Immunogenicity of PvCyRPA, PvCelTOS and Pvs25 chimeric recombinant protein of Plasmodium vivax in murine model.

In the Americas, P. vivax is the predominant causative species of malaria, a debilitating and economically significant disease. Due to the complexity of the malaria parasite life cycle, a vaccine formulation with multiple antigens expressed in various parasite stages may represent an effective approach. Based on this, we previously designed and constructed a chimeric recombinant protein, PvRMC-1, composed by PvCyRPA, PvCelTOS, and Pvs25 epitopes. This chimeric protein was strongly recognized by naturally acquired antibodies from exposed population in the Brazilian Amazon. However, there was no investigation about the induced immune response of PvRMC-1. Therefore, in this work, we evaluated the immunogenicity of this chimeric antigen formulated in three distinct adjuvants: Stimune, AddaVax or Aluminum hydroxide (Al(OH)3) in BALB/c mice. Our results suggested that the chimeric protein PvRMC-1 were capable to generate humoral and cellular responses across all three formulations. Antibodies recognized full-length PvRMC-1 and linear B-cell epitopes from PvCyRPA, PvCelTOS, and Pvs25 individually. Moreover, mice's splenocytes were activated, producing IFN-γ in response to PvCelTOS and PvCyRPA peptide epitopes, affirming T-cell epitopes in the antigen. While aluminum hydroxide showed notable cellular response, Stimune and Addavax induced a more comprehensive immune response, encompassing both cellular and humoral components. Thus, our findings indicate that PvRMC-1 would be a promising multistage vaccine candidate that could advance to further preclinical studies.

Testicular damage without clinical manifestations in BALB/c mice experimentally infected with Zika virus.

In 2015-2016, the Zika virus (ZIKV) caused a major epidemic in the Americas, increasing cases of microcephaly and Guillain-Barré syndrome. During this period, the discovery of ZIKV sexual transmission intensified studies on the impact of this virus on the reproductive organs. For this study, 2-month-old male BALB/c mice were infected with 1.26 x 106 PFU/mL of ZIKV in solution via the intravenous route. After three, seven, and fourteen days post-infection (DPI), blood and testicle samples were obtained to detect ZIKV RNA. The authors observed that the infected animals had slower weight gain than the control group. Viremia occurred only at 3DPI, and the ZIKV RNA was detected in one testis sample at 7DPI. The histopathological analysis of this organ revealed intense disorganization of the seminiferous tubules' structure, inflammatory infiltrate, necrosis, hemorrhage, fluid accumulation, congestion of blood vessels, and reduced sperm count. Ultrastructural analysis showed nuclear changes in tubule cells, activation of interstitial cells, and morphological changes in spermatozoa, in addition to fragmentation and decreased electron density of the genetic material of these cells. Thus, despite causing predominantly asymptomatic infections, ZIKV can cause significant subclinical and transient damage, including to male reproductive organs.

Immune response of BALB/c mice infected with two strains of Corynebacterium pseudotuberculosis.

This work evaluated aspects of the immune response of BALB/c mice infected with Corynebacterium pseudotuberculosis (T1 and C57). The fifteen BALB/c mice were euthanized after 70 days of infection and morphologically evaluated, also analyzing the innate and adaptive immune responses. The C57 strain induced more pronounced morphological changes than the T1 strain. There was an increase in CD4+ and CD8+ T cells identified during infection with the C57 strain. Cytokines of the inflammatory profile IL-1α and IL-6 and regulatory IL-13 and IL-10 presented significant differences. Cytokines IL-2, IL-4, INF-γ, IL-22, IL-21, and IL-27 did not differ significantly between groups. The obtained results contribute to a better understanding of the type of response and the immunological mechanisms involved during infection with different strains of C. pseudotuberculosis.

Assessment of the Virulence of the Burkholderia mallei Strain BAC 86/19 in BALB/c Mice.

