Evaluation of reference genes for gene expression analysis by real-time quantitative PCR (qPCR) in different tissues from mice infected by Ascaris suum.

Human ascariasis is the most prevalent helminth infection, affecting 445 million people worldwide. To better understand the impact of the immune system on the pathophysiology of individuals infected with Ascaris suum, mice have been used as experimental models. The RT-qPCR technique is a critical auxiliary tool of investigation used to quantify mRNA levels. However, proper normalization using reference genes is essential to ensure reliable outcomes to avoid analytical errors and false results. Despite the importance of reference genes for experimental A. suum infection studies, no specific reference genes have been identified yet. Therefore, we conducted a study to assess five potential reference genes (GAPDH, 18s, ACTB, B2M, and HPRT1) in different tissues (liver, lungs, small and large intestines) affected by A. suum larval migration in C57BL/6j mice. Tissue collection was carried out to analyze parasite burden and confirm the presence of larvae during the peak of migration in each tissue. Upon confirmation, we analyzed different genes in the tissues and found no common gene with stable expression. Our results highlight the importance of analyzing different genes and using different software programs to ensure reliable relative expression results. Based on our findings, B2M was ranked as the ideal reference gene for the liver, while 18S was the most stable gene in the lung and small intestine. ACTB, or a combination of ACTB with GAPDH, was deemed suitable as reference genes for the large intestine due to their stable expression and less variation between the control and infected groups. To further demonstrate the impact of using different reference genes, we normalized the expression of a chemokine gene (CXCL9) in all tissues. Significant differences in CXCL9 expression levels were observed between different groups in all tissues except for the large intestine. This underscores the importance of selecting appropriate reference genes to avoid overestimating target gene expression levels and encountering normalization-related issues that can lead to false results. In conclusion, our study highlights the significance of using reliable reference genes for accurate RT-qPCR analysis, especially in the context of A. suum infection studies in different tissues. Proper normalization is crucial to ensure the validity of gene expression data and avoid potential pitfalls in interpreting results.

The long-lasting Ascaris suum antigens in the lungs shapes the tissue adaptation modifying the pulmonary architecture and immune response after infection in mice.

Ascariasis is the most prevalent helminth affecting approximately 819 million people worldwide. The acute phase of Ascariasis is characterized by larval migration of Ascaris spp., through the intestinal wall, carried to the liver and lungs of the host by the circulatory system. Most of the larvae subsequently transverse the lung parenchyma leading to tissue injury, reaching the airways and pharynx, where they can be expectorated and swallowed back to the gastrointestinal tract, where they develop into adult worms. However, some larvae are trapped in the lung parenchyma inciting an inflammatory response that causes persistent pulmonary tissue damage long after the resolution of infection, which returns to tissue homeostasis. However, the mechanism by which chronic lung disease develops and resolves remains unknown. Here, using immunohistochemistry, we demonstrate that small fragments and larval antigens of Ascaris suum are deposited and retained chronically in the lung parenchyma of mice following a single Ascaris infection. Our results reveal that the prolonged presence of Ascaris larval antigens in the lung parenchyma contributes to the persistent immune stimulation inducing histopathological changes observed chronically following infection, and clearly demonstrate that larval antigens are related to all phases of tissue adaptation after infection: lung injury, chronic inflammation, resolution, and tissue remodeling, in parallel to increased specific humoral immunity and the recovery of lung function in mice. Additional insight is needed into the mechanisms of Ascaris antigen to induce chronic immune responses and resolution in the host lungs following larval migration.

Toxicological effects of the Morinda citrifolia L. fruit extract on maternal reproduction and fetal development in rats.

