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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.

No licensed vaccine exists against visceral leishmaniasis (VL), a disease caused by the Leishmania donovani parasite. We have previously reported both macrophages and dendritic cells play important role in the protection induced by a live attenuated centrin gene–deleted L. donovani (LdCen−/−) parasite vaccine. The role of neutrophils in orchestrating the initial innate response to pathogens is widely recognized. To investigate the early interaction of LdCen−/− with neutrophils, we immunized mice intradermally in the ear pinna with LdCen−/−. Compared with LdWT infection, LdCen−/− parasites induced higher recruitment of neutrophils to the ear dermis and ear draining lymph nodes (dLN) as early as 6–18 h after immunization, which were predominantly proinflammatory in nature. Neutrophils from ear dLN of LdCen−/−-immunized mice exhibited heightened expression of costimulatory molecules and attenuated expression of coinhibitory molecules necessary for higher T cell activation. Further phenotypic characterization revealed heterogeneous neutrophil populations containing Nα and Nβ subtypes in the ear dLN. Of the two, the parasitized Nα subset from LdCen−/−-immunized mice exhibited much stronger Ag-specific CD4+ T cell proliferation ex vivo. Adoptive transfer of neutrophils bearing LdCen−/− parasites induced an increased Th1 response in naive mice. Importantly, neutrophil depletion significantly abrogated Ag-specific CD4+ T cell proliferation in LdCen−/−-immunized mice and impaired protection against virulent challenge. Conversely, replenishing of neutrophils significantly restored the LdCen−/− -induced host-protective response. These results suggest that neutrophils are indispensable for protective immunity induced by LdCen−/− parasite vaccine.

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 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 generatedcentringene-deleted new worldL. mexicana(LmexCen−/−) parasites using CRISPR/Cas9 and showed that they protect mice against a homologousL. mexicanainfection that causes cutaneous disease. In this study, we tested whetherLmexCen−/−parasites can also protect against visceral leishmaniasis caused byL. donovaniin a hamster model. We showed that immunization withLmexCen−/−parasites is safe and does not cause lesions. Furthermore, such immunization conferred protection against visceral leishmaniasis caused by a needle-initiatedL. donovanichallenge, 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 mediatedL. donovanichallenge. Importantly, immunization withLmexCen−/−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 againstL. donovaniinfection. Taken together, our study demonstrates that immunization withLmexCen−/−parasites is safe and efficacious against the Old World visceral leishmaniasis.

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.

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.

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 betweenLeishmania, the host innate immune cells, and the adaptive immunity determine the severity of pathogenesis and disease development.Leishmaniaparasites 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 toLeishmaniainfection. 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 inLeishmaniapathogenesis. 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 efficaciousLeishmaniavaccines.

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.

Significance We report that suppression of the serine protease elastase reshapes innate responses induced by injected vaccines containing alum adjuvant. This reprogramming improves the induction of protective antibodies in the bloodstream and stimulates innate signals, which support the development of antibody responses in mucosal tissues. Our findings identify elastase as the innate regulator that blunts the adjuvant activity of alum. They also demonstrate that vaccination via mucosal routes is not an absolute requirement for antibody responses in mucosal tissues and secretions. Supplementation of an alum-based vaccine containing SARS-CoV-2 spike protein subunit 1 as antigen increased anti–SARS-CoV-2 immunity in the blood and mucosal secretions in mice. Thus, this strategy could help in the development of future protein-based vaccines against SARS-CoV-2., 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.

Parasitic infections pose a wide and varying threat globally, impacting over 25% of the global population with many more at risk of infection. These infections are comprised of, but not limited to, toxoplasmosis, malaria, leishmaniasis and any one of a wide variety of helminthic infections. While a great deal is understood about the adaptive immune response to each of these parasites, there remains a need to further elucidate the early innate immune response. Interleukin-33 is being revealed as one of the earliest players in the cytokine milieu responding to parasitic invasion, and as such has been given the name “alarmin.” A nuclear cytokine, interleukin-33 is housed primarily within epithelial and fibroblastic tissues and is released upon cellular damage or death. Evidence has shown that interleukin-33 seems to play a crucial role in priming the immune system toward a strong T helper type 2 immune response, necessary in the clearance of some parasites, while disease exacerbating in the context of others. With the possibility of being a double-edged sword, a great deal remains to be seen in how interleukin-33 and its receptor ST2 are involved in the immune response different parasites elicit, and how those parasites may manipulate or evade this host mechanism. In this review article we compile the current cutting-edge research into the interleukin-33 response to toxoplasmosis, malaria, leishmania, and helminthic infection. Furthermore, we provide insight into directions interleukin-33 research may take in the future, potential immunotherapeutic applications of interleukin-33 modulation and how a better clarity of early innate immune system responses involving interleukin-33/ST2 signaling may be applied in development of much needed treatment options against parasitic invaders.

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.

Parasitic infections are responsible for significant morbidity and mortality throughout the world. Management strategies rely primarily on antiparasitic drugs that have side effects and risk of drug resistance. Therefore, novel strategies are needed for treatment of parasitic infections. Host-directed therapy (HDT) is a viable alternative, which targets host pathways responsible for parasite invasion/survival/pathogenicity. Recent innovative combinations of genomics, proteomics and computational biology approaches have led to discovery of several host pathways that could be promising targets for HDT for treating parasitic infections. Herein, we review major advances in HDT for parasitic disease with regard to core regulatory pathways and their interactions.