Burkholderia mallei is an aerobic, Gram-negative, non-motile bacillus. As an obligate mammalian pathogen, it primarily affects solipeds. Although rarely transmitted to humans, the disease it causes, glanders, is classified as a zoonosis. The bacterium was officially eradicated in Brazil in 1969; however, it reemerged after three decades. This study aims to assess the virulence of a specific B. mallei strain, isolated in Brazil, in BALB/c mice through intranasal infection. The strain, B. mallei BAC 86/19, was obtained from the tracheal secretion of a young mare displaying positive serology but no clinical signs of glanders. Post-mortem examinations revealed macroscopic lesions consistent with the disease, however. In mice, the LD50 was determined to be approximately 1.59 × 105 colony-forming units (CFU)/animal. Mice exposed to either 0.1 × LD50 or 1 × LD50 displayed transient weight loss, which resolved after three or five days, respectively. B. mallei persisted within the liver and lung for five days post-infection and in the spleen for seven days. These findings underscore the detectable virulence of the Brazilian B. mallei BAC 86/19 strain in mice, which are relatively resilient hosts. This research points to the importance of the continued investigation of the virulence mechanisms and potential countermeasures associated with B. mallei infections, including their Brazilian isolates.

PML/RARa leukemia induced murine model for immunotherapy evaluation.

Even though leukemia murine models are valuable tools for new drug therapy studies, most of these models consist of immunocompromised mice, which do not exhibit immune responses. In order to obtain an adequate leukemia model, we established an acute promyelocytic leukemia transplantation-based model (PML/RARa) in immunocompetent BALB/c mice, thus making it possible to study drug-induced cellular immune responses in leukemia. The development of PML/RARa leukemia was confirmed by leukocytosis (76.27 ± 21.8 vs. 3.40 ± 1.06; P < 0.0001), anemia (7.46 ± 1.86 vs. 15.10 ± 0.96; P < 0.0001), and thrombocytopenia (131.85 ± 39.32 vs. 839.50 ± 171.20; P < 0.0001), and the presence of blasts in the peripheral blood of mice (approximately 50% blasts; P < 0.0001), 15 days after the transplants. These findings were corroborated through differential counts, flow cytometry, and in vivo imaging, which indicated increased number of immature cells in the bone marrow (15.75 ± 3.30 vs 6.69 ± 0.55; P < 0.001), peripheral blood (7.88 ± 2.67 vs 1.22 ± 0.89; P < 0.001), and spleen (35.21 ± 4.12 vs 1.35 ± 0.86; P < 0.0001), as well as promyelocytes in the bone marrow (41.23 ± 4.80 vs 5.73 ± 1.50; P < 0.0001), peripheral blood (46.08 ± 7.52 vs 1.10 ± 0.59; P < 0.0001) and spleen (35.31 ± 8.26 vs 2.49 ± 0.29; P < 0.0001) of PML/RARa mice. Compared to basal conditions of untransplanted mice, the PML/RARa mice exhibited frequencies of T lymphocytes CD4 helper = 14.85 ± 2.91 vs 20.77 ± 2.9 in the peripheral blood (P < 0.05); 12.75 ± 1.33 vs 45.90 ± 2.02 in the spleen (P < 0.0001); CD8 cytotoxic = 11.27 ± 3.44 vs 11.05 ± 1.22 in the peripheral blood (P > 0.05); 10.48 ± 1.16 vs 30.02 ± 1.80 in the spleen (P < 0.0001); natural killer (NK) cells = 3.68 ± 1.35 vs 6.84 ± 0.52 in the peripheral blood (P < 0.001); 4.43 ± 0.57 vs 6.40 ± 1.14 in the spleen (P < 0.05); B cells 2.50 ± 0.60 vs 15.20 ± 5.34 in the peripheral blood (P < 0.001); 17.77 ± 4.39 vs 46.90 ± 5.92 in the spleen (P < 0.0001); neutrophils = 5.97% ± 1.88 vs 31.57 ± 9.14 (P < 0.0001); and monocytes = 6.45 ± 2.97 vs 15.85 ± 2.57 (P < 0.001), selected as classical (3.33 ± 3.40 vs 57.80 ± 16.51, P < 0.0001), intermediate (57.42 ± 10.61 vs 21.75 ± 5.90, P < 0.0001), and non-classical monocytes (37.51 ± 10.85 vs 18.08 ± 7.13, P < 0.05) in the peripheral blood; and as classically activated (M1) within in the bone marrow (3.70 ± 0.94 vs 1.88 ± 0.39, P < 0.05) and spleen 15.19 ± 3.32 vs 9.47 ± 1.61, P < 0.05), in addition to alternatively activated (M2) macrophages within the bone marrow (23.06 ± 5.25 vs 1.76 ± 0.74, P < 0.0001) and spleen (46.51 ± 11.18 vs 30.58 ± 2.64, P < 0.05) compartments. All-trans retinoic acid (ATRA) treatment of PML/RARa mice reduced blast (immature cells) in the bone marrow (8.62 ± 1.81 vs 15.76 ± 1.25; P < 0.05) and spleen (8.75 ± 1.31 vs 35.21 ± 1.55; P < 0.0001) with no changes in the peripheral blood (10.13 ± 3.33 vs 7.88 ± 1.01; P > 0.05), as well as reduced promyelocytes in the bone marrow (19.79 ± 4.84 vs 41.23 ± 1.81; P < 0.05), peripheral blood (31.65 ± 3.92 vs 46.09 ± 2.84; P < 0.05) and spleen (24.84 ± 2.03 vs 41.46 ± 2.39; P < 0.001), and increased neutrophils of the peripheral blood (35.48 ± 7.24 vs 7.83 ± 1.40; P < 0.05) which was corroborated by reducing of immature cells and increase of neutrophil in the stained smears from PML/RARa mice, thus confirming that this model can be used in drug development studies. Our results show the effective induction of PML/RARa leukemia in BALB/c mice, thus producing a low-priced and reliable tool for investigating cellular immune responses in leukemia.

