Proteomic research on new urinary biomarkers of renal disease in canine leishmaniosis: Survival and monitoring response to treatment.

The objective of our study was to search for survival biomarkers (SB) and treatment response monitoring biomarkers (TRMB) in the urinary proteome of dogs with renal disease secondary to canine leishmaniosis (CanL), using UHPLC-MS/MS. The proteomic data are available via ProteomeXchange with identifier PXD042578. Initially, a group of 12 dogs was evaluated and divided into survivors (SG; n = 6) and nonsurvivors (NSG; n = 6). A total of 972 proteins were obtained from the evaluated samples. Then, bioinformatic analysis reduced them to 6 proteins like potential SB increased in the NSG, specifically, Haemoglobin subunit Alpha 1, Complement Factor I, Complement C5, Fibrinogen beta chain (fragment), Peptidase S1 domain-containing protein, and Fibrinogen gamma chain. Afterwards, SG was used to search for TRMB, studying their urine at 0, 30, and 90 days, and 9 proteins that decreased after treatment were obtained: Apolipoprotein E, Cathepsin B, Cystatin B, Cystatin-C-like, Lysozyme, Monocyte differentiation CD14, Pancreatitis-associated precursor protein, Profilin, and Protein FAM3C. Finally, enrichment analysis provided information about the biological mechanisms in which these proteins are involved. In conclusion, this study provides 15 new candidate urinary biomarkers and an improved understanding of the pathogenesis of kidney disease in CanL.

Clinical and laboratory characteristics of hemophagocytic lymphohistiocytosis induced by Leishmania infantum infection.

BACKGROUND: Visceral leishmaniasis (VL) could progress to secondary hemophagocytic lymphohistiocytosis (HLH), which is a rare but life-threatening condition with poor prognosis. So far, the clinical and laboratory characteristics of VL associated HLH have not been well elucidated. METHOD AND FINDINGS: In this study, we retrospectively analyzed the clinical and laboratory profiles between 17 patients with VL associated HLH and 27 patients with VL alone admitted at the Beijing Friendship Hospital, Capital Medical University from May 2016 to March 2021. In addition to the identification of Leishmania infection, hemophagocytosis was identified in bone marrow in the most cases of VL associated HLH (15/17). The patients with VL associated HLH had higher chances of bleeding, hepatomegaly, thrombocytopenia, hypertriglyceridemia, hyperferritinemia, hypofibrinogenemia, elevated secretion of soluble IL-2 receptor or lower NK cell activity compared to patients with VL only. Furthermore, patients with VL associated HLH had higher inflammation status associated with higher levels of Th1 (TNF-α, IFN-γ, IL-1beta, IL-6, IL-8, IL-12p70), Th2 (IL-4) and Th17 cytokines (IL-17, IL-23) in the peripheral blood, and higher parasite load (qPCR and parasite culture). All 27 VL cases were totally recovered after being treated with Sodium Stibogluconate, five of the 17 patients with VL associated HLH died even after timely treatment with anti-parasite and immunosuppressive chemotherapy. CONCLUSION: Without appropriate treatment, visceral leishmaniosis could develop to secondary HLH. The parasite culturing and qPCR detection of bone marrow samples facilitates the diagnosis of VL associated HLH in addition to other findings of HLH. Prompt treatment with anti-Leishmania and immunosuppressive chemotherapy is critical to reduce the mortality of VL associated HLH.

Diagnosis and clinical management of canine leishmaniosis by general veterinary practitioners: a questionnaire-based survey in Portugal.