Leishmaniasis includes a spectrum of diseases ranging from debilitating cutaneous to fatal visceral infections. This disease is caused by the parasitic protozoa of the genus Leishmania that is transmitted by infected sandflies. Over 1 billion people are at risk of leishmaniasis with an annual incidence of over 2 million cases throughout tropical and subtropical regions in close to 100 countries. Leishmaniasis is the only human parasitic disease where vaccination has been successful through a procedure known as leishmanization that has been widely used for decades in the Middle East. Leishmanization involved intradermal inoculation of live Leishmania major parasites resulting in a skin lesion that following natural healing provided protective immunity to re-infection. Leishmanization is however no longer practiced due to safety and ethical concerns that the lesions at the site of inoculation that can last for months in some people. New genome editing technologies involving CRISPR has now made it possible to engineer safer attenuated strains of Leishmania, which induce protective immunity making way for a second generation leishmanization that can enter into human trials. A major consideration will be how the test the efficacy of a vaccine in the midst of the visceral leishmaniasis elimination program. One solution will be to use the leishmanin skin test (LST) that was also used for decades to determine exposure and immunity to Leishmania. The LST involves injection of antigen from Leishmania in the skin dermis resulting in a delayed type hypersensitivity (DTH) immune reaction associated with a Th1 immune response and protection against visceral leishmaniasis. Reintroduction of novel approaches for leishmanization and the leishmanin skin test can play a major role in eliminating leishmaniasis.

The mechanisms that promote local inflammatory injury during lupus nephritis (LN) flare are largely unknown. Understanding the key immune cells that drive intrarenal inflammation will advance our knowledge of disease pathogenesis and inform the development of new therapeutics for LN management. In this study, we analyzed kidney biopsies from patients with proliferative LN and identified a novel inflammatory dendritic cell (infDC) population that is highly expressed in the LN kidney, but minimally present in healthy human kidneys. During an agnostic evaluation of immune transcript expression in the kidneys of patients with proliferative LN, the most abundantly overexpressed transcript from isolated glomeruli wasFCER1G, which encodes the Fc receptor gamma chain (FcRγ). To identify the cell types expressing FcRγ that infiltrate the kidney in LN, studies were done on kidney biopsies from patients with active LN using confocal immunofluorescence (IF) microscopy. This showed that FcRγ is abundantly present in the periglomerular (PG) region of the kidney and to a lesser extent in the tubulointerstitium (TI). Further investigation of the surface markers of these cells showed that they were FcRγ+, MHC II+, CD11c+, CD163+, CD5−, DC-SIGN+, CD64+, CD14+, CD16+, SIRPα+, CD206−, CD68−, CD123−, CD3−, and CD11b−, suggesting the cells were infDCs. Quantification of the infDCs showed an average 10-fold higher level of infDCs in the LN kidney compared to the healthy kidneys. Importantly, IF identified CD3+T cells to be adjacent to these infDCs in the PG space of the LN kidney, whereas both cell types are minimally present in the healthy kidney. Thus, we have identified a previously undescribed DC in lupus kidneys that may interact with intrarenal T cells and play a role in the pathogenesis of kidney injury during LN flare.

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.

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.

The vascular endothelium is a vital component in maintaining the structure and function of blood vessels. The endothelial cells (ECs) mediate vital regulatory functions such as the proliferation of cells, permeability of various tissue membranes, and exchange of gases, thrombolysis, blood flow, and homeostasis. The vascular endothelium also regulates inflammation and immune cell trafficking, and ECs serve as a replicative niche for many bacterial, viral, and protozoan infectious diseases. Endothelial dysfunction can lead to vasodilation and pro-inflammation, which are the hallmarks of many severe diseases. Exosomes are nanoscale membrane-bound vesicles that emerge from cells and serve as important extracellular components, which facilitate communication between cells and maintain homeostasis during normal and pathophysiological states. Exosomes are also involved in gene transfer, inflammation and antigen presentation, and mediation of the immune response during pathogenic states. Protozoa are a diverse group of unicellular organisms that cause many infectious diseases in humans. In this regard, it is becoming increasingly evident that many protozoan parasites (such as Plasmodium, Trypanosoma, Leishmania, and Toxoplasma) utilize exosomes for the transfer of their virulence factors and effector molecules into the host cells, which manipulate the host gene expression, immune responses, and other biological activities to establish and modulate infection. In this review, we discuss the role of the vascular endothelium and exosomes in and their contribution to pathogenesis in malaria, African sleeping sickness, Chagas disease, and leishmaniasis and toxoplasmosis with an emphasis on their actions on the innate and adaptive immune mechanisms of resistance.

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.

Chagas disease, caused by the intracellular protozoan parasite Trypanosoma cruzi, is a public health problem affecting 6 to 8 million people, mainly in Latin America. The role of microRNAs in the pathogenesis of Chagas disease has not been well described. Here, we investigate the role of microRNA-155 (miR-155), a proinflammatory host innate immune regulator responsible for T helper type 1 and type 17 (Th1 and Th17) development and macrophage responses during T. cruzi infection. For this, we compared the survival and parasite growth and distribution in miR-155(−/−) and wild-type (WT) C57BL/6 mice. The lack of miR-155 caused robust parasite infection and diminished survival of infected mice, while WT mice were resistant to infection. Immunological analysis of infected mice indicated that, in the absence of miR-155, there was decreased interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) production. In addition, we found that there was a significant reduction of CD8-positive (CD8(+)) T cells, natural killer (NK) cells, and NK-T cells and increased accumulation of neutrophils and inflammatory monocytes in miR-155(−/−) mice. Collectively, these data indicate that miR-155 is an important immune regulatory molecule critical for the control of T. cruzi infection.