NeuN distribution in brain structures of normal and Zika-infected suckling mice.

Microcephaly is the more severe brain malformation because of Zika virus infection. Increased vulnerability of neural stem and progenitor cells to Zika infection during prenatal neurodevelopment impairs the complete formation of cortical layers. Normal development of cerebellum is also affected. However, the follow-up of apparently healthy children born to Zika exposed mothers during pregnancy has revealed other neurological sequelae. This suggests Zika infection susceptibility remains in nervous tissue after neurogenesis end, when differentiated neuronal populations predominate. The neuronal nuclear protein (NeuN) is an exclusive marker of postmitotic neurons. Changes in NeuN expression are associated with neuronal degeneration. We have evaluated immunohistochemical expression of NeuN protein in cerebral cortex, hippocampus, and cerebellum of normal and Zika-infected neonatal Balb/c mice. The highest NeuN immunoreactivity was found mainly in neurons of all cortical layers, pyramidal layer of hippocampus, granular layer of dentate gyrus and in internal granular layer of cerebellum. Viral infection caused marked loss of NeuN immunostaining in all these brain areas. This suggests neurodegenerative effects of Zika virus infection during postmitotic neuron maturation and contribute to interpretation of neuropathogenic mechanisms of Zika.

In Vivo Antileishmanial Effect of 3,5-Diaryl-isoxazole Analogues Based on Veraguensin, Grandisin, and Machilin G: A Glance at a Preclinical Study.

New treatment approaches targeting cutaneous leishmaniasis (CL) are required since conventional drugs exhibit limitations due to their several adverse effects and toxicity. In this study, we aimed to evaluate the in vivo intralesional treatment efficacy of five isoxazole derivatives previously synthesized and effective in vitro against intracellular amastigote forms of Leishmania (L.) amazonensis. Among the tested analogues, 7 exhibited relevant in vivo therapeutic effects. The in silico predictions provided interesting information about the toxicity, suggesting the safety of analogue 7. Experiments performed with Salmonella typhimurium strains (TA98, TA100, and TA102) showed a non-mutagenicity profile of 7. The treatment of Leishmania-infected BALB/c mice with isoxazole 7 showed remarkably smaller CL lesions and decreased the parasitism (by 98.4%) compared to the control group. Hence, analogue 7 is a promising drug candidate and alternative treatment for CL caused by L. amazonensis.

Aloe vera and copaiba oleoresin-loaded chitosan films for wound dressings: microbial permeation, cytotoxicity, and in vivo proof of concept.