BACKGROUND: Canine leishmaniosis (CanL) can be appropriately managed following international recommendations. However, few studies have assessed the preferred protocols in real-life veterinary practice and whether these are in line with the guidelines. This survey aimed to investigate the current trends in the clinical management of CanL among veterinary practitioners in Portugal, taking into consideration different scenarios of infection/disease and the awareness of and application by veterinary practitioners of the current guidelines. METHODS: A questionnaire-based survey was conducted online using an electronic platform. The following topics were surveyed: (i) general characteristics of the responding veterinarian; (ii) the preferred protocols used for the diagnosis, treatment and prevention of CanL, considering different theoretical scenarios of infection/disease; and (iii) the responding veterinarian's current knowledge and application of the existing guidelines on CanL. After internal validation, the survey was distributed online, for 2 months, via Portuguese social network veterinary groups. Data were collected for descriptive analysis. RESULTS: Eighty-six replies were obtained. Analysis of the results showed that the preferred diagnostic techniques varied widely according to the theoretical scenario of infection/disease. In general daily practice, serology testing (enzyme-linked immunosorbent assay [ELISA]) was the most used tool (67.4%). The preferred matrices used for PCR test were lymph nodes (62.3%) and/or bone marrow (59.0%). Regarding treatment, for subclinical infection/stage I CanL, 51.2% of the respondents did not prescribe any medical treatment, but 98.8% proceeded with both monitoring and preventive measures. Among those who prescribed a treatment (n = 42), most chose domperidone (47.6%). For the treament of stages IIa, IIb and III CanL, allopurinol/meglumine antimoniate (MA) was chosen by 69.8, 73.3 and 51.2% of respondents, respectively, followed by allopurinol/miltefosine (20.9, 19.8 and 38.4%, respectively). In contrast, dogs with stage IV CanL were mostly treated with allopurinol/miltefosine (48.8%) rather than with allopurinol/MA (23.3%). The use of repellents was the preferred preventive strategy (98.8%). About 93.0% of responders were aware of the existence of guidelines, and most of these veterinarians consulted the guidelines of the LeishVet group and the Canine Leishmaniosis Working Group; however, 31.3% reported that they did not follow any specific recommendations. CONCLUSIONS: Of the veterinarians responding to the survey, most reported following international guidelines for the clinical management of CanL. While allopurinol/MA was the preferred therapeutic protocol for the treatment of stages II/III CanL, allopurinol/miltefosine was the first choice for the treatment of stage IV CanL, possibly due to the unpredictable effect of MA on renal function. This study contributes to a better understanding of the trends in practical approaches to the treatment of CanL in Portugal.

Complement receptor 3 mediates ruffle-like, actin-rich aggregates during phagocytosis of Leishmania infantum metacyclics.

The parasitic protozoan Leishmania infantum resides primarily in macrophages throughout mammalian infection. Infection is initiated by deposition of the metacyclic promastigote into the dermis of a mammalian host by the sand fly vector. Promastigotes enter macrophages by ligating surface receptors such as complement receptor 3 (CR3), inducing phagocytosis of the parasite. At the binding site of metacyclic promastigotes, we observed large asymmetrical aggregates of macrophage membrane with underlying actin, resembling membrane ruffles. Actin accumulation was observed at the point of initial contact, before phagosome formation and accumulation of peri-phagosomal actin. Ruffle-like structures did not form during phagocytosis of attenuated promastigotes or during phagocytosis of the intracellular amastigote form of L. infantum. Entry of promastigotes through massive actin accumulation was associated with a subsequent delay in fusion of the parasitophorous vacuole (PV) with the lysosomal markers LAMP-1 and Cathepsin D. Actin accumulation was also associated with entry through CR3, since macrophages from CD11b knockout (KO) mice did not form massive aggregates of actin during phagocytosis of metacyclic promastigotes. Furthermore, intracellular survival of L. infantum was significantly decreased in CD11b KO compared to wild type macrophages, although entry rates were similar. We conclude that both promastigote virulence and host cell CR3 are needed for the formation of ruffle-like membrane structures at the site of metacyclic promastigote phagocytosis, and that formation of actin-rich aggregates during entry correlates with the intracellular survival of virulent promastigotes.

Liver infusion tryptose (LIT): the best choice for growth, viability, and infectivity of Leishmania infantum parasites.

