Essential oil of oregano (Origanum vulgare L.) reduces infection and proliferation of Toxoplasma gondii in BeWo cells with induction of autophagy and death of tachyzoites through a mechanism similar to necrosis.

Toxoplasmosis poses a global health threat, ranging from asymptomatic cases to severe, potentially fatal manifestations, especially in immunocompromised individuals and congenital transmission. Prior research suggests that oregano essential oil (OEO) exhibits diverse biological effects, including antiparasitic activity against Toxoplasma gondii. Given concerns about current treatments, exploring new compounds is important. This study was to assess the toxicity of OEO on BeWo cells and T. gondii tachyzoites, as well as to evaluate its effectiveness in in vitro infection models and determine its direct action on free tachyzoites. OEO toxicity on BeWo cells and T. gondii tachyzoites was assessed by MTT and trypan blue methods, determining cytotoxic concentration (CC50), inhibitory concentration (IC50), and selectivity index (SI). Infection and proliferation indices were analyzed. Direct assessments of the parasite included reactive oxygen species (ROS) levels, mitochondrial membrane potential, necrosis, and apoptosis, as well as electron microscopy. Oregano oil exhibited low cytotoxicity on BeWo cells (CC50: 114.8 µg/mL ± 0.01) and reduced parasite viability (IC50 12.5 ± 0.06 µg/mL), demonstrating 9.18 times greater selectivity for parasites than BeWo cells. OEO treatment significantly decreased intracellular proliferation in infected cells by 84% after 24 h with 50 µg/mL. Mechanistic investigations revealed increased ROS levels, mitochondrial depolarization, and lipid droplet formation, linked to autophagy induction and plasma membrane permeabilization. These alterations, observed through electron microscopy, suggested a necrotic process confirmed by propidium iodide labeling. OEO treatment demonstrated anti-T. gondii action through cellular and metabolic change while maintaining low toxicity to trophoblastic cells.

Biogenic silver nanoparticles (AgNp-Bio) restore testosterone levels and increase TNF-α and IL-6 in Leydig cells infected with Toxoplasma gondii.

Toxoplasma gondii, a protozoan parasite, is responsible for toxoplasmosis. The available therapy for patients with toxoplasmosis involves a combination of pyrimethamine and sulfadiazine, which have several adverse effects, including bone marrow suppression, megaloblastic anemia, leukopenia, and granulocytopenia. The development of therapeutic alternatives is essential for the management of toxoplasmosis, emphasizing the recent advances in nanomedicine. This study aimed to evaluate the in vitro effects of biogenic silver nanoparticles (AgNp-Bio) on tachyzoite forms and Leydig cells infected with T. gondii. We observed that the AgNp-Bio reduced the viability of the tachyzoites and did not exhibit cytotoxicity against Leydig cells at low concentrations. Additionally, treatment with AgNp-Bio reduced the rate of infection and proliferation of the parasite, and lowered the testosterone levels in the infected cells. It increased the levels of IL-6 and TNF-α and reduced the levels of IL- 10. Among the morphological and ultrastructural changes, AgNp-Bio induced a reduction in the number of intracellular tachyzoites and caused changes in the tachyzoites with accumulation of autophagic vacuoles and a decrease in the number of tachyzoites inside the parasitophorous vacuoles. Collectively, our data demonstrate that the AgNp-Bio affect T. gondii tachyzoites by activating microbicidal and inflammatory mechanisms and could be a potential alternative treatment for toxoplasmosis.

Moringa oleifera extract promotes apoptosis-like death in Toxoplasma gondii tachyzoites in vitro.

Toxoplasma gondii is the causative agent of toxoplasmosis, and an important problem of public health. The current treatment for toxoplasmosis is the combination of pyrimethamine and sulphadiazine, which do not act in the chronic phase of toxoplasmosis and have several side-effects. This study evaluated the anti-T. gondii activity and potential mechanism of Moringa oleifera seeds' aqueous extract in vitro. The concentration of M. oleifera extract in HeLa cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assays. The presence of T. gondii was assessed by quantitative polymerase chain reaction and toluidine blue staining. Pyrimethamine and sulphadiazine were used as drug controls. Modifications in T. gondii morphology and ultrastructure were observed by electron microscopy. In vitro, the M. oleifera extract had no toxic effect on HeLa cells at concentrations below 50 µg mL−1. Moringa oleifera extract inhibits T. gondii invasion and intracellular proliferation with similar results for sulphadiazine + pyrimethamine, and also shows cellular nitric oxide production at a concentration of 30 µg mL−1. Electron microscopy analyses indicated structural and ultrastructural modifications in tachyzoites after treatment. We also observed an increase in reactive oxygen species production and a loss of mitochondrial membrane integrity. Nile Red staining assays demonstrated a lipid accumulation. Annexin V­fluorescein isothiocyanate and propidium iodide staining demonstrated that the main action of M. oleifera extract in T. gondii tachyzoites was compatible with late apoptosis. In conclusion, M. oleifera extract has anti-T. gondii activity in vitro and might be a promising substance for the development of a new anti-T. gondii drug.