Chitosan films are commonly used for wound dressing, provided that this polymer has healing, mucoadhesiveness and antimicrobial properties. These properties can be further reinforced by the combination of chitosan with polysaccharides and glycoproteins present in aloe vera, together with copaiba oleoresin's pharmacological activity attributed to sesquiterpenes. In this work, we developed chitosan films containing either aloe vera, copaiba oil or both, by casting technique, and evaluated their microbial permeation, antimicrobial activity, cytotoxicity, and in vivo healing potential in female adult rats. None of the developed chitosan films promoted microbial permeation, while the cytotoxicity in Balb/c 3 T3 clone A31 cell line revealed no toxicity of films produced with 2 % of chitosan and up to 1 % of aloe vera and copaiba oleoresin. Films obtained with either 0.5 % chitosan or 0.5 % copaiba oleoresin induced cell proliferation which anticipate their potential for closure of wound and for the healing process. The in vivo results confirmed that tested films (0.5 % copaiba-loaded chitosan film and 0.5 % aloe vera-loaded chitosan film) were superior to a commercial dressing film. For all tested groups, a fully formed epithelium was seen, while neoformation of vessels seemed to be greater in formulations-treated groups than those treated with the control. Our work confirms the added value of combining chitosan with aloe vera and copaiba oil in the healing process of wounds.

Eugenol-Rich Essential Oil from Pimenta dioica: In Vitro and In Vivo Potentialities against Leishmania amazonensis.

Pimenta dioica L. is one the most recognized species with diverse biological activities. In this study, in vitro activity and in vivo efficacy of essential oil from P. dioica (EO-Pd) was evaluated. The main compound was also included in the animal studies and its in silico prediction related to biological activities, molecular ligands, drug likeness, and ADME (absorption, distribution, metabolism, and excretion) properties are listed. The chemical composition analyzed by GC-MS retrieved 45 components, which the most abundant compound was the eugenol (80.1%). The EO-Pd was able to inhibit the growth of L. amazonensis (IC50 = 9.7 ± 0.7 and 11.3 ± 2.1 µg/mL, promastigotes and amastigotes, respectively). The cytotoxicity assay showed a CC50 of 104.5 ± 0.9 µg/mL and a selectivity index of 9. In the model of cutaneous leishmaniasis in BALB/c mice, the effect of EO-Pd and eugenol was observed after treatment at 30 mg/kg by intralesional route with 5 administrations every 4 days. In the in silico predictions, some targets that justified the antileishmanial activity of eugenol and good drug like properties for this compound, were obtained. This study showed for first time the potential of EO-Pd to inhibit L. amazonensis, which could be linked to the activity of major compound eugenol.

Primary infection of BALB/c mice with a dengue virus type 4 strain leads to kidney injury

BACKGROUND Dengue is a disease caused by dengue virus (DENV-1 through -4). Among the four serotypes, DENV-4 remains the least studied. Acute kidney injury is a potential complication of dengue generally associated with severe dengue infection. OBJECTIVES The goal of this study was to investigate the alterations caused by experimental dengue infection in the kidney of adult BALB/c mice. METHODS In this study, BALB/c mice were infected through the intravenous route with a DENV-4 strain, isolated from a human patient. The kidneys of the mice were procured and subject to histopathological and ultrastructural analysis. FINDINGS The presence of the viral antigen was confirmed through immunohistochemistry. Analysis of tissue sections revealed the presence of inflammatory cell infiltrate throughout the parenchyma. Glomerular enlargement was a common find. Necrosis of tubular cells and haemorrhage were also observed. Analysis of the kidney on a transmission electron microscope allowed a closer look into the necrotic tubular cells, which presented nuclei with condensed chromatin, and loss of cytoplasm. MAIN CONCLUSIONS Even though the kidney is probably not a primary target of dengue infection in mice, the inoculation of the virus in the blood appears to damage the renal tissue through local inflammation.

Moderate Toxicity of Potential Boron-containing Therapeutic, Dipotassium-trioxohydroxytetrafl uorotriborate - K2 (B3 O3 F4 OH) in Rats and Mice

Abstract Biological activity of boron-containing compounds (BCCs) has been well-known. Growing interest and numerous applications for BCCs have been reported. Boron and boron-containing acids show low acute toxicity in mammals but data on halogenated boroxine (HB) - dipotassium-trioxohydroxytetrafluorotriborate, K2(B3O3F4OH) acute toxicity have not been reported before. This compound, characterized as a potential therapeutic for skin changes, exhibits no observable genotoxicity in doses lower that 0.1 mg/ml in vitro and 55 mg/kg in vivo. It has also been confirmed as an antitumour agent both in vitro and in vivo as well as an inhibitor of enzymes involved in antioxidant mechanisms. The aim of this study was to assess the acute toxicity of HB and to determine the maximum tolerated dose as well as a dose free of any signs of toxicity in different test organisms. Acute toxicity of HB was tested in Sprague-Dawley and Wistar rats and BALB/c mice after single parenteral application of different doses. We determined doses free of any sign of toxicity and LD50 after single dose administration. LD50 of HB ranges from 63 to 75 mg/kg in different test models, meaning that HB shows moderate toxicity