Leishmania spp. parasites have a complex biological cycle presenting basically two different morphological stages, the amastigote and promastigote forms. In vitro cultivation allows a more complete study of the biological aspects of these parasites, indicating better conditions for infection, immunoassay tests, drug evaluations, and vaccines. Thus, we evaluated the three most used culture media for Leishmania spp., Grace's insect cell culture medium (Grace's), liver infusion tryptose (LIT), and Schneider's insect medium (Schneider's), without supplementation or supplemented with fetal calf serum (FCS) and bovine serum albumin (Albumin) to evaluate the growth, viability, and infectivity of the L. infantum promastigotes. It was observed that promastigote forms have a better growth in LIT and Schneider's with or without FCS when compared to that in Grace's. The supplementation with albumin promoted greater viability of the parasites independent of the medium. For in vitro infection of J774.A1 macrophages using light microscopy and flow cytometry analyses, FCS-supplemented LIT and Grace's promoted higher percentage of infected macrophages and parasite load compared with Schneider's media. Taken together, our results demonstrated that the supplementation of LIT culture medium with FCS is the most suitable strategy to cultivate Leishmania infantum parasites enabling the maintenance of growth and infective parasites for research uses.

Frequency, active infection and load of Leishmania infantum and associated histological alterations in the genital tract of male and female dogs.

Visceral leishmaniasis caused by the protozoan Leishmania infantum is a zoonosis. The domestic dog is the primary reservoir in urban areas. This study aimed to evaluate the frequency, active infection and load of L. infantum in the genital tract of male and female dogs seropositive for this parasite, as well as to identify histological genital alterations associated with this protozoan. We studied 45 male and 25 female L. infantum-seropositive noncastrated dogs from the same endemic area in Brazil. Tissue samples from the testis, epididymis, prostate, vulva, vagina, and uterus were examined by singleplex qPCR and parasitological tests (histopathology, immunohistochemistry, and parasitological culture). The latter were performed for the detection of active infection (parasites able to multiply and to induce lesions). Forty-four (98%) males and 25 (100%) females were positive for L. infantum in the genital tract (epididymis: 98%; vulva: 92%; vagina: 92%; testis: 91%; uterus: 84%; prostate: 66%). Active infection in the genital tract was confirmed in 69% of males and 64% of females (32% in the uterus). Parasite loads were similar in the testis, vulva, epididymis and vagina and lower in the prostate. Only the parasite load in the vagina was significantly associated with the number of clinical signs. Granulomatous inflammation predominated in all organs, except for the prostate. Only in the testis and epididymis was the inflammatory infiltrate significantly more intense among dogs with a higher parasite load in these organs. The high frequency, detection of active infection and similarity of L. infantum loads in the genital tract of infected males and females suggest the potential of venereal transmission of this parasite by both sexes and of vertical transmission by females in the area studied. Additionally, vertical transmission may be frequent since active L. infantum infection was a common observation in the uterus.

Lipophosphoglycans from dermotropic Leishmania infantum are more pro-inflammatory than those from viscerotropic strains.

Although Leishmania infantum is well-known as the aethiological agent of visceral leishmaniasis (VL), in some Central American countries it may cause atypical non-ulcerated cutaneous leishmaniasis (NUCL). However, the mechanisms favoring its establishment in the skin are still unknown. Lipophosphoglycan (LPG) is the major Leishmania multivirulence factor involved in parasite-host interaction. In the case of viscerotropic L. infantum, it causes an immunosuppression during the interaction with macrophages. Here, we investigated the biochemical and functional roles of LPGs from four dermotropic L. infantum strains from Honduras during in vitro interaction with murine macrophages. LPGs were extracted, purified and their repeat units analysed. They did not have side chains consisting of Gal(ß1,4)Man(α1)-PO4 common to all LPGs. Peritoneal macrophages from BALB/c and C57BL/6 were exposed to LPG for nitric oxide (NO) and cytokine (TNF-α and, IL-6) production. LPGs from dermotropic strains from Honduras triggered higher NO and cytokine levels compared to those from viscerotropic strains. In conclusion, LPGs from dermotropic strains are devoid of side-chains and exhibit high pro-inflammatory activity.