Morinda citrifolia L., also known as Noni, is widely used plant in folk medicine for various therapeutic purposes. However, reports on its effects during pregnancy are limited. Therefore, the objective of this study was to evaluate the effects of the M. citrifolia fruit extract on maternal performance and fetal development during pregnancy in rats. Pregnant Wistar rats (n = 12/group) were treated from gestational days (GD) 0-21 with water (control group) or the aqueous extract of M. citrifolia fruit at doses of 200, 400, or 750 mg/kg, orally. During pregnancy, clinical signs of toxicity, maternal weight, feed intake, and water consumption were noted. On GD 21, the rats were anesthetized and blood was collected to evaluate various biochemical parameters. During laparotomy, reproductive performance parameters were recorded, and fetuses were weighed and the anomalies analyzed. Reduced placental efficiency and fetal growth restriction were observed in the group treated with 400 mg/kg of M. citrifolia extract. The highest dose (750 mg/kg) augmented aspartate aminotransferase concentration and preimplantation losses, while reducing the number of live fetuses. Furthermore, both doses (400 and 750 mg/kg) of the plant extract caused fetal anomalies. In conclusion, consumption of high doses of the M. citrifolia aqueous extrac during pregnancy leads to maternal hepatotoxicity, anti-implantation effects, intrauterine growth restriction and fetal abnormalities, indicating that the plant fruit extract can be harmful to both the mother and the fetus.

IL-17RA receptor signaling contributes to lung inflammation and parasite burden during Toxocara canis infection in mice.

IL-17 is a cytokine produced by innate and acquired immunity cells that have an action against fungi and bacteria. However, its action in helminth infections is unclear, including in Toxocara canis infection. Toxocariasis is a neglected zoonosis representing a significant public health problem with an estimated seroprevalence of 19% worldwide. In the present study, we describe the immunopathological action of IL-17RA in acute T. canis infection. C57BL/6j (WT) and IL-17RA receptor knockout (IL-17RA-/-) mice were infected with 1000 T. canis eggs. Mice were evaluated 3 days post-infection for parasite load and white blood cell count. Lung tissue was harvested for histopathology and cytokine expression. In addition, we performed multiparametric flow cytometry in the BAL and peripheral blood, evaluating phenotypic and functional changes in myeloid and lymphoid populations. We showed that IL-17RA is essential to control larvae load in the lung; however, IL-17RA contributed to pulmonary inflammation, inducing inflammatory nodular aggregates formation and presented higher pulmonary IL-6 levels. The absence of IL-17RA was associated with a higher frequency of neutrophils as a source of IL-4 in BAL, while in the presence of IL-17RA, mice display a higher frequency of alveolar macrophages expressing the same cytokine. Taken together, this study indicates that neutrophils may be an important source of IL-4 in the lungs during T. canis infection. Furthermore, IL-17/IL-17RA axis is important to control parasite load, however, its presence triggers lung inflammation that can lead to tissue damage.

Phytochemical and antidiabetic analysis of Curatella americana L. aqueous extract on the rat pregnancy.

ETHNOPHARMACOLOGICAL RELEVANCE: Curatella americana L. is employed in popular medicine for treating diabetes. However, the understanding around its outcomes during pregnancy is unclear. AIM OF THE STUDY: To evaluate the phytochemical and hypoglycemic analysis of the C. americana extract and its maternal-fetal effect on diabetic rats. MATERIALS AND METHOD: Diabetes was chemically induced 24 h after birth in Wistar female newborn rats. At adulthood, after diabetes status confirmation, the rats were mated and randomized into four experimental groups: Nondiabetic (Control): given water; Treated: given C. americana extract; Diabetic, and Treated Diabetic rats. The aqueous extract of C. americana leaves (300 mg/kg) was administered daily through oral route during pregnancy. Maternal toxicity and biochemical profile, reproductive outcomes, fetal development, and phenolic composition and biogenic amines in aqueous extract were analyzed. RESULTS AND CONCLUSION: Phytochemical analysis revealed that the main phenolic components are 3-hydroxytyrosol, kaempferol, and quercetin, while tryptophan and putrescine derivatives were identified as the dominant amines. C. americana extract treatment improved the lipid profile, although no effect on hyperglycemic control in diabetic rats was observed. Maternal diabetes or C. americana extract caused embryo losses confirmed by the lower number of pre-embryos in early pregnancy and higher percentage of abnormal morphologically pre-embryos. C. americana extract previously caused premature pre-embryo fixation before implantation window in nondiabetic and diabetic mothers and intrauterine growth restriction in the fetuses of treated nondiabetic dams, complicating the embryo fetal development. These findings reinforce the caution of indiscriminate use of medicinal plants, especially during pregnancy.

Detrimental role of IL-33/ST2 pathway sustaining a chronic eosinophil-dependent Th2 inflammatory response, tissue damage and parasite burden during Toxocara canis infection in mice.