Head and neck squamous cell carcinoma (HNSCC) is a prevalent form of cancer with 5-years survival rates around 57%, and metastasis is a leading cause of mortality. Host-derived immunological factors that affect HNSCC tumor development and metastasis are not completely understood. We investigated the role of host-derived signal transducer and activator of transcription 4 (STAT4) during experimental HNSCC using an aggressive and metastatic HNSCC cell line, LY2, which was orthotopically injected into the buccal sulcus of wild type (WT) and STAT4 deficient (Stat4 -/-) BALB/c mice. Necropsies performed at terminal sacrifice revealed that Stat4 -/- mice displayed comparable primary tumor growth to the WT mice. However, the rate and extent of lymph node and lung metastasis among Stat4 -/- mice was significantly higher. Downstream analyses performed on primary tumors, draining lymph nodes, spleens and bone marrow revealed significant upregulation of lymphocytic immunosuppressive biomarkers as well as an accumulation of granulocytic MDSC subpopulations in draining lymph nodes of metastatic Stat4 -/- mice. Further, we observed a significant decrease in TH1, TH17, and cytotoxic activity in tumor bearing Stat4 -/- compared to WT mice. Our results demonstrate that STAT4 mediates resistance to HNSCC metastasis, and activation of STAT4 could potentially mitigate lymphatic metastasis in HNSCC patients.

Ly6Chi inflammatory monocytes (iMO) are critical for host defense against toxoplasmosis and malaria but their role in leishmaniasis is unclear. In this study, we report a detrimental role of Ly6Chi iMOs in visceral leishmaniasis (VL) caused by Leishmania donovani. We demonstrate that Ly6Chi iMOs are continuously recruited into the spleen and liver during L. donovani infection and they are preferential targets for the parasite. Using microarray-based gene expression profiling, we show that Ly6Chi iMOs isolated from the infected liver and spleen have distinct phenotypic and activation profiles. Furthermore, we demonstrate that blocking the recruitment of Ly6Chi iMOs into the liver and spleen during L. donovani infection using a CCR2 antagonist reduces the frequency of the pathogenic IFN-γ/IL10 dual producer CD4+ T cells in the spleen and leads to a significant reduction in parasite loads in the liver and spleen. Using STAT1−/− mice we show that STAT1 is critical for mediating the recruitment of Ly6Chi iMOs into organs during L. donovani infection, and adaptive transfer of wild type Ly6Chi iMOs into STAT1−/− recipients renders them susceptible to disease. Our findings reveal an unexpected pathogenic role for Ly6Chi iMOs in promoting parasite survival in VL and open the possibility of targeting this population for host-directed therapy during VL.

Differentiating acute pyelonephritis (APN) from acute rejection (AR) in renal allograft biopsies can sometimes be difficult because of overlapping clinical and histologic features, lack of positive urine cultures, and variable response to antibiotics. We wanted to study differential gene expression between AR and APN using biopsy tissue. Thirty-three biopsies were analyzed using NanoString multiplex platform and PCR (6 transplant baseline biopsies, 8 AR, 15 APN [8 culture positive, 7 culture negative], and 4 native pyelonephritis [NP]). Additional 22 biopsies were tested by PCR to validate the results. CXCL9, CXCL10, CXCL11, and IDO1 were the top differentially expressed genes, upregulated in AR. Lactoferrin (LTF) and CXCL1 were higher in APN and NP. No statistically significant difference in transcript levels was seen between culture-positive and culture-negative APN biopsies. Comparing the overall mRNA signature using Ingenuity pathway analysis, interferon-gamma emerged as the dominant upstream regulator in AR and allograft APN, but not in NP (which clustered separately). Our study suggests that chemokine pathways in graft APN may differ from NP and in fact resemble AR, due to a component of alloreactivity, resulting in variable response to antibiotic treatment. Therefore, cautious addition of steroids might help in resistant cases of graft APN.

Background and objectives Staphylococcus infection–associated GN (SAGN) is a well recognized disease entity, particularly because of the frequent IgA-dominant glomerular immunoglobulin staining on kidney biopsy. Biopsy features can resemble two other disease entities – primary IgA nephropathy and Henoch-Schonlein purpura nephritis – posing a diagnostic pitfall. This is clinically relevant because of the crucial difference in the therapeutic approach. The diagnosis of SAGN is further complicated by the variability in the degree of glomerular IgA (and C3) staining, the extent of electron dense immune-type deposits, and positive ANCA serology in some patients. Design, setting, participants, & measurements We performed a thorough histopathologic review of our single-center cohort of 78 culture-proven SAGN biopsies to assess the spectrum of IgA staining, prevalence of ANCA serology, prevalence of subepithelial “humps,” and other histologic features to distinguish from primary IgA nephropathy. Results Among the 78 SAGN biopsies, IgA staining was trace in 25%, mild in 19%, moderate in 44%, and strong in 12% of the cases. C3 was frequently moderate-to-strong but was trace in 14% of the biopsies. Concomitantly trace IgA, IgG, and C3 (pauci-immune pattern) was seen in 13%. Crescents were present in 35% of the SAGN biopsies. Out of 41 patients tested for ANCA, nine (22%) were positive, including patients with endocarditis and other infections. Subepithelial humps were identified in only 31% of the SAGN biopsies. Conclusions SAGN biopsies show marked variability in IgA immunofluorescence staining and low frequency of subepithelial humps compared with poststreptococcal GN. Occasional ANCA positivity is present in cases of SAGN, even in infections other than endocarditis. Therefore, biopsy diagnosis can be difficult particularly when clinical symptoms of infection are subtle. Both the pathologist and the nephrologist should be aware of these diagnostic pitfalls.

