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.

Genetic diversity of Toxoplasma gondii in South America: occurrence, immunity, and fate of infection.

Toxoplasma gondii is an intracellular parasite with a worldwide distribution. Toxoplasma gondii infections are of great concern for public health, and their impact is usually most severe in pregnant women and their foetuses, and in immunocompromised individuals. Displaying considerable genetic diversity, T. gondii strains differ widely according to geographical location, with archetypal strains predominantly found in the Northern Hemisphere and non-archetypal (atypical) strains, with highly diverse genotypes, found mainly in South America. In this review, we present an overview of the identification and distribution of non-archetypal strains of T. gondii. Special attention is paid to the strains that have been isolated in Brazil, their interaction with the host immunological response, and their impact on disease outcomes. The genetic differences among the strains are pivotal to the distinct immunological responses that they elicit. These differences arise from polymorphisms of key proteins released by the parasite, which represent important virulence factors. Infection with divergent non-archetypal strains can lead to unusual manifestations of the disease, even in immunocompetent individuals.

Immunogenic mapping of rDyn-1 and rKDDR-plus proteins and selection of oligopeptides by immunoblotting for the diagnosis of Leishmania infantum-infected dogs.

Endemic in Brazil, visceral leishmaniasis (VL) is a zoonotic infection that is among the most important parasitic diseases transmitted by vectors. Dogs are the main reservoirs of canine leishmaniasis (CanL) and their identification is used in some countries as part of disease prevention and control measures in the canine and human population. In this context, serological tests are necessary, composed of antigens capable of correctly identifying infected dogs, minimizing the number of false-negative cases. This study aimed to identify more immunoreactive peptides derived from two previously described whole proteins (rDyn-1 and rKDDR-plus) and compare their performance to the control antigens rK39 and the crude extract for the detection of dogs infected with L. infantum, especially the asymptomatic ones. The three selected peptides and a mixture of them, along with the rDyn-1, rKDDR-plus, rK39, and crude extract antigens were evaluated using indirect ELISA with sera samples from 186 dogs with CanL, being asymptomatic (n = 50), symptomatic (n = 50), co-infected (n = 19), infected with Babesia sp. (n = 7), Ehrlichia sp. (n = 6), T. cruzi (n = 20) and uninfected (n = 34). The results showed that the rDyn-1 protein and the peptide mixture had the highest sensitivity (100% and 98.32%, respectively) and specificity (97.01 and 98.51, respectively). A high degree of kappa agreement was found for rDyn-1 protein (0.977), mixed peptides (0.965), rKDDR-plus protein (0.953), K-plus peptide 1 (0.930) and Dyn-1 peptide (0.893). The mixture of peptides showed the highest likelihood (65.87). The ELISA using the mixture of peptides and the rDyn-1 protein showed high performance for CanL serodiagnosis. More mix combinations of the peptides and additional extended field tests with a larger sample size are recommended.

Chronic infection by atypical Toxoplasma gondii strain induces disturbance in microglia population and altered behaviour in mice.