BALB/c and C57BL/6 mouse strains influence gastric function outcomes with administration of cisplatin and dexamethasone

Abstract Our aim was to evaluate the effects of cisplatin and dexamethasone alone and combined on gastric contractility and histomorphometry of BALB/c and C57BL/6 mice. BALB/c and C57BL/6 male mice (8-week-old) were randomly separated into: Control; Cisplatin (7.5 mg/Kg); Dexamethasone (2.0 mg/Kg); and Dexamethasone plus Cisplatin (2.0 mg/Kg of dexamethasone 1-hour prior to 7.5 mg/Kg of cisplatin). Drugs were administered intraperitoneally for three days. Body weight and food intake were evaluated on 2nd day. Alternating Current Biosusceptometry technique was employed to measure gastric contractions on 3rd day. Afterward, mice were killed for gastric histomorphometric analysis. Cisplatin decreased food intake and caused bradygastria in BALB/c mice; however, the amplitude of gastric contractions decreased in both BALB/c and C57BL/6. Dexamethasone and cisplatin combined restored the gastric frequency and food intake only in BALB/c, but drug combination reduced the gastric amplitude of contractions in both strains. Dexamethasone alone increased gastric mucosa thickness in C57BL/6 and decreased muscular thickness in BALB/c. In conclusion, the mouse strains presented differences in acute effects of cisplatin and dexamethasone alone and combined on gastric function. This reinforces the importance of choosing the appropriate mouse strain for studying the acute effects of drugs on the gastrointestinal tract.

Eficacia de la artemisina en modelos murinos de leishmaniasis cutánea experimental causada por Leishmania amazonensis / Artemisine efficacy in murine models of experimental cutaneous leishmaniasis caused by Leishmania amazonensis

Los limitados tratamientos disponibles para enfrentar la leishmaniasis requieren el desarrollo de investigaciones para buscar nuevos agentes terapéuticos. Una estrategia recomendada es el reposicionamiento farmacológico, en el que la artemisina figura como un posible candidato. El objetivo de este estudio es evaluar las potencialidades de la artemisina en dos modelos murinos de leishmaniasis cutánea experimental. Para ello, se emplearon ratones BALB/c (susceptibles) y C57BL/6 (resistentes) infectados con Leishmania amazonensis. El tratamiento se realizó por vía oral o intralesional con cinco dosis de artemisina a 30 mg/kg cada 4 días. Se determinó el comportamiento del peso, la evolución del tamaño de la lesión y la carga parasitaria. En ambos modelos animales se observó que el tratamiento con artemisina (oral e intralesional) disminuyó el tamaño de la lesión y la carga parasitaria con respecto a los grupos infectados sin tratamiento (p 0,05). Los ratones C57BL/6 tratados por vía oral fueron los únicos capaces de controlar las lesiones hasta el final del experimento. Se demuestra la eficacia in vivo de la artemisina en dos modelos de leishmaniasis cutánea inducida por L. amazonensis y se destaca la administración por vía oral en el control de la enfermedad. Se sugiere el futuro desarrollo de este fármaco para el tratamiento de la leishmaniasis cutánea.

The limited treatments available for leishmaniasis require the development of research for new therapeutic agents. One recommended strategy is the pharmacological repositioning, where artemisinin stands out as a possible candidate. The aim of this study is to evaluate the potential of artemisinin in two murine models of experimental cutaneous leishmaniasis. For this purpose, BALB/c (susceptible) and C57BL/6 (resistant) mice infected with Leishmania amazonensis were used. Oral or intralesional treatment was performed with five doses of artemisinin at 30 mg/kg every four days. Weight behavior, evolution of lesion size, and parasitic load were determined. In both animal models it was observed that treatment with artemisinin (oral and intralesional) decreased lesion size and parasitic load with respect to the untreated infected groups (p 0.05). Orally treated C57BL/6 mice were the only ones able to control lesions until the end of the experiment. The in vivo efficacy of artemisinin in two models of cutaneous leishmaniasis induced by L. amazonensis is demonstrated and oral administration is highlighted in the control of the disease. Further development of this drug for the treatment of cutaneous leishmaniasis is suggested.