Toxocariasis is a neglected disease that affects people around the world. Humans become infected by accidental ingestion of eggs containing Toxocara canis infective larvae, which upon reaching the intestine, hatch, penetrate the mucosa and migrate to various tissues such as liver, lungs and brain. Studies have indicated that Th2 response is the main immune defense mechanism against toxocariasis, however, there are still few studies related to this response, mainly the IL-33/ST2 pathway. Some studies have reported an increase in IL-33 during helminth infections, including T. canis. By binding to its ST2 receptor, IL-33 stimulating the Th2 polarized immune cell and cytokine responses. Thus, we aimed to investigate the role of the IL-33/ST2 pathway in the context of T. canis larval migration and the immunological and pathophysiological aspects of the infection in the liver, lungs and brain from Wild-Type (WT) BALB/c background and genetically deficient mice for the ST2 receptor (ST2-/-). The most important findings revealed that the IL-33/ST2 pathway is involved in eosinophilia, hepatic and cerebral parasitic burden, and induces the formation of granulomas related to tissue damage and pulmonary dysfunction. However, ST2-/- mice, the immune response was skewed to Th1/Th17 type than Th2, that enhanced the control of parasite burden related to IgG2a levels, tissue macrophages infiltration and reduced lung dysfunction. Collectively, our results demonstrate that the Th2 immune response triggered by IL-33/ST2 pathway mediates susceptibility to T. canis, related to parasitic burden, eosinophilia and granuloma formation in which consequently contributes to tissue inflammation and injury.

Concomitant experimental coinfection by Plasmodium berghei NK65-NY and Ascaris suum downregulates the Ascaris-specific immune response and potentiates Ascaris-associated lung pathology.

BACKGROUND: Ascariasis and malaria are highly prevalent parasitic diseases in tropical regions and often have overlapping endemic areas, contributing to high morbidity and mortality rates in areas with poor sanitary conditions. Several studies have previously aimed to correlate the effects of Ascaris-Plasmodium coinfections but have obtained contradictory and inconclusive results. Therefore, the present study aimed to investigate parasitological and immunopathological aspects of the lung during murine experimental concomitant coinfection by Plasmodium berghei and Ascaris suum during larvae ascariasis. METHODS: C57BL/6J mice were inoculated with 1 × 104 P. berghei strain NK65-NY-infected red blood cells (iRBCs) intraperitoneally and/or 2500 embryonated eggs of A. suum by oral gavage. P. berghei parasitaemia, morbidity and the survival rate were assessed. On the seventh day postinfection (dpi), A. suum lung burden analysis; bronchoalveolar lavage (BAL); histopathology; NAG, MPO and EPO activity measurements; haematological analysis; and respiratory mechanics analysis were performed. The concentrations of interleukin (IL)-1ß, IL-12/IL-23p40, IL-6, IL-4, IL-33, IL-13, IL-5, IL-10, IL-17A, IFN-γ, TNF and TGF-ß were assayed by sandwich ELISA. RESULTS: Animals coinfected with P. berghei and A. suum show decreased production of type 1, 2, and 17 and regulatory cytokines; low leukocyte recruitment in the tissue; increased cellularity in the circulation; and low levels of NAG, MPO and EPO activity that lead to an increase in larvae migration, as shown by the decrease in larvae recovered in the lung parenchyma and increase in larvae recovered in the airway. This situation leads to severe airway haemorrhage and, consequently, an impairment respiratory function that leads to high morbidity and early mortality. CONCLUSIONS: This study demonstrates that the Ascaris-Plasmodium interaction is harmful to the host and suggests that this coinfection may potentiate Ascaris-associated pathology by dampening the Ascaris-specific immune response, resulting in the early death of affected animals.

A treatment with a boiled aqueous extract of Hancornia speciosa Gomes leaves improves the metabolic status of streptozotocin-induced diabetic rats.