Biogenic silver nanoparticles reduce adherence, infection, and proliferation of toxoplasma gondii RH strain in HeLa cells without inflammatory mediators induction.

The highlights of biogenic silver nanoparticles (AgNp-Bio) include low toxicity - depending on size and concentration - and efficient antiparasitic activity. Therefore, the objective of this study was to assess the action of the AgNp-Bio on HeLa cells in an infection with strain of RH Toxoplasma gondii. Firstly, we performed a cellular viability test and characterized the AgNp-Bio to proceed with the infection of HeLa cells with T. gondii to be treated using AgNp-Bio or conventional drugs. Subsequently, we determined the level of standard cytokines Th1/Th2 as well as the content of nitric oxide (NO) and reactive oxygen species (ROS). Results indicated a mean size of 69 nm in diameter for the AgNp-Bio and obtained a dose-dependent toxicity. In addition, the concentrations of 3 and 6 µM promoted a significant decrease in adherence, infection, and intracellular proliferation. We also found lower IL-8 and production of inflammatory mediators. Thus, the nanoparticles reduced the adherence, infection, and proliferation of ROS and NO, in addition to immunomodulating the IL-8. Therefore, our data proved relevant to introduce a promising therapeutic alternative to toxoplasmosis.

Activity of rosuvastatin in tachyzoites of Toxoplasma gondii (RH strain) in HeLa cells.

Due to the toxicity of conventional medication in toxoplasmosis, some drugs are being studied for treating this infection, such as statins, especially rosuvastatin compound, which is efficient in inhibiting the initial isoprenoid biosynthesis processes in humans and the parasite. The goal of this study was to assess the activity of rosuvastatin in HeLa cells infected with the RH strain of T. gondii. In the experiment, HeLa cells (1 × 105) were infected with tachyzoites of T. gondii (5 × 105). After the experimental infection, we assessed the number of infected cells and the amount of intracellular tachyzoites. In addition, culture supernatants were collected to determine the amount of cytokines by cytometric bead array. We observed that there was no cytotoxicity in the concentrations tested in this cell line. The effect of rosuvastatin showed a significant reduction in both the number of infected cells and the proliferation index of the intracellular parasite, when compared with the conventional treatment combining sulfadiazine and pyrimethamine for toxoplasmosis. There were also reduced levels of cytokines IL-6 and IL-17. Therefore, it was concluded that rosuvastatin exhibited antiproliferative activity. The data presented are significant to promote further studies and the search for alternative treatment for toxoplasmosis.

Leishmania amazonensis infection regulates oxidate stress in hyperglycemia and diabetes impairing macrophage's function and immune response.

Leishmaniasis is a group of infectious diseases caused by protozoa of the Leishmania genus and its immunopathogenesis results from an unbalanced immune response during the infection. Diabetes is a chronic disease resulting from dysfunction of the body's production of insulin or the ability to use it properly, leading to hyperglycemia causing tissue damage and impairing the immune system. AIMS: The objective of this work was to evaluate the effects of hyperglycemia and diabetes during Leishmania amazonensis infection and how these conditions alter the immune response to the parasite. METHODS: An in vitro hyperglycemic stimulus model using THP-1-derived macrophages and an in vivo experimental diabetes with streptozotocin (STZ) in C57BL/6 mice was employed to investigate the impact of diabetes and hyperglicemia in Leishmania amazonensis infection. RESULTS: We observed that hyperglycemia impair the leishmanicidal capacity of macrophages derived from THP-1 cells and reverse the resistance profile that C57BL/6 mice have against infection by L. amazonensis, inducing more exacerbated lesions compared to non-diabetic animals. In addition, the hyperglycemic stimulus favored the increase of markers related to the phenotype of M2 macrophages. The induction of experimental diabetes in C57BL/6 mice resulted in a failure in the production of nitric oxide (NO) in the face of infection and macrophages from diabetic animals failed to process and present Leishmania antigens, being unable to activate and induce proliferation of antigen-specific lymphocytes. CONCLUSION: Together, these data demonstrate that diabetes and hyperglycemia can impair the cellular immune response, mainly of macrophages, against infection by parasites of the genus Leishmania.