The macrophage microtubule network acts as a key cellular controller of the intracellular fate of Leishmania infantum.

The parasitophorous vacuoles (PVs) that insulate Leishmania spp. in host macrophages are vacuolar compartments wherein promastigote forms differentiate into amastigote that are the replicative form of the parasite and are also more resistant to host responses. We revisited the biogenesis of tight-fitting PVs that insulate L. infantum in promastigote-infected macrophage-like RAW 264.7 cells by time-dependent confocal laser multidimensional imaging analysis. Pharmacological disassembly of the cellular microtubule network and silencing of the dynein gene led to an impaired interaction of L. infantum-containing phagosomes with late endosomes and lysosomes, resulting in the tight-fitting parasite-containing phagosomes never transforming into mature PVs. Analysis of the shape of the L. infantum parasite within PVs, showed that factors that impair promastigote-amastigote differentiation can also result in PVs whose maturation is arrested. These findings highlight the importance of the MT-dependent interaction of L. infantum-containing phagosomes with the host macrophage endolysosomal pathway to secure the intracellular fate of the parasite.

Chemical inhibition of ß-glucocerebrosidase does not affect phagocytosis and early containment of Leishmania by murine macrophages.

Gaucher disease is a lysosomal storage disease in which a genetic deficiency in ß-glucocerebrosidase leads to the accumulation of glycosphingolipids in lysosomes. Macrophages are amongst the cells most severely affected in Gaucher disease patients. One phenotype associated with Gaucher macrophages is the impaired capacity to fight bacterial infections. Here, we investigate whether inhibition of ß-glucocerebrosidase activity affects the capacity of macrophages to phagocytose and act on the early containment of human pathogens of the genus Leishmania. Towards our aim, we performed in vitro infection assays on macrophages derived from the bone marrow of C57BL/6 mice. To mimic Gaucher disease, macrophages were incubated with the ß-glucocerebrosidase inhibitor, conduritol B epoxide (CBE), prior to contact with Leishmania. This treatment guaranteed that ß-glucocerebrosidase was fully inhibited during the contact of macrophages with Leishmania, its enzymatic activity being progressively recovered along the 48 h that followed removal of the inhibitor. Infections were performed with L. amazonensis, L. infantum, or L. major, so as to explore potential species-specific responses in the context of ß-glucocerebrosidase inactivation. Parameters of infection, recorded immediately after phagocytosis, as well as 24 and 48 h later, revealed no noticeable differences in the infection parameters of CBE-treated macrophages relative to non-treated controls. We conclude that blocking ß-glucocerebrosidase activity during contact with Leishmania does not interfere with the phagocytic capacity of macrophages and the early onset of leishmanicidal responses.

Phenotypical Characterization of Spleen Remodeling in Murine Experimental Visceral Leishmaniasis.

Background: Visceral leishmaniasis (VL) is caused by Leishmania infantum or L. donovani infection. One of the main problems related to this disease is the emergence of severe clinical forms with a lethality of 5-20%, even while under specific treatment. In humans and other species susceptible to fatal VL, such as dogs and hamsters, the disruption of splenic white pulp (WP) is accompanied by disease progression. Control of VL progression is seen in BALB/c mice, as evidenced by a mild clinical presentation and controlled parasite replication in the liver and spleen. In this study, we investigated the features involved in the morphological remodeling of splenic compartments associated with the control of VL progression to death. Methods: We evaluated cohorts of BALB/c mice after 30, 60, and 90 days of infection by L. infantum. Spleen morphology, cell population subsets and cytokine production were studied in the spleen using flow- and histo-cytometry. Results: Intraperitoneal infection with 108 promastigotes of L. infantum led to progressive increases in spleen size at 60 and 90 days after infection. Splenomegaly was the only clinical sign of disease observed. At 30 days after infection, hyperplasia in the WP and decreased numbers of plasmacytoid dendritic cells were observed. The WP hyperplasia subsided at 60 days post-infection. However, the splenomegaly remained in association with increased numbers of macrophages, B and T lymphocytes and plasma cells. An increased number of lymphoid tissue inducer (LTi) cells was observed; these were distributed around the periarteriolar lymphoid sheath in control mice and scattered throughout the red pulp in the Leishmania-infected mice. After 90 days of infection, increased IL-6 and IFN-γ production was seen in the spleen, as well as higher frequencies of follicular and plasmacytoid dendritic cells. Conclusion: The data presented herein emphasizes the potential role of spleen remodeling in the control of severe forms of VL and highlights features potentially involved in this process.