The outcome of visceral leishmaniasis, caused by parasite Leishmania donovani, depends on the recruitment of leishmanicidal Th1 cells. Chemokine receptor CXCR3, preferentially expressed by Th1 cells, is critical for migration of these T cells during infection. During chronic VL, there is a decrease in the presence of CXCR3-expressing CD4+ T cells in the spleen, which is associated with high parasitic burden in this organ. We therefore examined whether T cell-specific expression of CXCR3 in mice (CXCR3Tg) would promote resistance to VL. L. donovani infected CXCR3Tg mice showed increased accumulation of T cells in the spleens compared to WT littermates (CXCR3+/+). However, CXCR3+ T cells from CXCR3Tg mice showed low CD69 expression and these mice developed fewer granulomas. Additionally, both groups of mice showed similar cytokine profiles and parasitic burdens during the course of infection. In summary, although T cell-specific expression of CXCR3 promoted the accumulation of CXCR3-expressing T cells during L. donovani infection, this did not enhance resistance to VL.

Cutaneous leishmaniasis (CL) is the most common form of leishmaniasis and is caused by several species of Leishmania parasite. Clinical presentation of CL varies from a self-healing infection to a chronic form of the disease determined by the virulence of infecting Leishmania species and host immune responses to the parasite. Mouse models of CL show contradictory roles of lymphocytes in pathogenesis, while acquired immune responses are responsible for host protection from diseases. To reconcile the inconclusive roles of acquired immune responses in pathogenesis, we infected mice from various genetic backgrounds with two pathogenic strains of Leishmania major, Friedlin or 5ASKH, and assessed the outcome of the infections. Our findings showed that the genetic backgrounds of L. major determine the impact of lymphocytes for pathogenesis. In the absence of lymphocytes, L. major Friedlin induced the lowest inflammatory reaction and pathology at the site of infection, while 5ASKH infection induced a strong inflammatory reaction and severe pathology. Lymphocytes ameliorated 5ASKH mediated pathology, while it exacerbated pathology during Friedlin infection. Excess inflammatory reactions, like the recruitment of macrophages, neutrophils, eosinophils and production of pro-inflammatory cytokines, together with uncontrolled parasite growth in the absence of lymphocytes during 5ASKH infection may induce severe pathology development. Taken together our study provides insight into the impact of differences in the genetic background of Leishmania on CL pathogenesis., PLoS neglected tropical diseases, 13(11), e0007865; 2019

CD4(+) T helper 1 (Th1) cells producing interferon gamma (IFN-γ) are critical for the resolution of visceral leishmaniasis (VL). MicroRNA 155 (miR155) promotes CD4(+) Th1 responses and IFN-γ production by targeting suppressor of cytokine signaling-1 (SOCS1) and Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP-1) and therefore could play a role in the resolution of VL. To determine the role of miR155 in VL, we monitored the course of Leishmania donovani infection in miR155 knockout (miR155KO) and wild-type (WT) C57BL/6 mice. miR155KO mice displayed significantly higher liver and spleen parasite loads than WT controls and showed impaired hepatic granuloma formation. However, parasite growth eventually declined in miR155KO mice, suggesting the induction of a compensatory miR155-independent antileishmanial pathway. Leishmania antigen-stimulated splenocytes from miR155KO mice produced significantly lower levels of Th1-associated IFN-γ than controls. Interestingly, at later time points, levels of Th2-associated interleukin-4 (IL-4) and IL-10 were also lower in miR155KO splenocyte supernatants than in WT mice. On the other hand, miR155KO mice displayed significantly higher levels of IFN-γ, iNOS, and TNF-α gene transcripts in their livers than WT mice, indicating that distinct organ-specific antiparasitic mechanisms were involved in control of L. donovani infection in miR155KO mice. Throughout the course of infection, organs of miR155KO mice showed significantly more PDL1-expressing Ly6C(hi) inflammatory monocytes than WT mice. Conversely, blockade of Ly6C(hi) inflammatory monocyte recruitment in miR155KO mice significantly reduced parasitic loads, indicating that these cells contributed to disease susceptibility. In conclusion, we found that miR155 contributes to the control of L. donovani but is not essential for infection resolution.