Impact of Toxoplasma gondii infection on TM3 Leydig cells: Alterations in testosterone and cytokines levels.

Leydig cells play pivotal roles in eliciting male characteristics by producing testosterone and any damage to these cells can compromise male fertility Toxoplasma gondii (T. gondii) is an intracellular parasite capable to invade any nucleated cell, including cells from male reproductive system. Herein, we evaluated the capacity of RH strain of T. gondii to infect TM3 Leydig cells and the impact of this infection on testosterone and inflammatory mediators production. We first, by performing adherence, infection, and intracellular proliferation assays, we found a significant increase in the number of infected Leydig cells, peaking 48 h after the infection with T. gondii. Supernatants of TM3 infected cells exhibited, in a time-dependent manner, increased levels of testosterone as well as monocyte chemoattractant protein-1 (MCP-1) and interferon-γ (IFN-γ), which is correlated with the robust T. gondii infection. In conclusion, our study provides new insights regarding the harmful effects of T. gondii infection on male reproductive system.

Biogenic silver nanoparticles (AgNp-Bio) reduce Toxoplasma gondii infection and proliferation in HeLa cells, and induce autophagy and death of tachyzoites by apoptosis-like mechanism.

Toxoplasma gondii is a protozoan parasite that can cause severe and debilitating diseases, especially in immunocompromised individuals. The available treatment is based on drugs that have low efficacy, high toxicity, several adverse effects, and need long periods of treatment. Thus, the search for therapeutic alternatives is urgently needed. Biogenic silver nanoparticles (AgNp-Bio) have been associated with several biological effects, as antiproliferative, pro-apoptotic, antioxidant, antiviral, antifungal, and antiprotozoal activity. Thus, the objective was evaluating AgNp-Bio effect on HeLa cells infected with T. gondii (RH strain). First, nontoxic AgNp-Bio concentrations for HeLa cells (1.5 - 6 µM) were determined, which were tested on cells infected with T. gondii. A significant reduction in infection, proliferation, and intracellular parasitic load was observed, also an increase in ROS and IL-6. Additionally, the evaluation of the action mechanisms of the parasite showed that AgNp-Bio acts directly on tachyzoites, inducing depolarization of the mitochondrial membrane, ROS increase, and lipid bodies accumulation, as well as triggering an autophagic process, causing damage to the parasite membrane, and phosphatidylserine exposure. Based on this, it was inferred that AgNp-Bio affects T. gondii by inducing immunomodulation and microbicidal molecules produced by infected cells, and acts on parasites, by inducing autophagy and apoptosis.

Impairment of effector molecules response in diabetes induces susceptibility to Leishmania amazonensis infection.

Type 2 Diabetes is a chronic disease resulting from insulin dysfunction that triggers a low-grade inflammatory state and immune impairment. Leishmaniasis is an infectious disease characterized by chronic inflammation resulted from the parasite's immunomodulation ability. Thus, due to the delicate immune balance required in the combat and resistance to Leishmania infection and the chronic deregulation of the inflammatory response observed in type 2 diabetes, we evaluated the response of PBMC from diabetic patients to in vitro Leishmania amazonensis infection. For that, peripheral blood was collected from 25 diabetic patients and 25 healthy controls matched for age for cells extraction and subsequent experimental infection for 2 or 24 h and analyzed for phagocytic and leishmanicidal capacity by optical microscopy, oxidative stress by GSSG generation, labeling of intracellular mediators by enzyme-Linked immunosorbent assay, and cytokines measurement with cytometric beads array technique. We found that the diabetic group had a higher percentage of infected cells and a greater number of amastigotes per cell. Also, even inducing NF-kB phosphorylation and increasing TNF production after infection, cells from diabetic patients were unable to downregulate NRF2 and generate oxidative stress, which may be associated with the exacerbated levels of IL-6 observed. PBMC of diabetic individuals are more susceptible to infection by L. amazonensis and fail to control the infection over time due to the inability to generate effector microbicidal molecules.

A 21st Century Evil: Immunopathology and New Therapies of COVID-19.