Toxoplasma gondii chronic infection is characterized by the establishment of tissue cysts in the brain and increased levels of IFN-γ, which can lead to brain circuitry interference and consequently abnormal behaviour in mice. In this sense, the study presented here sought to investigate the impact of chronic infection by two T. gondii strains in the brain of infection-resistant mice, as a model for studying the involvement of chronic neuroinflammation with the development of behavioural alterations. For that, male BALB/c mice were divided into three groups: non-infected (Ni), infected with T. gondii ME49 clonal strain (ME49), and infected with TgCkBrRN2 atypical strain (CK2). Mice were monitored for 60 days to establish the chronic infection and then submitted to behavioural assessment. The enzyme-linked immunosorbent assay was used for measurement of specific IgG in the blood and levels of inflammatory cytokines and neurotrophic factors in the brain, and the cell's immunophenotype was determined by multiparametric flow cytometry. Mice infected with ME49 clonal strain displayed hyperlocomotor activity and memory deficit, although no signs of depressive- and/or anxiety-like behaviour were detected; on the other hand, chronic infection with CK2 atypical strain induced anxiety- and depressive-like behaviour. During chronic infection by CK2 atypical strain, mice displayed a higher number of T. gondii brain tissue cysts and inflammatory infiltrate, composed mainly of CD3+ T lymphocytes and Ly6Chi inflammatory monocytes, compared to mice infected with the ME49 clonal strain. Infected mice presented a marked decrease of microglia population compared to non-infected group. Chronic infection with CK2 strain produced elevated levels of IFN-γ and TNF-ɑ in the brain, decreased NGF levels in the prefrontal cortex and striatum, and altered levels of fractalkine (CX3CL1) in the prefrontal cortex and hippocampus. The persistent inflammation and the disturbance in the cerebral homeostasis may contribute to altered behaviour in mice, as the levels of IFN-γ were shown to be correlated with the behavioural parameters assessed here. Considering the high incidence and life-long persistence of T. gondii infection, this approach can be considered a suitable model for studying the impact of chronic infections in the brain and how it impacts in behavioural responses.

Development of chimeric protein as a multivalent vaccine for human Kinetoplastid infections: Chagas disease and leishmaniasis.

Leishmania spp. and Trypanosoma cruzi are parasitic kinetoplastids of great medical and epidemiological importance since they are responsible for thousands of deaths and disability-adjusted life-years annually, especially in low- and middle-income countries. Despite efforts to minimize their impact, current prevention measures have failed to fully control their spread. There are still no vaccines available. Taking into account the genetic similarity within the Class Kinetoplastida, we selected CD8+ T cell epitopes preserved among Leishmania spp. and T. cruzi to construct a multivalent and broad-spectrum chimeric polyprotein vaccine. In addition to inducing specific IgG production, immunization with the vaccine was able to significantly reduce parasite burden in the colon, liver and skin lesions from T. cruzi, L. infantum and L. mexicana challenged mice, respectively. These findings were supported by histopathological analysis, which revealed decreased inflammation in the colon, a reduced number of degenerated hepatocytes and an increased proliferation of connective tissue in the skin lesions of the corresponding T. cruzi, L. infantum and L. mexicana vaccinated and challenged mice. Collectively, our results support the protective effect of a polyprotein vaccine approach and further studies will elucidate the immune profile associated with this protection. Noteworthy, our results act as conceptual proof that a single multi-kinetoplastida vaccine can be used effectively to control different infectious etiologies, which in turn can have a profound impact on the development of a new generation of vaccines.

Biological activity of 1,2,3-triazole-2-amino-1,4-naphthoquinone derivatives and their evaluation as therapeutic strategy for malaria control.

Malaria can be caused by several Plasmodium species and the development of an effective vaccine is challenging. Currently, the most effective tool to control the disease is the administration of specific chemotherapy; however, resistance to the frontline antimalarials is one of the major problems in malaria control and thus the development of new drugs becomes urgent. The study presented here sought to evaluate the antimalarial activities of compounds derived from 2-amino-1,4-naphthoquinones containing 1,2,3-triazole using in vivo and in vitro models. 1H-1,2,3-Triazole 2-amino-1,4-naphthoquinone derivatives were synthesized and evaluated for antimalarial activity in vitro, using P. falciparum W2 chloroquine (CQ) resistant strain and in vivo using the murine-P. berghei ANKA strain. Acute toxicity was determined as established by the OECD (2001). Cytotoxicity was evaluated against HepG2 and Vero mammalian cell lines. Transmission electron microscopy of the Plasmodium falciparum trophozoite (early and late stages) was used to evaluate the action of compounds derived at ultra-structural level. The compounds displayed low cytotoxicity CC50 > 100 µM, neither did they cause hemolysis at the tested doses and nor the signs of toxicity in the in vivo acute toxicity test. Among the five compounds tested, one showed IC50 values in submicromolar range of 0.8 µM. Compounds 7, 8 and 11 showed IC50 values < 5 µM, and selectivity index (SI) ranging from 6.8 to 343 for HepG2, and from 13.7 to 494.8 for Vero cells. Compounds 8 and 11 were partially active against P. berghei induced parasitemia in vivo. Analysis of the ultrastructural changes associated with the treatment of these two compounds, showed trophozoites with completely degraded cytoplasm, loss of membrane integrity, organelles in the decomposition stage and possible food vacuole deterioration. Our results indicated that compounds 8 and 11 may be considered hit molecules for antimalarial drug discovery platform and deserve further optimization studies.