Made available in DSpace on 2019-10-04T11:57:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-04-01 Nova Southeastern University President Faculty Research Development Grant National Institutes of Health Krakow Award for Excellence in Research from the Harvard School of Dental Medicine Locally produced osteoclastogenic factor RANKL plays a critical role in the development of bone resorption in periradicular periodontitis. However, because RANKL is also required for healthy bone remodeling, it is plausible that a costimulatory molecule that upregulates RANKL production in inflammatory periradicular periodontitis may be involved in the pathogenic bone loss processes. We hypothesized that macrophage migration inhibitory factor (MIF) would play a role in upregulating the RANKL-mediated osteoclastogenesis in the periradicular lesion. In response to pulp exposure, the bone loss and level of MIF mRNA increased in the periradicular periodontitis, which peaked at 14 d, in conjunction with the upregulated expressions of mRNAs for RANKL, proinflammatory cytokines (TNF-alpha, IL-6, and IL-1 beta), chemokines (MCP-1 and SDF-1), and MIF's cognate receptors CXCR4 and CD74. Furthermore, expressions of those mRNAs were found significantly higher in wild-type mice compared with that of MIF-/- mice. In contrast, bacterial LPS elicited the production of MIF from ligament fibroblasts in vitro, which, in turn, enhanced their productions of RANKL and TNF-alpha. rMIF significantly upregulated the number of TRAP(+) osteoclasts in vitro. Finally, periapical bone loss induced in wild-type mice were significantly diminished in MIF-/- mice. Altogether, the current study demonstrated that MIF appeared to function as a key costimulatory molecule to upregulate RANKL-mediated osteoclastogenesis, leading to the pathogenically augmented bone resorption in periradicular lesions. These data also suggest that the approach to neutralize MIF activity may lead to the development of a therapeutic regimen for the prevention of pathogenic bone loss in periradicular periodontitis. Harvard Univ, Sch Dent Med, 188 Longwood Ave, Boston, MA 02115 USA King Abdulaziz Univ, Fac Dent, Jeddah 21589, Saudi Arabia Forsyth Inst, Cambridge, MA 02142 USA Nova Southeastern Univ, Coll Dent Med, 320X S Univ Dr, Ft Lauderdale, FL 33324 USA Univ Michigan, Sch Dent, Ann Arbor, MI 48109 USA Sao Paulo State Univ, Sch Dent, BR-15015050 Aracatuba, SP, Brazil Ohio State Univ, Dept Microbiol, 484 W 12th Ave, Columbus, OH 43210 USA Tohoku Univ, Grad Sch Dent, Sendai, Miyagi 9808575, Japan Sao Paulo State Univ, Sch Dent, BR-15015050 Aracatuba, SP, Brazil National Institutes of Health: AG-053615 National Institutes of Health: DE-18499 National Institutes of Health: DE -19917 National Institutes of Health: DE -007327 National Institutes of Health: DE -027851

Helminth parasites modulate immune responses in their host to prevent their elimination and to establish chronic infections. Our previous studies indicate that Taenia crassiceps-excreted/secreted antigens (TcES) downregulate inflammatory responses in rodent models of autoimmune diseases, by promoting the generation of alternatively activated-like macrophages (M2) in vivo. However, the molecular mechanisms triggered by TcES that modulate macrophage polarization and inflammatory response remain unclear. Here, we found that, while TcES reduced the production of inflammatory cytokines (IL-6, IL-12, and TNFα), they increased the release of IL-10 in LPS-induced bone marrow-derived macrophages (BMDM). However, TcES alone or in combination with LPS or IL-4 failed to increase the production of the canonical M1 or M2 markers in BMDM. To further define the anti-inflammatory effect of TcES in the response of LPS-stimulated macrophages, we performed transcriptomic array analyses of mRNA and microRNA to evaluate their levels. Although the addition of TcES to LPS-stimulated BMDM induced modest changes in the inflammatory mRNA profile, it induced the production of mRNAs associated with the activation of different receptors, phagocytosis, and M2-like phenotype. Moreover, we found that TcES induced upregulation of specific microRNAs, including miR-125a-5p, miR-762, and miR-484, which are predicted to target canonical inflammatory molecules and pathways in LPS-induced BMDM. These results suggest that TcES can modulate proinflammatory responses in macrophages by inducing regulatory posttranscriptional mechanisms and hence reduce detrimental outcomes in hosts running with inflammatory diseases.

Oral cancer kills about 1 person every hour each day in the United States and is the sixth most prevalent cancer worldwide. The pro-inflammatory cytokine 'macrophage migration inhibitory factor' (MIF) has been shown to be expressed in oral cancer patients, yet its precise role in oral carcinogenesis is not clear. In this study, we examined the impact of global Mif deletion on the cellular and molecular process occurring during oral carcinogenesis using a well-established mouse model of oral cancer with the carcinogen 4-nitroquinoline-1-oxide (4NQO). C57BL/6 Wild-type (WT) and Mif knock-out mice were administered with 4NQO in drinking water for 16 weeks, then regular drinking water for 8 weeks. Mif knock-out mice displayed fewer oral tumor incidence and multiplicity, accompanied by a significant reduction in the expression of pro-inflammatory cytokines Il-1β, Tnf-α, chemokines Cxcl1, Cxcl6 and Ccl3 and other molecular biomarkers of oral carcinogenesis Mmp1 and Ptgs2. Further, systemic accumulation of myeloid-derived tumor promoting immune cells was inhibited in Mif knock-out mice. Our results demonstrate that genetic Mif deletion reduces the incidence and severity of oral carcinogenesis, by inhibiting the expression of chronic pro-inflammatory immune mediators. Thus, targeting MIF is a promising strategy for the prevention or therapy of oral cancer.

Leishmaniasis is a disease caused by the Leishmania parasites. The injection of the parasites into the host occurs when a sand fly, which is the vector, bites the skin of the host. The parasites, which are obligate, take advantage of the immune system response and invade both the classically activated macrophages (M1) and the alternatively activated macrophages (M2). In this paper, we develop a mathematical model to explain the evolution of the disease. Simulations of the model show that, M2 macrophages steadily increase and M1 macrophages steadily decrease, while M1+M2 reach a steady state which is approximately the same as at healthy state of the host. Furthermore, the ratio of Leishmania parasites to macrophages depends homogeneously on their ratio at the time of the initial infection, in agreement with in vitro experimental data. The model is used to simulate treatment by existing or potential new drugs, and to compare the efficacy of different schedules of drug delivery.