Coronavirus Disease 2019 (COVID-19) has been classified as a global threat, affecting millions of people and killing thousands. It is caused by the SARS-CoV-2 virus, which emerged at the end of 2019 in Wuhan, China, quickly spreading worldwide. COVID-19 is a disease with symptoms that range from fever and breathing difficulty to acute respiratory distress and death, critically affecting older patients and people with previous comorbidities. SARS-CoV-2 uses the angiotensin-converting enzyme 2 (ACE2) receptor and mainly spreads through the respiratory tract, which it then uses to reach several organs. The immune system of infected patients has been demonstrated to suffer important alterations, such as lymphopenia, exhausted lymphocytes, excessive amounts of inflammatory monocytes and macrophages, especially in the lungs, and cytokine storms, which may contribute to its severity and difficulty of establishing an effective treatment. Even though no specific treatment is currently available, several studies have been investigating potential therapeutic strategies, including the use of previously approved drugs and immunotherapy. In this context, this review addresses the interaction between SARS-CoV-2 and the patient's host immune system during infection, in addition to discussing the main immunopathological mechanisms involved in the development of the disease and potential new therapeutic approaches.

Pravastatin and Simvastatin Pretreatment in Combination with Pyrimethamine and Sulfadiazine Reduces Infection Process of Toxoplasma gondii Tachyzoites (RH Strain) in HeLa Cells.

PURPOSE: Toxoplasma gondii is a protozoan from phylum Apicomplexa, which causes the toxoplasmosis infection; this one exhibits an apicoplast organelle which assists in the metabolism of isoprenoids and other pivotal mediators for the parasite survival. Statins are drugs that inhibit cholesterol synthesis, blocking the conversion of the substrate HMG-CoA to mevalonate, thus preventing the initial processes of the biosynthesis of these precursors, both in humans and parasite. Our goal was to verify whether the Toxoplasma gondii (RH strain) tachyzoites form pretreated with pravastatin and simvastatin in association with pyrimethamine and sulfadiazine at low concentrations could affect the infection processes, suggesting direct action on protozoa intracellular proliferation through the inhibition of isoprenoids in the parasite's apicoplast. METHODS: To have the adhesion, infection, and parasite proliferation during experimental infection investigated, HeLa cells (105) were subjected to a 24-hour infection by T. gondii tachyzoites forms of RH strain (5 × 105) pretreated for 30 min with pravastatin and/or simvastatin combined or not with pyrimethamine and sulfadiazine. RESULTS: Combined with conventional drugs at low concentrations pravastatin and simvastatin inhibit the adhesion, invasion, and intracellular proliferation of T. gondii in HeLa cells which are similar to the positive control. CONCLUSION: Pravastatin and simvastatin in association with pyrimethamine and sulfadiazine at low concentrations can be regarded as a promising, effective alternative to toxoplasmosis treatment with reduced side effects.

Pravastatin and simvastatin inhibit the adhesion, replication and proliferation of Toxoplasma gondii (RH strain) in HeLa cells.

The conventional treatment for toxoplasmosis with pyrimethamine and sulfadiazine shows toxic effects to the host, and it is therefore necessary to search for new drugs. Some studies suggest the use of statins, which inhibit cholesterol synthesis in humans and also the initial processes of isoprenoid biosynthesis in the parasite. Thus, the objective of this study was to evaluate the activity of the statins pravastatin and simvastatin in HeLa cells infected in vitro with the RH strain of T. gondii. HeLa cells (1×105) were infected with T. gondii tachyzoites (5×105) following two different treatment protocols. In the first protocol, T. gondii tachyzoites were pretreated with pravastatin (50 and 100µg/mL) and simvastatin (1.56 and 3.125µg/mL) for 30min prior to infection. In the second, HeLa cells were first infected (5×105) with tachyzoites and subsequently treated with pravastatin and simvastatin for 24h at the concentrations noted above. Initially, we evaluated the cytotoxicity of drugs by the MTT assay, number of tachyzoites adhered to cells, number of infected cells, and viability of tachyzoites by trypan blue exclusion. The supernatant of the cell cultures was collected post-treatment for determination of the pattern of Th1/Th2/Th17 cytokines by cytometric bead array. There was no cytotoxicity to HeLa cells with 50 and 100µg/mL pravastatin and 1.56 and 3.125µg/mL simvastatin. There was no change in the viability of tachyzoites that received pretreatment. Regarding the pre- and post-treatment of the cells with pravastatin and simvastatin alone, there was a reduction in adhesion, invasion and proliferation of cells to T. gondii. As for the production of cytokines, we found that IL-6 and IL-17 were significantly reduced in cells infected with T. gondii and treated with pravastatin and simvastatin, when compared to control. Based on these results, we can infer that pravastatin and simvastatin alone possess antiproliferative effects on tachyzoites forms of T. gondii, giving these drugs new therapeutic uses.