The balancing act: Immunology of leishmaniosis.

Immune control of Leishmania infantum, the causative agent of most canine leishmaniosis (CanL), requires a balancing act between inflammatory and regulatory responses. This balance is specifically between the proinflammatory T helper 1 type (Th1) CD4+ T cells that are responsible for controlling parasite replication and T regulatory 1 cells which mediate an immunosuppressive, regulatory, response needed to dampen overabundant inflammation but if predominant, result in CanL progression. How this delicate immune cell interaction occurs in the dog will be highlighted in this review, focusing on the progressive changes observed within myeloid lineage cells (predominantly macrophages), B cells and T cells. After exposure to parasites, macrophages should become activated, eliminating L. infantum through release of reactive oxygen species. Unfortunately, multiple parasite and host factors can prevent macrophage activation allowing parasites to persist within them. T cells balance between a productive TH1 type CD4+ response capable of producing IFN-γ which aids macrophage activation versus T cell exhaustion which reduces T cell proliferation, IFN-γ production and allows parasite expansion within macrophages. Neutrophils and Th17 cells add to the inflammatory state, aiding in parasite removal, but also leading to pathology. A regulatory B cell population increases IL-10 production and down regulates the TH1 response allowing parasite growth. All of these immune challenges affect the balance between progression to clinical disease and maintaining sub-clinical disease. Vaccines and immunotherapies targeted at recovering or maintaining T and B cell function can be important factors in mending the immune balance required to survive CanL.

Feline leishmaniosis: diagnosis, treatment and outcome in 16 cats.

OBJECTIVES: Leishmaniosis is a vector-borne disease and in European countries is caused by Leishmania infantum. Cats are considered secondary reservoirs of the infection in endemic areas. The objective of this retrospective study is to describe the clinical findings, diagnosis, treatment and outcome of feline leishmaniosis (FeL) in 16 cats in Spain. METHODS: Medical records of cats diagnosed with leishmaniosis were retrospectively reviewed for cases that met the following inclusion criteria: identification of Leishmania organisms and/or DNA on cytological and/or histological specimens and/or a high anti-Leishmania antibody titre, compatible clinical findings and pathological abnormalities. RESULTS: Sixteen cats met the inclusion criteria, all of which were living in areas endemic for canine leishmaniosis. Systemic signs were present in 11 cases (68.8%). The most common clinical signs on presentation included cutaneous lesions in 12 cats (75%), ocular disease in six cats (37.5%) and anorexia in six cats (37.5%). A polyclonal gammopathy was noted in 12 cats (85.7%). Non-regenerative anaemia and renal abnormalities were present in six (37.5%) and five patients (31.3%), respectively. In nine cats (56.3%), immunosuppressive conditions/comorbidities were identified. The diagnosis was made in eight of the cats (50%) by cytology, but a combination of diagnostic tests was needed for definitive diagnosis in the remaining patients. Twelve cats (75%) were treated specifically for leishmaniosis. Five of the 12 cats (41.7%) did not improve with treatment. The median survival time in the group of patients treated specifically for leishmaniosis was 17 months. Median survival of patients treated with concomitant diseases was 13 months vs 41 months in those without, although this was not statistically significant (P = 0.557). CONCLUSIONS AND RELEVANCE: Presentation of FeL appears to be similar to canine leishmaniosis but with some specific features: ulcerative and nodular skin lesions are the predominant cutaneous signs; cats with immunosuppressive conditions or coexisting diseases were more commonly present than typically seen in dogs (mainly feline immunodeficiency virus). A combination of diagnostic tests may be needed for definitive diagnosis.