BACKGROUND: Hancornia speciosa is usually used in Brazilian folk medicine to treat diabetes. The hypothesis of the present study is that this medicinal plant exerts beneficial effects on hyperglycemia, preventing diabetic complications. Therefore, the aim of this study was to evaluate the treatment effect of the aqueous extract of H. speciosa leaves on metabolic parameters of diabetic rats. METHODS: The H. speciosa extract (400 mg/Kg) was administered to both nondiabetic and severely diabetic female Wistar rats by gavage. The Oral Glucose Tolerance Test was performed and the area under the curve (AUC) was estimated on day 17 of pregnancy. After 21 days of treatment, the animals were anesthetized and killed to obtain organ weights. Blood samples were collected for an analysis of serum biochemical parameters. RESULTS: After treatment with the H. speciosa extract, the parameters of nondiabetic rats remained unchanged. In treated diabetic rats, glycemia, AUC, dyslipidemia parameters, and relative organ weights were decreased compared with nontreated diabetic rats. Severely diabetic rats showed decompensated hyperglycemia, polydipsia, hyperphagia and dyslipidemia. However, the aqueous extract of H. speciosa leaves decreased diabetes complications (indicating a lack of toxicity), reduced blood glucose levels, and exerced lipid-lowering effects. CONCLUSION: Based on or findings, the H. speciosa leaf extract may be a safe and promising candidate treatment for diabetes and other diseases.

Beneficial effects of Hibiscus rosa-sinensis L. flower aqueous extract in pregnant rats with diabetes.

PURPOSE: The Hibiscus rosa-sinensis flower is widely used in Brazilian traditional medicine for the treatment of diabetes and has shown antifertility activity in female Wistar rats. However, there is no scientific confirmation of its effect on diabetes and pregnancy. The aim of this study was evaluate the effect of aqueous extract of H. rosa-sinensis flowers on maternal-fetal outcome in pregnant rats with diabetes. METHODS: Diabetes was induced by streptozotocin (STZ, 40 mg/kg) in virgin, adult, female Wistar rats. After diabetes induction, the rats were mated. The pregnant rats were distributed into four groups (n minimum = 11 animals/group): non-diabetic, non-diabetic treated, diabetic, and diabetic treated. Oral aqueous extract of Hibiscus rosa-sinensis was administered to rats in the treatment groups during pregnancy. At term pregnancy, maternal reproductive outcomes, fetal parameters, and biochemical parameters were analyzed. RESULTS: The non-diabetic treated group showed decreased high density lipoprotein cholesterol, increased atherogenic index (AI) and coronary artery risk index (CRI), and increased preimplantation loss rate compared to the non-diabetic group. Although treatment with H. rosa-sinensis led to no toxicity, it showed deleterious effects on cardiac and reproductive functions. However, the diabetic treated group showed increased maternal and fetal weights, reduced AI and CRI, and reduced preimplantation loss rate compared to the untreated diabetic group. CONCLUSION: Our results demonstrate beneficial effects of this flower only in pregnant rats with diabetes and their offspring. Although these findings cannot be extrapolated to human clinical use, they show that the indiscriminate intake of H. rosa-sinensis may be harmful to healthy individuals and its use should be completely avoided in pregnancy.

Medicinal Plants for Diabetes Treatment During Pregnancy.

Diabetes mellitus is a syndrome of great importance that affects an increasing number of people every day. In particular, diabetes is a common and important disease during pregnancy and is marked by complications, both fetal and maternal, that increase the risks of morbidity and mortality for diabetic pregnant women and their offspring. Drugs such as insulin and hypoglycemic drugs are given to treat diabetes, but regular exercise and adequate diet have also been indicated. Furthermore, coadjutant therapies such as medicinal plants are popularly used to reduce diabetes-induced hyperglycemia, either within or outside the context of pregnancy. However, studies examining plant use for diabetes treatment are necessary to confirm its possible effects and its safety for the mother and fetus. The objective of this literature review was to conduct a survey of plant species that are utilized worldwide and their stated therapeutic uses. A literature search was performed using the terms "diabetes and pregnancy", which resulted in the identification of 31,272 articles. Of these studies, only 12 (0.0038%) were related to medicinal plants, demonstrating that there has been little investigation into this issue. Of the papers analyzed in this review, half evaluated plant leaves, indicating that these scientific studies attempted to reproduce the preparations commonly used by various populations, i.e., in the form of tea. Additionally, more than 90% of studies utilized experimental animals to evaluate the maternal-fetal safety of medicinal plant substances that may potentially be dangerous for humans. Thus, once confidence levels for plant-derived substances are established based on toxicological analyses and safety is confirmed, it is possible that plants will be used to complement conventional diabetes therapies.