Serodiagnosis of leishmaniasis in asymptomatic and symptomatic dogs by use of the recombinant dynamin-1-like protein from Leishmania infantum: A preliminary study.

Visceral leishmaniasis (VL) is a fatal manifestation of an infection caused by intracellular protozoa of the Leishmania genus. In New World countries, VL is classified as a zoonotic disease with domestic dogs acting as its main reservoir. Asymptomatic dogs are as competent to transmit Leishmania to the vectors as symptomatic dogs, however current diagnostic tests are limited and present low sensitivity for this important group. The development of accurate tests is fundamental to the early diagnosis, treatment, and control of canine leishmaniasis. In this study, we investigated the use of a recombinant protein (dynamin-1-like protein, Dyn-1) from L. infantum, as a potential target antigen for leishmaniasis serodiagnosis in both symptomatic and asymptomatic dogs. The antigenic performance of the protein was evaluated by means of ELISA assays using sera from symptomatic (n = 25), asymptomatic (n = 34) and non-infected dogs (n = 36) using ELISA. In addition, sera from dogs experimentally infected with Trypanosoma cruzi (n = 49) and naturally infected with Babesia sp. (n = 8) were tested to evaluate possible cross-reactivity. A crude soluble antigen (CSA) of Leishmania was used as an antigen control and K39 and K26 were used as reference antigens because they are already widely used in commercial tests. rDyn-1-based assay showed the highest sensitivity (97%) compared to the antigens K39 (88%), K26 (86%) and crude extract (95%). The highest specificity among the tests was also obtained with the protein rDyn-1 (94%), compared with the other antigens K39 (81%), K26 (87%), and crude extract (77%). This study showed that the rDyn-1 ELISA assay was able to identify 100% of asymptomatic dogs, establishing its potential as a target for the diagnosis of canine leishmaniasis.

Vaccination with Formulation of Nanoparticles Loaded with Leishmania amazonensis Antigens Confers Protection against Experimental Visceral Leishmaniasis in Hamster

Visceral leishmaniasis (VL) is a fatal disease caused by the protozoa Leishmania infantum for which dogs are the main reservoirs. A vaccine against canine visceral leishmaniasis (CVL) could be an important tool in the control of human and CVL by reducing the infection pressure of L. infantum. Despite the CVL vaccine available on the market, the Brazilian Ministry of Health did not implement the use of it in their control programs. In this sense, there is an urgent need to develop more efficient vaccines. In this study, the association between two polymeric nanoformulations, (poly (D, L-lactic) acid (PLA) polymer) loading Leishmania amazonensis antigens, was evaluated as a potential immunobiological agent against VL using golden hamsters as an experimental model. The results indicated that no significant adverse reactions were observed in animals vaccinated with LAPSmP. LAPSmP presented similar levels of total anti-Leishmania IgG as compared to LAPSmG. The LAPSmP and LAPSmG groups showed an intense reduction in liver and spleen parasitic load by qPCR. The LAPSmP and LAPSmG vaccines showed exceptional results, indicating that they may be promising candidates as a VL vaccine.

Nanoformulations with Leishmania braziliensis Antigens Triggered Controlled Parasite Burden in Vaccinated Golden Hamster (Mesocricetus auratus) against Visceral Leishmaniasis.