Cancer Progression Is not Different in Mice of Different Gender Inoculated With Cells of the Triple-Negative 4T1 Breast Cancer Model.

Background: Breast cancer in men is a rare and poorly studied disease, and its treatment is based on women breast cancer studies. However, clinical outcome is not the same in men and women. Basic studies and clinical trials in animal models provide detailed information on cancer, origin, development, cell signaling pathways, sites of metastasis, and target molecules. It is necessary to explore the biology of breast cancer in male animal models that allow observing their similarity. Methods: The triple-negative 4T1 breast cancer model was developed in both male and female mice and studied weekly during 4 weeks. For that, twenty 8-week-old female and male BALB/c mice were used. Sixteen mice (eight males and eight females) were inoculated into the second left thoracic mammary pad with 20,000 4T1 cells, resuspended in 20 µL phosphate-buffered saline (PBS). All samples were processed for immunodetection, characterized histopathologically and immunohistochemically. Results: In this work, we describe the development of a triple-negative 4T1 breast cancer model in male BALB/c mice. Breast tumors were characterized histopathologically at different time points and corresponded to a moderately differentiated invasive ductal carcinoma, estrogen receptor ER-/progesterone receptor PR-/human epidermal growth factor receptor 2 HER2-/Ki67+, with histological grade II (moderately differentiated; a solid mass with occasional duct formation and moderate to severe nuclear pleomorphism), infiltrating the adipose and muscular tissue, and metastasis to lungs. From the results, we did not observe differences in the time of tumor development, necrosis, color change of tumor tissue, and lung metastasis between male and female mice. Even though we did not find histological differences, response to treatment and molecular signaling may be different. Conclusions: The histogenesis of male breast tumors was similar to that of female BALB/c mice. The histological and immunohistochemical characteristics of male tumors also match the features reported for stage IV human breast cancer of men and women. The murine male breast cancer model described here can be a significant tool to explore the molecular mechanisms involved in male breast cancer tumorigenesis and metastasis and may bring new approaches for clinical treatment of triple-negative breast cancer in men.

TPEN/TPGS (T2) combo dramatically reduces Philadelphia chromosome-positive pro-lymphoblastic B leukemia in BALB/c mice.

Acute lymphoblastic leukemia (ALL) is hematological neoplasia that affects human beings from early life to adulthood. Although ALL treatment has been effective, an important percentage of ALL patients are resilient to treatment. Therefore, there is an urgent need for testing a new combination of compounds for the treatment of this disease. Recently, combined TPEN and TPGS (T2 combo) have shown selective cytotoxic effects in vitro leukemia cells such as Jurkat, K562, and Ba/F3 cells. In this study, we aimed to test the effect of combined TPEN and TPGS agents (T2 combo) at a fixed dose (TPEN 5 mg/kg: TPGS 100 mg/kg) on leukemic Ba/F3-BCR-ABL P210 BALB-c mice model. We found that 4 successive 2-day apart intravenous injections of T2 combo showed a statistically significant reduction of Ba/F3 BCR-ABL leukemia cells (- 69%) in leukemia BALB/c mice (n = 6) compared to untreated leukemia group (n = 6). Moreover, the T2 combo was innocuous to non-leukemia BALB/c mice (n = 3) compared to untreated non-leukemia mice (control, n = 3). After treatments (day 42), all mice were left to rest until day 50. Outstandingly, the leukemia BALB/c mice treated with the T2 combo showed a lower percentage of Ba/F3-BCR-ABL P210 cells (- 84%) than untreated leukemia BALB/c mice. Furthermore, treatment of leukemia and non-leukemia mice with T2 combo showed no significant tissue alteration/damage according to the histopathological analysis of brain, heart, liver, kidney, and spleen samples; however, T2 combo significantly reduced the number of leukocytes in the bone marrow of treated leukemia mice. We conclude that the T2 combo specifically affects leukemia cells but no other tissue/organs. Therefore, we anticipate that the T2 combo might be a potential pro-oxidant combination for the treatment of leukemia patients.

A specific Leishmania infantum polyepitope vaccine triggers Th1-type immune response and protects against experimental visceral leishmaniasis.

