infection, although macrophages from both T. crassiceps -infected STAT1 +/+ and STAT1 -/- mice responded to IL-4 in vitro, and both groups of mice were able to produce the Th2 cytokine IL-13. This suggests that CD11b + CCR2 + Ly6C hi Ly6G - cells give rise to M2 macrophages in this infection. In summary, a lack of STAT1 resulted in impaired recruitment of CD11b + CCR2 + Ly6C hi Ly6G - cells, failure to develop M2 macrophages, and increased resistance against T. crassiceps infection.

MicroRNA-21 (miR-21) inhibits IL-12 expression and impairs the Th1 response necessary for control of Leishmania infection. Recent studies have shown that Leishmania infection induces miR-21 expression in dendritic cells and macrophages, and inhibition of miR-21 restores IL-12 expression. Because miR-21 is known to be expressed due to inflammatory stimuli in a wide range of hematopoietic cells, we investigated the role of miR-21 in regulating immune responses during visceral leishmaniasis (VL) caused by Leishmania donovani infection. We found that miR-21 expression was significantly elevated in dendritic cells, macrophages, inflammatory monocytes, polymorphonuclear neutrophils, and in the spleen and liver tissues after L. donovani infection, concomitant with an increased expression of disease exacerbating IL-6 and STAT3. Bone marrow dendritic cells from miR-21 knockout (miR-21KO) mice showed increased IL-12 production and decreased production of IL-10. On L. donovani infection, miR-21KO mice exhibited significantly greater numbers of IFN-γ- and TNF-α-producing CD4 + and CD8 + T cells in their organs that was associated with increased production of Th1-associated IFN-γ, TNF-α, and NO from the splenocytes. Finally, miR-21KO mice displayed significantly more developing and mature hepatic granulomas leading to reduction in organ parasitic loads compared with wild type counterparts. Similar results were noted in L. donovani -infected wild type mice after transient miR-21 depletion. These observations indicate that miR-21 plays a critical role in pathogenesis of VL by suppressing IL-12- and Th1-associated IFN-γ and also inducing disease-promoting induction of the IL-6 and STAT-3 signaling pathway. miR-21 could therefore be used as a potential target for developing host-directed treatment for VL., (Copyright © 2021 by The American Association of Immunologists, Inc.)

The genetic diversity of Leishmania spp. in North Eastern Pakistan remains undetermined despite increased cases of cutaneous leishmaniasis (CL). This study was designed to decipher the molecular characterization and genetic diversity of Leishmania spp. in North Eastern Pakistan. Out of 13761 CL suspected cases, 567 cases were microscopically positive and confirmed as Leishmania spp. by internal transcribed spacer (ITS) gene amplification through the PCR- RFLP technique. Further, isolates were directly sequenced to conduct phylogenetic analysis for genetic diversity. Among suspected CL cases, Mirpur showed the highest proportion of CL infection with 4.85% (118/2431) of the cases, while the Neelum district showed the lowest percentage at 3.29% (9/273). The slide positivity rate, annual blood examination rate, and annual parasite incidence rate were 3.84, 0.27, and 0.01% respectively, and the incidence of CL in the age group 1-20 years old was higher in males (50.92%) than females (25.75%). The RFLP analysis and sequencing confirmed the occurrence of Leishmania tropica, Leishmania major, and Leishmania infantum. Leishmania tropica (p = 0.02) confirmed significantly higher nucleotides variation than L. major (p = 0.05). Current findings confirmed the prior assumption that anthroponotic CL is the primary CL form present in North Eastern Pakistan. Moreover, this is the first report based on molecular identification of L. major, and L. infantum from North Eastern Pakistan. This remarkable heterogeneity in the Leishmania spp. is the leading cause of treatment failure and emergence of new haplotypes. Therefore more extensive investigations are recommended from all geographical regions of North Eastern Pakistan, especially those using a large sample size., (Copyright © 2021. Published by Elsevier B.V.)