Immune cells that infiltrate a tumor may be a prognostic factor for patients who have had surgically resected hepatocellular carcinoma (HCC). The density of intratumoral total (CD3+) and cytotoxic (CD8+) T lymphocytes was measured in the tumor interior and in the invasive margin of 65 stage I to IV HCC tissue specimens from a single cohort. Immune cell density in the interior and margin was converted to a binary score (0, low; 1, high), which was correlated with tumor recurrence and relapse-free survival (RFS). In addition, the expression of programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) was correlated with the density of CD3+ and CD8+ cells and clinical outcome. High densities of both CD3+ and CD8+ T cells in both the interior and margin, along with corresponding Immunoscores, were significantly associated with a low rate of recurrence (P = 0.007) and a prolonged RFS (P = 0.002). In multivariate logistic regression models adjusted for vascular invasion and cellular differentiation, both CD3+ and CD8+ cell densities predicted recurrence, with odds ratios of 5.8 [95% confidence interval (CI), 1.6–21.8] for CD3+ and 3.9 (95% CI, 1.1–14.1) for CD8+. Positive PD-L1 staining was correlated with high CD3 and CD8 density (P = 0.024 and 0.005, respectively) and predicted a lower rate of recurrence (P = 0.034), as well as prolonged RFS (P = 0.029). Immunoscore and PD-L1 expression, therefore, are useful prognostic markers in patients with HCC who have undergone primary tumor resection. Cancer Immunol Res; 4(5); 419–30. ©2016 AACR.

Leishmania donovani is an intracellular parasite that infects professional phagocytes and causes visceral leishmaniasis (VL). The immune response during VL has been extensively studied in the context of T-helper (Th)1 and Th2 responses. Immunity against this parasite is dependent on IFN-γ production and subsequent macrophage activation, and the Th2 response promotes granuloma formation. The cytokine IL-17A is associated with neutrophilic inflammation. Depletion of neutrophils during experimental VL results in enhanced parasitic loads. Furthermore, although patients resistant to VL showed enhanced levels of IL-17A in circulation, little is known about the role of IL-17A during VL infection. Here, we used IL-17A-deficient mice and IL-17A reporter mice to address the role of IL-17A during VL. IL-17A−/− mice were highly resistant to VL infection, showing decreased parasites in the liver and spleen. This unexpected phenotype was associated with enhanced IFN-γ production by T cells and decreased accumulation of neutrophils and monocytes, resulting in reduced number of granulomas. We also found γδ T and Th17 cells as the main IL-17A+ cells during VL infection. Our data reveal an unexpected role of IL-17A rendering susceptibility against L. donovani by regulating the IFN-γ response and promoting detrimental inflammation.—Terrazas, C., Varikuti, S., Kimble, J., Moretti, E., Boyaka, P. N., Satoskar, A. R. IL-17A promotes susceptibility during experimental visceral leishmaniasis caused by Leishmania donovani.

Intestinal epithelial cells (IECs) are known to regulate allergic sensitization. We addressed the role of the intrinsic IKKβ signaling in IECs in the effector phase of allergy following oral allergen challenge and its impact on the severity of responses is poorly. Upon orally sensitization by co-administration of ovalbumin with cholera toxin as adjuvant, wild-type and mice lacking IKKβ in IECs (IKKβΔIEC mice) developed similar levels of serum IgE and allergen-specific secretory IgA in the gut. However, subsequent allergen challenges in the gut promoted allergic lower responses in KKβΔIEC mice. Analysis of cytokines and chemokines in serum and gut tissues after oral allergen challenge revealed impaired eotaxin responses in IKKβΔIEC mice, which correlated with lower frequencies of eosinophils in the gut lamina propria. We also determined that IECs were a major source of eotaxin and that impaired eotaxin production was due to the lack of IKKβ signaling in IECs. Oral administration of CCL11 to IKKβΔIEC mice during oral allergen challenge enhanced allergic responses to levels in wild-type mice, confirming the role of IEC-derived eotaxin as regulator of the effector phase of allergy following allergen challenge. Our results identified targeting IEC-derived eotaxin as potential strategy to limit the severity of allergic responses to food antigens.

It is well established that dendritic cells and macrophages play a role in antigen presentation to B and T cells and in shaping B and T cell responses via cytokines they produce. We have previously reported that depletion of neutrophils improves the production of mucosal IgA after sublingual immunization with Bacillus anthracis edema toxin as adjuvant. These past studies also demonstrated that an inverse correlation exists between the number of neutrophils and production of IgA by B cells. Using specific inhibitors of elastase, we addressed whether the elastase activity of neutrophil could be the factor that interferes with production of IgA and possibly other immunoglobulin isotypes. We found that murine splenocytes and mesenteric lymph node cells cultured for 5 days in the presence of neutrophil elastase inhibitors secreted higher levels of IgG and IgA than cells cultured in the absence of inhibitor. The effect of the inhibitors was dose-dependent and was consistent with increased frequency of CD138(+) cells expressing IgG or IgA. Finally, neutrophil elastase inhibitors increased transcription of mRNA for AID, IL-10, BAFF and APRIL, factors involved in B cell differentiation. These findings identify inhibitors of elastase as potential adjuvants for increasing production of antibodies.

CXCR3, an X-linked gene, is subject to X chromosome inactivation (XCI), but it is unclear whether CXCR3 escapes XCI in immune cells. We determined whether CXCR3 escapes XCI in vivo, evaluated the contribution of allelic CXCR3 expression to the phenotypic properties of T cells during experimental infection with Leishmania, and examined the potential implications to sex differences in immune responses. We used a bicistronic CXCR3 dual-reporter mouse, with each CXCR3 allele linked to a green or red fluorescent reporter without affecting endogenous CXCR3 expression. Our results show that CXCR3 escapes XCI, biallelic CXCR3-expressing T cells produce more CXCR3 protein than monoallelic CXCR3-expressing cells, and biallelic CXCR3-expressing T cells produce more IFN-γ, IL-2, and CD69 compared with T cells that express CXCR3 from one allele during Leishmania mexicana infection. These results demonstrate that XCI escape by CXCR3 potentially contributes to the sex-associated bias observed during infection.