The development of an immunogenic, effective, and safe vaccine is essential as an alternative for disease control. The present study aimed to evaluate the immunogenicity and efficacy potential of a polyepitope T-cell antigen candidate against visceral leishmaniasis in a murine model. BALB/c mice were immunized with three doses subcutaneously with Poly-T Leish alone or adjuvanted with Saponin plus Monophosphoryl lipid A, with 15-day intervals between doses, and challenged with 107 stationary-phase Leishmania infantum promastigotes via tail vein. Immunogenicity and parasitism in spleen and liver of immunized mice were evaluated 45 days post-challenge. Our results revealed that the immunization with Poly-T Leish and Poly-T Leish/SM increases the percentage of specific T (CD4+ and CD8+) lymphocytes proliferation in vitro after antigen-specific stimulation. Also, Poly-T Leish and Poly-T Leish/SM groups showed a high percentage of IFN-γ and TNF-α-producing T cells, meanwhile, the Poly-T Leish/SM group also showed an increased percentage of multifunctional T cells producing double and triple-positive (IFN-γ+TNF-α+IL-2+) cytokines. The immunization with Poly-T Leish or Poly-T Leish/SM stimulated a decreased IL-4 and IL-10 compared to the Saline and adjuvant group. Poly-T Leish/SM immunized mice exhibit a noteworthy reduction in the parasite burden (spleen and liver) through real-time PCR (96%). Moreover, we observed higher nitrite secretion in 120-hour stimulated-culture supernatant using Griess method. We demonstrated that the Poly-T Leish/SM candidate was potentially immunogenic, providing enhancement of protective immune mechanisms, and conferred protection reducing parasitism. Our candidate was considered potential against visceral leishmaniasis, and eventually, could be tested in phase I and II clinical trials in dogs.

Comparison of probiotic Lactobacillus strains isolated from dairy and Iranian traditional food products with those from human source on intestinal microbiota using BALB/C mice model.

This study compares the probiotic Lactobacillus strains isolated from dairy and Iranian traditional food products with those from human sources on intestinal microbiota using BALB/C mice model. First, Lactiplantibacillus plantarum (M11), Limosilactobacillus fermentum (19SH), Lactobacillus acidophilus (AC2), and Lactobacillus gasseri (52b) strains, isolated from either Iranian traditionally fermented products or human (healthy woman vaginal secretions), identified with molecular methods and selected based on the surface hydrophobicity, auto- and co-aggregation, were investigated for their probiotic properties and compared with their standard probiotic strains in vitro. The native strains and their mixtures (MIX) were then orally fed to five groups of female inbred BALB/C mice over the course of 38 days by gavage at 0.5 and 4 McFarland, respectively, equal to 1.5 × 108 and 1 × 109 cfu/ml. Feeding paused for 6 days to test the bacteria's adhesion in vivo. According to the findings, the probiotic Lactobacillus strain isolated from human source (52b) exhibited the best in vitro and in vivo adhesion ability. Probiotic Lactobacillus strains isolated from Iranian traditional food products (19SH and AC2) had the most co-aggregation with Listeria monocytogenes (ATTC 7644), Salmonella enterica subsp. enterica (ATCC 13,076), and Escherichia coli (NCTC 12,900 O157:H7) in vitro. These strains produced the most profound decreasing effect on the mice intestinal microbiota and pathogens in vivo. The difference in the strains and their probiotic potential is related to the sources from which they are isolated as well as their cell walls. The results suggest that (19SH and 52b strains) are the best candidates to investigate the cell wall and its effect on the host immune system.

Parasitological and immunological evaluation of a quinoline derivative salt incorporated into a polymeric micelle formulation against Leishmania infantum infection.