Alum, used as an adjuvant in injected vaccines, promotes T helper 2 (Th2) and serum antibody (Ab) responses. However, it fails to induce secretory immunoglobulin (Ig) A (SIgA) in mucosal tissues and is poor in inducing Th1 and cell-mediated immunity. Alum stimulates interleukin 1 (IL-1) and the recruitment of myeloid cells, including neutrophils. We investigated whether neutrophil elastase regulates the adjuvanticity of alum, and whether a strategy targeting neutrophil elastase could improve responses to injected vaccines. Mice coadministered a pharmacological inhibitor of elastase, or lacking elastase, developed high-affinity serum IgG and IgA antibodies after immunization with alum-adsorbed protein vaccines, including the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). These mice also developed broader antigen-specific CD4 + T cell responses, including high Th1 and T follicular helper (Tfh) responses. Interestingly, in the absence of elastase activity, mucosal SIgA responses were induced after systemic immunization with alum as adjuvant. Importantly, lack or suppression of elastase activity enhanced the magnitude of anti-SARS-CoV-2 spike subunit 1 (S1) antibodies, and these antibodies reacted with the same epitopes of spike 1 protein as sera from COVID-19 patients. Therefore, suppression of neutrophil elastase could represent an attractive strategy for improving the efficacy of alum-based injected vaccines for the induction of broad immunity, including mucosal immunity., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Leishmaniasis is a neglected tropical disease that affects 12 million people worldwide. The disease has high morbidity and mortality rates and is prevalent in over 80 countries, leaving more than 300 million people at risk of infection. Of all of the manifestations of this disease, cutaneous leishmaniasis (CL) is the most common form and it presents as ulcerating skin lesions that can self-heal or become chronic, leading to disfiguring scars. This review focuses on the different pathologies and disease manifestations of CL, as well as their varying degrees of severity. In particular, this review will discuss self-healing localized cutaneous leishmaniasis (LCL), leishmaniasis recidivans (LR), mucocutaneous leishmaniasis (MCL), anergic diffuse cutaneous leishmaniasis (ADCL), disseminated leishmaniasis (DL), and Post Kala-azar Dermal Leishmaniasis (PKDL), which is a cutaneous manifestation observed in some visceral leishmaniasis (VL) patients after successful treatment. The different clinical manifestations of CL are determined by a variety of factors including the species of the parasites and the host's immune response. Specifically, the balance between the pro and anti-inflammatory mediators plays a vital role in the clinical presentation and outcome of the disease. Depending upon the immune response, Leishmania infection can also transition from one form of the disease to another. In this review, different forms of cutaneous Leishmania infections and their immunology are described., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Volpedo, Pacheco-Fernandez, Holcomb, Cipriano, Cox and Satoskar.)