Leishmaniasis is a widespread vector-borne disease in Brazil, with Leishmania (Leishmania) infantum as the primary etiological agent of visceral leishmaniasis (VL). Dogs are considered the main reservoir of this parasite, whose treatment in Brazil is restricted to the use of veterinary medicines, which do not promote a parasitological cure. Therefore, efficient vaccine development is the best approach to Canine Visceral Leishmaniasis (CVL) control. With this in mind, this study used hamsters (Mesocricetus auratus) as an experimental model in an anti-Leishmania preclinical vaccine trial to evaluate the safety, antigenicity, humoral response, and effects on tissue parasite load. Two novel formulations of nanoparticles made from poly(D, L-lactic) acid (PLA) polymer loading Leishmania braziliensis crude antigen (LB) exhibiting two different particle sizes were utilized: LBPSmG (570 nm) and LBPSmP (388 nm). The results showed that the nanoparticles were safe and harmless to hamsters and were antigenic with the induction in LBSap, LBPSmG, and LBPSmG groups of total anti-Leishmania IgG antibodies 30 days after challenge, which persists 200 days in LBSap and LBPSmP. At the same time, a less pronounced hepatosplenomegaly in LBSap, LBPSmG, and LBPSmP was found when compared to control groups, as well as a less pronounced inflammatory infiltrate and granuloma formation in the spleen. Furthermore, significant reductions of 84%, 81%, and 90% were observed in spleen parasite burden accessed by qPCR in the LBSap, LBPSmG, and LBPSmP groups, respectively. In this way, LBSap, LBPSmG, and LBPSmP formulations showed better results in vaccinated and L. infantum-challenged animals in further reducing parasitic load in the spleen and attenuating lesions in liver and splenic tissues. This results in safe, harmless nanoformulation vaccines with significant immunogenic and infection control potential. In addition, animals vaccinated with LBPSmP had an overall reduction in parasite burden in the spleen, indicating that a smaller nanoparticle could be more efficient in targeting antigen-presenting cells.

Accessing the Variability of Multicopy Genes in Complex Genomes using Unassembled Next-Generation Sequencing Reads: The Case of Trypanosoma cruzi Multigene Families.

Repetitive elements cause assembly fragmentation in complex eukaryotic genomes, limiting the study of their variability. The genome of Trypanosoma cruzi, the parasite that causes Chagas disease, has a high repetitive content, including multigene families. Although many T. cruzi multigene families encode surface proteins that play pivotal roles in host-parasite interactions, their variability is currently underestimated, as their high repetitive content results in collapsed gene variants. To estimate sequence variability and copy number variation of multigene families, we developed a read-based approach that is independent of gene-specific read mapping and de novo assembly. This methodology was used to estimate the copy number and variability of MASP, TcMUC, and Trans-Sialidase (TS), the three largest T. cruzi multigene families, in 36 strains, including members of all six parasite discrete typing units (DTUs). We found that these three families present a specific pattern of variability and copy number among the distinct parasite DTUs. Inter-DTU hybrid strains presented a higher variability of these families, suggesting that maintaining a larger content of their members could be advantageous. In addition, in a chronic murine model and chronic Chagasic human patients, the immune response was focused on TS antigens, suggesting that targeting TS conserved sequences could be a potential avenue to improve diagnosis and vaccine design against Chagas disease. Finally, the proposed approach can be applied to study multicopy genes in any organism, opening new avenues to access sequence variability in complex genomes. IMPORTANCE Sequences that have several copies in a genome, such as multicopy-gene families, mobile elements, and microsatellites, are among the most challenging genomic segments to study. They are frequently underestimated in genome assemblies, hampering the correct assessment of these important players in genome evolution and adaptation. Here, we developed a new methodology to estimate variability and copy numbers of repetitive genomic regions and employed it to characterize the T. cruzi multigene families MASP, TcMUC, and transsialidase (TS), which are important virulence factors in this parasite. We showed that multigene families vary in sequence and content among the parasite's lineages, whereas hybrid strains have a higher sequence variability that could be advantageous to the parasite's survivability. By identifying conserved sequences within multigene families, we showed that the mammalian host immune response toward these multigene families is usually focused on the TS multigene family. These TS conserved and immunogenic peptides can be explored in future works as diagnostic targets or vaccine candidates for Chagas disease. Finally, this methodology can be easily applied to any organism of interest, which will aid in our understanding of complex genomic regions.