Background Small glomerular IgA deposits have been reported in patients with liver cirrhosis, mainly as an incidental finding in autopsy studies. We recently encountered nine cirrhotic patients who presented with acute proliferative glomerulonephritis with unusually large, exuberant glomerular immune complex deposits, in the absence of systemic lupus erythematosus (SLE) or monoclonal gammopathy-related kidney disease. Deposits were typically IgA dominant/codominant. Our aim was to further elucidate the etiology, diagnostic pitfalls, and clinical outcomes. Methods We present clinical features and kidney biopsy findings of nine cirrhotic patients with an unusual acute immune complex glomerulonephritis. We also identified native kidney biopsies from all patients with liver cirrhosis at our institution over a 13-year period (January 2004 to December 2016) to evaluate presence of glomerular IgA deposits in them (n = 118). Results Six of nine cirrhotic patients with the large immune deposits had a recent/concurrent acute bacterial infection, prompting a diagnosis of infection-associated glomerulonephritis and treatment with antibiotics. In the remaining three patients, no infection was identified and corticosteroids were initiated. Three of nine patients recovered kidney function (one recovered kidney function after liver transplant); three patients developed chronic kidney disease but remained off dialysis; two patients became dialysis-dependent and one patient developed sepsis and expired shortly after biopsy. Within the total cohort of 118 patients with cirrhosis, 67 others also showed IgA deposits, albeit small; and 42 patients had no IgA deposits. Conclusions These cases provide support to the theory that liver dysfunction may compromise clearance of circulating immune complexes, enabling deposition in the kidney. At least in a subset of cirrhotic patients, a superimposed bacterial infection may serve as a “second-hit” and lead to acute glomerulonephritis with exuberant immune complex deposits. Therefore, a trial of antibiotics is recommended and caution is advised before immunosuppressive treatment is offered. Unfortunately, most of these patients have advanced liver failure; therefore both diagnosis and management remain a challenge.

Visceral leishmaniasis (VL), caused by the protozoan parasite Leishmania donovani, is a global health problem affecting millions of people worldwide. Treatment of VL largely depends on therapeutic drugs such as pentavalent antimonials, amphotericin B, and others, which have major drawbacks due to drug resistance, toxicity, and high cost. In this study, for the first time, we have successfully demonstrated the synthesis and antileishmanial activity of the novel sterol pentalinonsterol (PEN), which occurs naturally in the root of a Mexican medicinal plant, Pentalinon andrieuxii. In the experimental BALB/c mouse model of VL induced by infection with L. donovani, intravenous treatment with liposome-encapsulated PEN (2.5 mg/kg) led to a significant reduction in parasite burden in the liver and spleen. Furthermore, infected mice treated with liposomal PEN showed a strong host-protective TH1 immune response characterized by IFN-γ production and formation of matured hepatic granulomas. These results indicate that PEN could be developed as a novel drug against VL.

Host–pathogen interaction is an area of considerable interest. Intracellular parasites such as Leishmania reside inside phagocytes such as macrophages, dendritic cells and neutrophils. Macrophages can be activated by cytokines such as IFN-γ and Toll like receptor (TLR) agonists resulting in enhanced microbicidal activity. Leishmania parasites hijack the microbicidal function of macrophages, mainly by interfering with intracellular signaling initiated by IFN-γ and TLR ligands. Here we used transgenic Leishmania donovani parasites expressing the red fluorescent protein DsRed2 and imaging-flow cytometry technology to evaluate parasitic loads inside the macrophage in vitro. Further, this methodology enables us to visualize impairment in NFκB translocation to the nucleus in L. donovani infected macrophages. Additionally we show that uninfected bystander macrophages have a similar impairment in NFκB translocation as in L. donovani infected macrophages in response to the TLR4 agonist LPS. This evidence suggests a possible immunosuppressive role for infected macrophages in regulating the activation of uninfected bystander macrophages.

A spate of recent publications describes a newly recognized form of glomerulonephritis associated with active staphylococcal infection. The key kidney biopsy findings, glomerular immunoglobulin A (IgA) deposits dominant or codominant with IgG deposits, resemble those of IgA nephritis. Many authors describe this condition as "postinfectious" and have termed it "poststaphylococcal glomerulonephritis." However, viewed through the prism of poststreptococcal glomerulonephritis, the prefix "post" in poststaphylococcal glomerulonephritis is historically incorrect, illogical, and misleading with regard to choosing therapy. There are numerous reports describing the use of high-dose steroids to treat poststaphylococcal glomerulonephritis. The decision to use steroid therapy suggests that the treating physician believed that the dominant problem was a postinfectious glomerulonephritis, not the infection itself. Unfortunately, steroid therapy in staphylococcus-related glomerulonephritis can precipitate severe staphylococcal sepsis and even death and provides no observable benefits. Poststreptococcal glomerulonephritis is an authentic postinfectious glomerulonephritis; poststaphylococcal glomerulonephritis is not. Making this distinction is important from the perspective of history, pathogenesis, and clinical management.