Interferon (IFN)-γ is indispensable in the resolution of cutaneous leishmaniasis (CL), while the Th2 cytokines IL-4, IL-10, and IL-13 mediate susceptibility. A recent study found that miR155, which promotes CD4 + Th1 response and IFN-γ production, is dispensable in the control of Leishmania donovani infection. Here, the role of miR155 in CL caused by L. major was investigated using miR155-deficient (miR155 -/- ) mice. Infection was controlled significantly quicker in the miR155 -/- mice than in their wild-type (WT) counterparts, indicating that miR155 contributes to the pathogenesis of CL. Faster resolution of infection in miR155 -/- mice was associated with increased levels of Th1-associated IL-12 and IFN-γ and reduced production of Th2- associated IL-4, IL-10, and IL-13. Concentrations of IFN-γ + CD8 + T cells and natural killer cells in draining lymph nodes were significantly higher in the L. major-infected miR155 -/- mice than in the infected WT mice, as indicated by flow-cytometry. After in vitro IFN-γ stimulation, nitric oxide and IL-12 production were increased, IL-10 production was decreased, and parasite clearance was enhanced in L. major-infected miR155 -/- DCs compared to those in WT DCs. Furthermore, IFN-γ production from activated miR155 -/- T cells was significantly enhanced in L. major-infected miR155 -/- DCs. Together, these findings demonstrate that miR155 promotes susceptibility to CL caused by L. major by promoting Th2 response and inhibiting DC function., (Copyright © 2021 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Leishmaniasis is a vector-borne Neglected Tropical Disease (NTD) transmitted by the sand fly and is a major public health problem worldwide. Infections caused by Leishmania clinically manifest as a wide range of diseases, such as cutaneous (CL), diffuse cutaneous (DCL), mucosal (MCL) and visceral leishmaniasis (VL). The host innate and adaptative immune responses play critical roles in the defense against leishmaniasis. However, Leishmania parasites also manipulate the host immune response for their survival and replication. In addition, other factors such as sand fly salivary proteins and microbiota also promote disease susceptibility and parasite spread by modulating local immune response. Thus, a complex interplay between parasite, sand fly and the host immunity governs disease severity and outcome. In this review, we discuss the host immune response during Leishmania infection and highlight the factors associated with resistance or susceptibility., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Tacrolimus (TAC) is an immunosuppressive drug, optimally used for liver, kidney, and heart transplant to avoid immune rejection. In retrospect, a multitude of studies have reported effects of TAC, such as nephrotoxicity, diabetes, and other complications. However, limited information is available regarding short-term exposure of TAC on the liver. Therefore, the present study was designed to unravel the effects of short-term exposure of TAC on a rat model. The animal model was established by TAC administration for 6, 12, 24, and 48 h time points. Liver histopathological changes were observed with PAS-D, reticulin stain, and immunostaining of PCNA and CK-7 coupled with glycogen quantification in a liver homogenate. TUNEL assay was performed to evaluate the DNA damage in the liver. Concentration of GSH and activities of SOD and CAT in the serum were measured to assess the antioxidant status, whereas liver tissue MDA level was measured as a biomarker of oxidative stress. Hepatic gene expression analysis of IL-10, IL-13, SOCS-2, and SOCS-3 was performed by RT-PCR. Results revealed marked changes in liver architecture of all TAC-treated groups, as evidenced by sinusoid dilation, hepatocyte derangement, glycogen deposition, and collapsed reticulin fibers. Significant increase in PCNA and CK-7 immunostaining along with the presence of TUNEL-positive cells was revealed in treatment groups as compared to the control group. Serum antioxidant enzyme status was markedly decreased, whereas the liver MDA level was increased in TAC treatment groups indicating oxidative stress induction. The gene expression profile of cytokines was significantly upregulated in treatment groups highlighting an inflammatory response. In conclusion, results of the current study propose that even a short-term TAC exposure can induce change in antioxidant status and lipid peroxidation. Therefore, these factors should be considered to avoid and minimize immunosuppression-related issues in a prolonged course of treatment., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 N. Fatima et al.)

Sanliurfa, a city in southeastern Turkey, is host to 477,166 Syrian refugees. The incidence of cutaneous leishmaniasis (CL) may be on the rise in areas affected by a refugee crisis, like Sanliurfa; thus, consequently, local uncommon species of Leishmania may be encountered in these regions. This might potentially make diagnosis and treatment more challenging over time. In this study, it was aimed to identify the causative agents of CL in clinical samples. A total of 154 patients (60 Syrian and 94 Turkish) who were diagnosed with CL via microscopical examination and PCR were enrolled this study. All of the samples were analyzed using internal transcribed spacer 1 genes, restriction fragment length polymorphism, DNA-sequencing, and phylogenetic analyses. In this study, Leishmania tropica was determined to be the predominant species in 140 of the patients (90.9%), followed by Leishmania major in 12 patients (7.8%), and Leishmania infantum in 2 patients (1.3%). Of the 94 Turkish patients, 94.7% were infected with L. tropica and 5.3% were infected with L. major, while none were infected with L. infantum. However, of the 60 Syrian patients, 85% were infected with L. tropica, 11.7% were infected with L. major, and 3.3% were infected with L. infantum. There was a significant association between the Leishmania species and the nations (Turkish-Syrian) (P < 0.001). The sequences were numbered from MH347941 to MH347953 and submitted to GenBank. This study confirmed that L. tropica, L. major, and L. infantum coexisted in Sanliurfa. This was the first time that the species L. infantum was reported among recent immigrants from Syria in Sanliurfa. Therefore, it is necessary to discriminate the Leishmania species for diagnosis, treatment, and controlled studies in hyper-endemic regions.