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.

Nitric oxide contributes to liver inflammation and parasitic burden control in Ascaris suum infection.

BACKGROUND: Human ascariasis is one of the most prevalent neglected tropical diseases worldwide. The immune response during human ascariasis is characterized by Th2 polarization and a mixed Th2/Th17 response during the pathogenesis of experimental larval ascariasis. Cytokines and other pro-inflammatory mediators, such as nitric oxide (NO), are involved in helminthic infections. However, the role of NO in ascariasis remains unclear. OBJECTIVES: Given the importance of NO in inflammation, we aimed to determine the immunological and histopathological alterations in the livers of C57BL/6 iNOS-/- mice during A. suum infection. METHODS: In this study, parasitic load was evaluated in the livers of wild type C57BL/6 and C57BL/6 iNOS-/- mice infected with A. suum. Histopathological and morphometric analyses and analysis of serum cytokines via Cytometric Bead Array were performed, and the activity of eosinophil peroxidase and myeloperoxidase of neutrophils in the tissues were determined. RESULTS: The results showed that NO is important for controlling parasitic load during infection by A. suum. C57BL/6iNOS-/- mice showed reduced inflammatory processes and less tissue damage during liver larval migration of A. suum, which is associated with a reduction in serum levels of pro-inflammatory cytokines. CONCLUSIONS: We demonstrated that NO is a crucial inflammatory molecule during Ascaris sp. infection and controls the establishment of the parasite and the development of the host immune response in the liver.

Reduced vitamin D receptor (VDR) and cathelicidin antimicrobial peptide (CAMP) gene expression contribute to the maintenance of inflammatory immune response in leprosy patients.

Leprosy is an infectious disease influenced by genetic, immunological, and environmental factors. Reduced gene expressions may be associated with the immunological response pattern and leprosy susceptibility. We investigated the direct and indirect effects of Vitamin D Receptor (VDR) and Cathelicidin Antimicrobial Peptide (CAMP) gene expressions on the serum levels of vitamin D, Cathelicidin, and cytokines in newly-diagnosed leprosy patients and post-six-months of multidrug therapy (MDT). Thirty-four leprosy patients were assessed, paucibacillary (PB; n = 14) and multibacillary (MB; n = 20) cases, untreated or having received six months of MDT, 18 healthy controls, and 25 household contacts. VDR and CAMP gene expression levels were strongly correlated to some important cytokines in both, untreated leprosy patients (PB, r = 0.9319; MB, r = 0.9569) and patients who had undergone MDT (PB, r = 0.9667; MB, r = 0.9569). We observed that both gene expressions directly influenced IL-2, IFN-γ, and IL-17F serum levels in leprosy patients compared to the household contacts and healthy individuals. VDR and CAMP gene expressions induced a persistent inflammatory response in PB and MB leprosy patients, even after six months of MDT, to fight the Mycobacterium leprae infection. Due to the persistent inflammatory profile, multidrug therapy is suggested to be maintained for more than six months, especially for MB patients. Vitamin D supplementation is recommended from the onset as a transcription factor to improve VDR and CAMP gene expression in leprosy patients.

Genetic Background Affects the Mucosal Secretory IgA Levels, Parasite Burden, Lung Inflammation, and Mouse Susceptibility to Ascaris suum Infection.