Combined in vitro IL-12 and IL-15 stimulation promotes cellular immune response in dogs with visceral leishmaniasis.

Domestic dogs are the main reservoir of Leishmania infantum, a causative agent of visceral leishmaniasis (VL). The number of human disease cases is associated with the rate of canine infection. Currently available drugs are not efficient at treating canine leishmaniasis (CanL) and months after the treatment most dogs show disease relapse, therefore the development of new drugs or new therapeutic strategies should be sought. In CanL, dogs lack the ability to mount a specific cellular immune response suitable for combating the parasite and manipulation of cytokine signaling pathway has the potential to form part of effective immunotherapeutic methods. In this study, recombinant canine cytokines (rcaIL-12, rcaIL-2, rcaIL-15 and rcaIL-7) and soluble receptor IL-10R1 (rcasIL-10R1), with antagonistic activity, were evaluated for the first time in combination (rcaIL-12/rcaIL-2, rcaIL-12/rcaIL-15, rcaIL-12/rcasIL-10R1, rcaIL-15/rcaIL-7) or alone (rcasIL-10R1) to evaluate their immunomodulatory capacity in peripheral blood mononuclear cells (PBMCs) from dogs with leishmaniasis. All the combinations of recombinant proteins tested were shown to improve lymphoproliferative response. Further, the combinations rcaIL-12/rcaIL-2 and rcaIL-12/rcaIL-15 promoted a decrease in programmed cell death protein 1 (PD-1) expression in lymphocytes. These same combinations of cytokines and rcaIL-12/rcasIL-10R1 induced IFN-γ and TNF-α production in PBMCs. Furthermore, the combination IL-12/IL-15 led to an increased in T-bet expression in lymphocytes. These findings are encouraging and indicate the use of rcaIL-12 and rcaIL-15 in future in vivo studies aimed at achieving polarization of cellular immune responses in dogs with leishmaniasis, which may contribute to the development of an effective treatment against CanL.

Transcriptional analysis of THP-1 cells infected with Leishmania infantum indicates no activation of the inflammasome platform.

Leishmaniasis is caused by intracellular parasites transmitted to vertebrates by sandfly bites. Clinical manifestations include cutaneous, mucosal or visceral involvement depending upon the host immune response and the parasite species. To assure their survival inside macrophages, these parasites developed a plethora of highly successful strategies to manipulate various immune system pathways. Considering that inflammasome activation is critical for the establishment of a protective immune response in many parasite infections, in this study we determined the transcriptome of THP-1 cells after infection with L. infantum, with a particular focus on the inflammasome components. To this end, the human cell line THP-1, previously differentiated into macrophages by PMA treatment, was infected with L. infantum promastigotes. Differentiated THP-1 cells were also stimulated with LPS to be used as a comparative parameter. The gene expression signature was determined 8 hours after by RNA-seq technique. Infected or uninfected THP-1 cells were stimulated with nigericin (NIG) to measure active caspase-1 and TNF-α, IL-6 and IL-1ß levels in culture supernatants after 8, 24 and 48 hours. L. infantum triggered a gene expression pattern more similar to non-infected THP-1 cells and very distinct from LPS-stimulated cells. Some of the most up-regulated genes in L. infantum-infected cells were CDC20, CSF1, RPS6KA1, CD36, DUSP2, DUSP5, DUSP7 and TNFAIP3. Some up-regulated GO terms in infected cells included cell coagulation, regulation of MAPK cascade, response to peptide hormone stimulus, negative regulation of transcription from RNA polymerase II promoter and nerve growth factor receptor signaling pathway. Infection was not able to induce the expression of genes associated with the inflammasome signaling pathway. This finding was confirmed by the absence of caspase-1 activation and IL-1ß production after 8, 24 and 48 hours of infection. Our results indicate that L. infantum was unable to activate the inflammasomes during the initial interaction with THP-1 cells.