Background: Leishmaniasis is a neglected tropical disease affecting millions of people worldwide. Emerging drug resistance of Leishmania species poses threaten to the effective control and elimination programme of this neglected tropical disease., Methods: In this work, we conducted drug-resistance testing, whole genome resequencing and proteome profiling for a recently reported clinical isolate with supposed drug resistance (HCZ), and two reference sensitive strains (DD8 and 9044) of Leishmania donovani, to explore molecular mechanisms underlying drug resistance in this parasite., Results: With reference to DD8 and 9044 strains, HCZ isolate showed higher-level virulence and clear resistance to antimonials in promastigote culture, infected macrophages and animal experiment. Pairwise genomic comparisons revealed genetic variations (86 copy number variations, 271 frameshift mutations in protein-coding genes and two site mutations in non-coding genes) in HCZ isolate that were absent from the reference sensitive strains. Proteomic analysis indicated different protein expression between HCZ isolate and reference strains, including 69 exclusively detected proteins and 82 consistently down-/upregulated molecules in the HCZ isolate. Integrative analysis showed linkage of 12 genomic variations (gene duplication, insertion and deletion) and their protein expression changes in HCZ isolate, which might be associated with pathogenic and antimony-resistant phenotype. Functional annotation analyses further indicated that molecules involved in nucleotide-binding, fatty acid metabolism, oxidation-reduction and transport might play a role in host-parasite interaction and drug-resistance., Conclusions: This comprehensive integrative work provided novel insights into the genetic basis underlying virulence and resistance, suggesting new aspects to be investigated for a better intervention against L. donovani and associated diseases.

Triple-negative breast cancer (TNBC), defined as loss of estrogen, progesterone, and Her2 receptors, is a subtype of highly aggressive breast cancer with worse prognosis and poor survival rate. Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory cytokine aberrantly expressed in many solid tumors and known to promote tumor progression and metastasis. However, its role in TNBC progression and metastasis is unexplored. Here we have shown that in TNBC patients, MIF expression was significantly enriched in the tumor compared to adjacent normal tissue. Using publically available patient datasets, we showed that MIF overexpression correlates with worse survival in TNBC compared to other hormonal status. Orthotopic implantation of TNBC cells into MIF knockout mice showed reduced tumor growth compared to wild-type mice. In addition, we have shown that MIF downregulation inhibits TNBC growth and progression in a syngeneic mouse model. We further showed that CPSI-1306, a small-molecule MIF inhibitor, inhibits the growth of TNBC cells in vitro. Mechanistic studies revealed that CPSI-1306 induces intrinsic apoptosis by alteration in mitochondrial membrane potential, cytochrome c (Cyt c) release, and activation of different caspases. In addition, CPSI-1306 inhibits the activation of cell survival and proliferation-related molecules. CPSI-1306 treatment also reduced the tumor growth and metastasis in orthotopic mouse models of mammary carcinoma. CPSI-1306 treatment of tumor-bearing mice significantly inhibited TNBC growth and pulmonary metastasis in a dose-dependent manner. Histological analysis of xenograft tumors revealed a higher number of apoptotic cells in CPSI-1306-treated tumors compared to vehicle controls. Our studies, for the first time, show that MIF overexpression in TNBC enhances growth and metastasis. Taken together, our results indicate that using small molecular weight MIF inhibitors could be a promising strategy to inhibit TNBC progression and metastasis.