The anti‐tumor role and mechanisms of Cannabidiol (CBD), a non‐psychotropic cannabinoid compound, are not well studied especially in triple‐negative breast cancer (TNBC). In the present study, we analyzed CBD's anti‐tumorigenic activity against highly aggressive breast cancer cell lines including TNBC subtype. We show here ‐for the first time‐that CBD significantly inhibits epidermal growth factor (EGF)‐induced proliferation and chemotaxis of breast cancer cells. Further studies revealed that CBD inhibits EGF‐induced activation of EGFR, ERK, AKT and NF‐kB signaling pathways as well as MMP2 and MMP9 secretion. In addition, we demonstrated that CBD inhibits tumor growth and metastasis in different mouse model systems. Analysis of molecular mechanisms revealed that CBD significantly inhibits the recruitment of tumor‐associated macrophages in primary tumor stroma and secondary lung metastases. Similarly, our in vitro studies showed a significant reduction in the number of migrated RAW 264.7 cells towards the conditioned medium of CBD‐treated cancer cells. The conditioned medium of CBD‐treated cancer cells also showed lower levels of GM‐CSF and CCL3 cytokines which are important for macrophage recruitment and activation. In summary, our study shows ‐for the first time‐that CBD inhibits breast cancer growth and metastasis through novel mechanisms by inhibiting EGF/EGFR signaling and modulating the tumor microenvironment. These results also indicate that CBD can be used as a novel therapeutic option to inhibit growth and metastasis of highly aggressive breast cancer subtypes including TNBC, which currently have limited therapeutic options and are associated with poor prognosis and low survival rates.

Cutaneous leishmaniasis, caused mainly by Leishmania major , an obligate intracellular parasite, is a disfiguring disease characterized by large skin lesions and is transmitted by a sand fly vector. We previously showed that the chemokine receptor CXCR3 plays a critical role in mediating resistance to cutaneous leishmaniasis caused by Leishmania major . Furthermore, T cells from L. major -susceptible BALB/c but not L. major -resistant C57BL/6 mice fail to efficiently upregulate CXCR3 upon activation. We therefore examined whether transgenic expression of CXCR3 on T cells would enhance resistance to L. major infection in susceptible BALB/c mice. We generated BALB/c and C57BL/6 transgenic mice, which constitutively overexpressed CXCR3 under a CD2 promoter, and then examined the outcomes with L. major infection. Contrary to our hypothesis, transgenic expression of CXCR3 (CXCR3 Tg ) on T cells of BALB/c mice resulted in increased lesion sizes and parasite burdens compared to wild-type (WT) littermates after L. major infection. Restimulated lymph node cells from L. major -infected BALB/c-CXCR3 Tg mice produced more interleukin-4 (IL-4) and IL-10 and less gamma interferon (IFN-γ). Cells in draining lymph nodes from BALB/c-CXCR3 Tg mice showed enhanced Th2 and reduced Th1 cell accumulation associated with increased neutrophils and inflammatory monocytes. However, monocytes displayed an immature phenotype which correlated with increased parasite burdens. Interestingly, transgenic expression of CXCR3 on T cells did not impact the outcome of L. major infection in C57BL/6 mice, which mounted a predominantly Th1 response and spontaneously resolved their infection similar to WT littermates. Our findings demonstrate that transgenic expression of CXCR3 on T cells increases susceptibility of BALB/c mice to L. major .

Introduction: Brodifacoum (BDF) is a superwarfarin that is used primarily as a rodenticide. There have been increasing numbers of reports of human cases of accidental or intentional BDF ingestion with high mortality rate. Its broad availability and high lethality suggest that BDF should be considered a potential chemical threat. Currently, there is no biomarker for early detection of BDF ingestion in humans; patients typically present with severe coagulopathy. Since we demonstrated earlier that warfarin can induce acute kidney injury with hematuria, we tested whether BDF would also lead to change in urinary biomarkers. Material and Methods: BDF was administered to Sprague Dawley rats via oral gavage. N-acetylcysteine (NAC) was given per os in drinking water 24 h prior to BDF. Urinalysis was performed at different times after BDF administration. Anticoagulation and serum creatinine levels were analyzed in the blood. Results: We observed that within a few hours the animals developed BDF-dose-dependent transient hemoglobinuria, which ceased within 24 h. This was accompanied by a transient decrease in hematocrit, gross hemolysis and an increase in free hemoglobin in the serum. At later times, animals developed true hematuria with red blood cells in the urine, which was associated with BDF anticoagulation. NAC prevented early hemoglobinuria, but not late hematuria associated with BDF. Conclusions: We propose that transient early hemoglobinuria (associated with oxidative stress) with consecutive late hematuria (associated with anticoagulation) are novel biomarkers of BDF poisoning, and they can be used in clinical setting or in mass casualty with BDF to identify poisoned patients.

Ox40 ligand (Ox40L)-Ox40 pathway has been shown to enhance Th2 responses and play a role in pathogenesis of cutaneous leishmaniasis (CL) caused by Leishmania major. Using Ox40l(-/-) BALB/c mice we analyzed the role of this pathway in determining the outcome to CL caused by L. mexicana and compared to L. major. Contrary to our expectations, Ox40l(-/-) mice were highly susceptible to both L. major (LV39) and L. mexicana (M379) and developed large non-healing lesions containing parasites comparable to Ox40l(+/+) BALB/c mice. Interestingly, upon in vitro stimulation with Leishmania antigen (LmAg), the lymph node cells from L. major infected Ox40l(-/-) mice produced significantly less IL-4 and IL-10 compared to Ox40l(+/+) mice. L. mexicana infected Ox40l(-/-) and Ox40l(+/+) mice did not show any difference in the production of IL-4 and IL-10. No difference was noted in the amount of Th1 cytokines IFN-ү and IL-12 produced by Ox40l(-/-) and Ox40l(+/+) mice infected with either parasite. These results indicate that the Ox40L-Ox40 pathway promotes Th2 bias only in L. major infection but not L. mexicana infection and this pathway is not critical for susceptibility to CL.