Lipophosphoglycans from dermotropic Leishmania infantum are more pro-inflammatory than those from viscerotropic strains

Although Leishmania infantum is well-known as the aethiological agent of visceral leishmaniasis (VL), in some Central American countries it may cause atypical non-ulcerated cutaneous leishmaniasis (NUCL). However, the mechanisms favoring its establishment in the skin are still unknown. Lipophosphoglycan (LPG) is the major Leishmania multivirulence factor involved in parasite-host interaction. In the case of viscerotropic L. infantum, it causes an immunosuppression during the interaction with macrophages. Here, we investigated the biochemical and functional roles of LPGs from four dermotropic L. infantum strains from Honduras during in vitro interaction with murine macrophages. LPGs were extracted, purified and their repeat units analysed. They did not have side chains consisting of Gal(β1,4)Man(α1)-PO4 common to all LPGs. Peritoneal macrophages from BALB/c and C57BL/6 were exposed to LPG for nitric oxide (NO) and cytokine (TNF-α and, IL-6) production. LPGs from dermotropic strains from Honduras triggered higher NO and cytokine levels compared to those from viscerotropic strains. In conclusion, LPGs from dermotropic strains are devoid of side-chains and exhibit high pro-inflammatory activity.

Transcriptome Analysis Identifies Immune Markers Related to Visceral Leishmaniasis Establishment in the Experimental Model of BALB/c Mice.

Visceral leishmaniasis (VL) caused by Leishmania donovani and L. infantum is a potentially fatal disease. To date there are no registered vaccines for disease prevention despite the fact that several vaccines are in preclinical development. Thus, new strategies are needed to improve vaccine efficacy based on a better understanding of the mechanisms mediating protective immunity and mechanisms of host immune responses subversion by immunopathogenic components of Leishmania. We found that mice vaccinated with CPA162-189-loaded p8-PLGA nanoparticles, an experimental nanovaccine, induced the differentiation of antigen-specific CD8+ T cells in spleen compared to control mice, characterized by increased dynamics of proliferation and high amounts of IFN-γ production after ex vivo re-stimulation with CPA162-189 antigen. Vaccination with CPA162-189-loaded p8-PLGA nanoparticles resulted in about 80% lower parasite load in spleen and liver at 4 weeks after challenge with L. infantum promastigotes as compared to control mice. However, 16 weeks after infection the parasite load in spleen was comparable in both mouse groups. Decreased protection levels in vaccinated mice were followed by up-regulation of the anti-inflammatory IL-10 production although at lower levels in comparison to control mice. Microarray analysis in spleen tissue at 4 weeks post challenge revealed different immune-related profiles among the two groups. Specifically, vaccinated mice were characterized by similar profile to naïve mice. On the other hand, the transcriptome of the non-vaccinated mice was dominated by increased expression of genes related to interferon type I, granulocyte chemotaxis, and immune cells suppression. This profile was significantly enriched at 16 weeks post challenge, a time-point which is relative to disease establishment, and was common for both groups, further suggesting that type I signaling and granulocyte influx has a significant role in disease establishment, pathogenesis and eventually in decreased vaccine efficacy for stimulating long-term protection. Overall, we put a spotlight on host immune networks during active VL as potential targets to improve and design more effective vaccines against disease.

Infection by Leishmania (Leishmania) infantum chagasi causes intestinal changes B-1 cells dependent.

Evaluating the histopathological and morphometric changes caused by Leishmania (Leishmania) infantum chagasi infection either in the presence or absence of B-1 cells. Wild-type Balb/c and XID mice were used. Half of XID mice received B-1 cells adoptive transfer (XID + B1). Five animals from each group were infected (Balb/c I, XID I and XID + B1 I), totalizing six groups (n = 5). After 45 days of infection, the ileum was collected for histological processing and analysis. After infection, the XID animals showed an increase in the thickness of the intestinal layers, in the depth and width of the crypt and in the villi width. However, the Balb/c I group showed a reduction in almost all these parameters, whereas the villi width was increased. The villi height decreased in the infected XID animals; however, it was increased in the XID + B1 I group. Leishmania (L) infantum chagasiinfection caused a decrease in the number of Paneth cells; however, their area was increased. Finally, goblet cells and enterocytes presented different change profiles among groups. This study showed that the parasite infection causes structural and histopathological alterations in the intestine. These changes might be influenced by the absence of B-1 cells.