Listeria monocytogenes is a facultative intracellular pathogen responsible for the life-threatening disease listeriosis. The pore-forming toxin listeriolysin O (LLO) is a critical virulence factor that plays a major role in the L. monocytogenes intracellular lifecycle and is indispensable for pathogenesis. LLO is also a dominant antigen for T cells involved in sterilizing immunity and it was proposed that LLO acts as a T cell adjuvant. In this work, we generated a novel full-length LLO toxoid (LLO T ) in which the cholesterol-recognition motif, a threonine-leucine pair located at the tip of the LLO C-terminal domain, was substituted with two glycine residues. We showed that LLO T lost its ability to bind cholesterol and to form pores. Importantly, LLO T retained binding to the surface of epithelial cells and macrophages, suggesting that it could efficiently be captured by antigen-presenting cells. We then determined if LLO T can be used as an antigen and adjuvant to protect mice from L. monocytogenes infection. Mice were immunized with LLO T alone or together with cholera toxin or Alum as adjuvants. We found that mice immunized with LLO T alone or in combination with the Th2-inducing adjuvant Alum were not protected against L. monocytogenes. On the other hand, mice immunized with LLO T along with the experimental adjuvant cholera toxin, were protected against L. monocytogenes, as evidenced by a significant decrease in bacterial burden in the liver and spleen three days post-infection. This immunization regimen elicited mixed Th1, Th2, and Th17 responses, as well as the generation of LLO-neutralizing antibodies. Further, we identified T cells as being required for immunization-induced reductions in bacterial burden, whereas B cells were dispensable in our model of non-pregnant young mice. Overall, this work establishes that LLO T is a promising vaccine antigen for the induction of protective immunity against L. monocytogenes by subunit vaccines containing Th1-driving adjuvants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in occasional skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen -/- ). Notably, LmCen -/- is a genetically engineered centrin gene knock-out mutant strain that is antibiotic resistant marker free and does not have detectable off-target mutations. Mice immunized with LmCen -/- have no visible lesions following challenge with L. major-infected sand flies, while non-immunized animals develop large and progressive lesions with a 2-log fold higher parasite burden. LmCen -/- immunization results in protection and an immune response comparable to leishmanization. LmCen -/- is safe since it is unable to cause disease in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.

The present study aimed on the site specific delivery and enhanced in-vivo efficacy of antimonial drugs against the visceral leishmaniasis via macrophage targeted mannose anchored thiomer based nanoparticles. Mannose anchored thiolated nanoformulation [M-(CS-g-PEI)-TGA] was developed and evaluated in terms particle size, zeta-potential and entrapment efficacy. The TEM and EDX analysis was carried out to evaluate the morphology and successful entrapment of antimonial drug. Mucodhesion, permeation enhancement, oral pharmacokinetics, and in-vivo anti-leishmanial activity were carried out. The M-(CS-g-PEI)-TGA were found to be spherical having particle size of 287 ± 20 nm. Ex-vivo permeation indicated a 7.39-fold enhanced permeation of Meglumine Antimoniate with M-(CS-g-PEI)-TGA across Caco-2 cells compared to the Glucantime. Evaluation of in-vitro reduction in the parasitic burden via flow cytometric analysis indicated a 5.7-fold lower IC 50 for M-(CS-g-PEI)-TGA compared to Glucantime. A 6.1-fold improvement in the oral bioavailability and 5.2-fold reduced parasitic burden in the L. donovani infected BALB/c mice model was observed with M-(CS-g-PEI)-TGA compared to Glucantime. The results encouraged the concept of M-(CS-g-PEI)-TGA nanoformulations as a promising strategy for oral therapy against visceral leishmaniasis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)