Leishmaniasis is a parasitic disease caused by Leishmania protozoa, which presents a large spectrum of clinical manifestations. In the present study, a quinoline derivative salt named N-(2-((7-chloroquinolin-4-yl)amino)ethyl)-N-(prop-2-yn-1-yl)prop-2-yn-1-aminium chloride or QDS3 was in vitro and in vivo tested against L. infantum by means of its incorporation in Poloxamer 407-based polymeric micelles (QDS3/M). The in vitro antileishmanial activity of QDS3 and QDS3/M was investigated in L. infantum promastigotes, axenic amastigotes and infected macrophages. BALB/c mice were infected with L. infantum, and parasitological parameters were evaluated 1 and 15 days post-treatment by determining the parasite load by a limiting dilution assay, besides a quantitative PCR (qPCR) method. Immunological response was assessed based on production of cellular cytokines, as well as by quantification of nitrite levels and specific antibodies. In vitro results showed that QDS3 free or in micelles presented effective antileishmanial action against both parasite stages, being more effective in amastigotes. In vivo data showed that treatment using QDS3 or QDS3/M reduced the parasite load in the livers, spleens, draining lymph nodes (dLN) and bone marrows of the treated animals, 1 and 15 days after treatment, when compared to values found in the control groups. Additionally, treated mice developed a polarized Th1-type immune response, with higher levels of IL-12, IFN-γ, GM-CSF and nitrite, besides high production of specific IgG2a antibodies, when compared to the controls. Parasitological and immunological data obtained using the micellar composition were better than the others. In conclusion, QDS3, mainly when applied in a delivery adjuvant system, could be considered for future studies as therapeutic candidate against VL.

Assessment of the Route of Exposure to Ovalbumin and Cow's Milk Proteins on the Induction of IgE Responses in BALB/c Mice.

BALB/c mice can be orally sensitized to food proteins under acid suppressive medication, mimicking human exposure and triggering a human-like allergic immune response. However, the reproducibility of such an oral food allergy model remains questionable. Our aim was to evaluate the IgE responses triggered against ovalbumin (OVA) and cow's milk proteins (CMP) after intragastric (IG), either under gastric-acid suppression or not, or intraperitoneal (IP) sensitization in BALB/c mice. OVA (0.2 mg) and different concentrations of CMP were administered with/without the antacid sucralfate by the IG route. For IP sensitization, OVA or CMP (0.5 mg) were administered. ELISA was used to evaluate IgE responses. The IP sensitization protocols triggered more robust and consistent anti-OVA or anti-CMP IgE responses than the intragastric ones (with/without sucralfate) (p < 0.05). 2.7% (1/36), and 5.5% (3/54) of the mice that underwent the sucralfate-assisted IG protocol triggered IgE responses against OVA or CMP, respectively. All the mice were administered OVA or CMP via IP triggered detectable IgE responses. The IP sensitization model is more reliable than the IG one for evaluating the intrinsic sensitizing and/or allergenic potential of food proteins, even if IG immunizations are carried out under gastric-acid suppression.

Effects of Migonemyia migonei salivary gland homogenates on Leishmania (Viannia) braziliensis infection in BALB/c mice.

Cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis is the most widespread clinical form of leishmaniasis in the Americas. Migonemyia migonei is a widely distributed phlebotomine sand fly species in Brazil and has been implicated as a vector for L. (V.) braziliensis. In the present study, we investigated the effects of salivary gland homogenates (SGH) of Mg. migonei on the course of L. (V.) braziliensis infection in BALB/c mice. Mice were separated into four groups (six mice per group): CTRL (uninfected mice); SGH (mice inoculated with Mg. migonei SGH); SGH+LEISH (mice inoculated with Mg. migonei SGH plus L. (V.) braziliensis promastigotes); LEISH (mice inoculated with L. (V.) braziliensis promastigotes). Mice were followed up for 8 weeks and the cellular immune response was evaluated by flow cytometry at the end of the experiment. Analysis of cytokine production by splenic cells stimulated with 0.5 SGH, 0.25 SGH of Mg. migonei or L. (V.) braziliensis soluble antigen stimulation (LSA) demonstrated that upon stimulation with SGH 0.25, the production of IL-17A and TNF was not sustained in the SGH group, with decreasing levels of these cytokines after 5 days compared to 3 days of incubation. Analyzing the production of cytokines after LSA stimulation, we observed lower levels of IL-17A in the SGH group after 5 days compared to 3 days. The same was observed for IFN-γ in the SGH group. Yet, the levels of TNF were significantly higher in the LEISH group after 5 days compared to 3 days. Among SGH+LEISH and LEISH mice, three animals in each group developed skin lesions on the tail, the mean lesion size was significantly higher in the LEISH group. Our study suggests that Mg. migonei SGH may modulate BALB/c immune response, as reflected by the low production or early decrease of pro-inflammatory cytokines in splenic cell cultures following stimulation with L. (V.) braziliensis antigen. Our data also suggest that Mg. migonei saliva may reduce the lesion size in BALB/c mice, but further research with a larger sample size is needed to confirm this hypothesis.