New Insights on the Adjuvant Properties of the Leishmania infantum Eukaryotic Initiation Factor.

Vaccination is the most effective tool against infectious diseases. Subunit vaccines are safer compared to live-attenuated vaccines but are less immunogenic and need to be delivered with an adjuvant. Adjuvants are essential for enhancing vaccine potency by improving humoral and cell-mediated immune responses. Only a limited number of adjuvants are licensed for human vaccines, and their mode of action is still not clear. Leishmania eukaryotic initiation factor (LeIF) has been described having a dual role, as a natural adjuvant and as an antigen that possesses advantageous immunomodulatory properties. In this study, we assessed the adjuvant properties of recombinant Leishmania infantum eukaryotic initiation factor (LieIF) through in vitro and in vivo assays. LieIF was intraperitoneally administered in combination with the protein antigen ovalbumin (OVA), and the widely used alum was used as a reference adjuvant. Our in vitro studies using J774A.1 macrophages showed that LieIF induced stimulatory effects as demonstrated by the enhanced surface expression of CD80 and CD86 co-stimulatory molecules and the induced production of the immune mediators NO and MIP-1α. Additionally, LieIF co-administration with OVA in an in vivo murine model induced a proinflammatory environment as demonstrated by the elevated expression of TNF-α, IL-1ß, and NF-κB2 genes in peritoneal exudate cells (PEC). Furthermore, PEC derived from OVA-LieIF-immunized mice exhibited elevated expression of CD80 molecule and production of NO and MIP-1α in culture supernatants. Moreover, LieIF administration in the peritoneum of mice resulted in the recruitment of neutrophils and monocytes at 24 h post-injection. Also, we showed that this immunopotentiating effect of LieIF did not depend on the induction of uric acid danger signal. These findings suggest the potential use of LieIF as adjuvant in new vaccine formulations against different infectious diseases.

In vitro antileishmanial effects of Physalis angulata root extract on Leishmania infantum.

OBJECTIVE: In the present study, we evaluated the effects of the aqueous extract of Physalis angulata root (AEPa) on Leishmania infantum proliferation, morphology, and the driving mechanism in leishmanicidal activity and modulatory action on macrophages. METHODS: L. infantum promastigotes were treated with 50 and 100 µg/mL AEPa for 72 h and then antipromastigote assay was performed by counts in a Newbauer chamber, morphological changes were analyzed by transmission electron microscopy and the mechanism of the leishmanicidal activity was detected. In addition, macrophages were infected with L. infantum and were used to evaluate anti-amastigote activity of AEPa and effects of AEPa on cytokine secretion after 72-hour treatment. RESULTS: Treatment with AEPa reduced the numbers of L. infantum promastigotes (50% inhibitory concentration (IC50) = 65.9 µg/mL; selectivity index (SI) = 22.1) and amastigotes (IC50 = 37.9 µg/mL; SI = 38.5) compared with the untreated control. Amphotericin B reduced 100% of the promastigote numbers after 72 h of treatment (IC50 = 0.2 µg/mL). AEPa induced several morphological changes and increased the production of reactive oxygen species and apoptotic death in promastigotes after treating for 72 h. AEPa (100 µg/mL) promoted tumor necrosis factor-α secretion in macrophages infected with L. infantum after 72 h of treatment, but did not induce an increase in this cytokine in noninfected macrophages. In addition, AEPa showed no cytotoxic effect on J774-A1 cells (50% cytotoxic concentration >1000 µg/mL). CONCLUSION: AEPa presented antileishmanial activity against the promastigotes and amastigotes of L. infantum without macrophage cytotoxicity; these results show that natural products such as P. angulata have leishmanicidal potential and in the future may be an alternative treatment for leishmaniasis.