Perfil clínico de pacientes com diagnóstico de tuberculose atendidos no Hospital Universitário de Londrina, Paraná / Clinical profile of patients diagnosed with tuberculosis treated at the University Hospital of Londrina, Paraná

O objetivo deste estudo consistiu em analisar o perfil clínico de pacientes positivos para tuberculose através de um estudo epidemiológico, descritivo e retrospectivo, com base em dados secundários contidos em prontuários dos pacientes atendidos no Hospital Universitário de Londrina compreendendo de janeiro de 2010 a dezembro de 2014. Dos prontuários disponíveis para a análise no momento de estudo 86 casos eram positivos para tuberculose, sendo a maioria do sexo masculino (65/76%) com faixa etária compreendida entre 2 e 91 anos. Os principais sinais clínicos apresentados foram tosse (50/58%), febre (45/52%) e perda de peso (34/40%). Em relação à forma clinica, 58/67% dos pacientes apresentaram a forma pulmonar, e 28/33% a forma não pulmonar. Casos positivos para tuberculose em associação com HIV/AIDS corresponderam a 32/37%. Também foram relatados hábitos prejudiciais dos pacientes nos quais 30/34% eram tabagistas, 20/23% usuários de drogas e 14/16% etilistas. Diante da escassez de dados publicados referentes à tuberculose na nossa região e sua relevância para a saúde pública, nosso estudo contribui com os aspectos epidemiológicos principalmente em relação ao elevado número de casos de coinfecção com Vírus da Imunodeficiência Humana e pacientes com evolução a óbito, auxiliando assim, o desenvolvimento e implementação de campanhas ou projetos que visem o diagnóstico e tratamento precoce.

The objective of this study was to analyze the clinical profile of patients positive for tuberculosis through an epidemiological study, descriptive and retrospective, based on secondary data contained in medical records of patients that attended the Hospital Universitário de Londrina comprising from January 2010 to December 2014. From the medical records available for analysis at the moment of study 86 cases were positive for tuberculosis, being the majority male (65/76%) with age group comprised between 2 to 91 years. The main symptoms presented were cough (50/58%), fever (45/52%) and weight loss (34/40%). Regarding the clinical form, 58/67% of the patients presented the pulmonary form, and 28/33% the non-pulmonary form. Positive cases for tuberculosis in association with HIV/AIDS corresponded to 32/37%. Harmful habits were also reported in patients in which 30/34% were smokers, 20/23% drug users and 14/16% were alcoholics. Against scarcity of published data referring to tuberculosis in our region and its relevance to public health, our study contributes to the epidemiological aspects mainly in relation to the high number of cases of coinfection with Human Immunodeficiency Virus and patients with evolution to death, assisting the development and implementation of campaigns or projects aimed at early diagnosis and treatment.

Coproparasitological survey of intestinal parasites in the city of Londrina, Parana, Brazil: a retrospective analysis

Intestinal parasites are a major public health problem. It is important to inform and educate the public about these infections, especially where such data are scarce. This study aimed to determine the prevalence of intestinal parasites from the analysis of medical records of individuals of the city of Londrina. We analyzed 11,641 fecal reports from February 2009 to December 2012. Data were cataloged after the completion of parasitological testing of Hoffmann, Pons & Janer, Faust and Kato-Katz. From 11,641 reports, 19.1% were positive for intestinal parasites. Among those, 52.1% pertained to females and 47.9% to males, with predominance of positivity of 27.1% among children 0-10 years. For the regions studied, the northern region stood out with 35.4% of cases and prevalence of 6.8%. Among the pathogenic protozoa, reports of Giardia lamblia comprised 19.1% of positivity, while hookworms were the most frequent among helminths, comprising 7.8% of positive cases. It follows that poor conditions of basic sanitation contribute to the dissemination of these parasites. Early diagnosis is a determinant of successful treatment. Additionally, epidemiological data may be used to study the risk factors for transmission and may result in measures applicable to improving living conditions in the community