Ascariasis is a neglected tropical disease that is widespread in the world and has important socioeconomic impacts. The presence of various stages of worm development in the pulmonary and intestinal mucosae induces a humoral and cellular immune response. However, although there is much evidence of the protective role of mucosal immunity against various pathogens, including helminths, there is still a gap in the knowledge about the immune response and the mechanisms of action that are involved in protection against diseases, especially in the initial phase of ascariasis. Thus, the aim of this study was to evaluate the kinetic aspects of the immune parasitological parameters in intestinal and pulmonary mucosae in male mice with early ascariasis. Therefore, two mouse strains that showed different susceptibilities to ascariasis (BALB/c and C57BL/6J) when experimentally infected with 2,500 infective eggs of Ascaris suum from time point 0 were examined: the immune parasitological parameters were evaluated each 2 days after infection over a period of 12 days. The results were suggestive of a synergetic action of intestinal and pulmonary secretory IgA (S-IgA) contributing to protection against early ascariasis by reducing the amount of migrating larvae as well as the influx of leukocytes in the lung and the consequent impairment of pulmonary capacity.

Eosinophils mediate SIgA production triggered by TLR2 and TLR4 to control Ascaris suum infection in mice.

Human ascariasis is the most prevalent but neglected tropical disease in the world, affecting approximately 450 million people. The initial phase of Ascaris infection is marked by larval migration from the host's organs, causing mechanical injuries followed by an intense local inflammatory response, which is characterized mainly by neutrophil and eosinophil infiltration, especially in the lungs. During the pulmonary phase, the lesions induced by larval migration and excessive immune responses contribute to tissue remodeling marked by fibrosis and lung dysfunction. In this study, we investigated the relationship between SIgA levels and eosinophils. We found that TLR2 and TLR4 signaling induces eosinophils and promotes SIgA production during Ascaris suum infection. Therefore, control of parasite burden during the pulmonary phase of ascariasis involves eosinophil influx and subsequent promotion of SIgA levels. In addition, we also demonstrate that eosinophils also participate in the process of tissue remodeling after lung injury caused by larval migration, contributing to pulmonary fibrosis and dysfunction in re-infected mice. In conclusion, we postulate that eosinophils play a central role in mediating host innate and humoral immune responses by controlling parasite burden, tissue inflammation, and remodeling during Ascaris suum infection. Furthermore, we suggest that the use of probiotics can induce eosinophilia and SIgA production and contribute to controlling parasite burden and morbidity of helminthic diseases with pulmonary cycles.

The increased presence of repetitive motifs in the KDDR-plus recombinant protein, a kinesin-derived antigen from Leishmania infantum, improves the diagnostic performance of serological tests for human and canine visceral leishmaniasis.

Visceral leishmaniasis (VL) is caused by protozoa belonging to the Leishmania donovani complex and is considered the most serious and fatal form among the different types of leishmaniasis, if not early diagnosed and treated. Among the measures of disease control stand out the management of infected dogs and the early diagnosis and appropriate treatment of human cases. Several antigens have been characterized for use in the VL diagnosis, among them are the recombinant kinesin-derived antigens from L. infantum, as rK39 and rKDDR. The main difference between these antigens is the size of the non-repetitive kinesin region and the number of repetitions of the 39 amino acid degenerate motif (6.5 and 8.5 repeats in rK39 and rKDDR, respectively). This repetitive region has a high antigenicity score. To evaluate the effect of increasing the number of repeats on diagnostic performance, we designed the rKDDR-plus antigen, containing 15.3 repeats of the 39 amino acid degenerate motif, besides the absence of the non-repetitive portion from L. infantum kinesin. Its performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and rapid immunochromatographic test (ICT), and compared with the kinesin-derived antigens (rKDDR and rK39). In ELISA with human sera, all recombinant antigens had a sensitivity of 98%, whereas the specificity for rKDDR-plus, rKDDR and rK39 was 100%, 96% and 71%, respectively. When evaluated canine sera, the ELISA sensitivity was 97% for all antigens, and the specificity for rKDDR-plus, rKDDR and rK39 was 98%, 91% and 83%, respectively. Evaluation of the ICT/rKDDR-plus, using human sera, showed greater diagnostic sensitivity (90%) and specificity (100%), when compared to the IT LEISH (79% and 98%, respectively), which is based on the rK39 antigen. These results suggest that the increased presence of repetitive motifs in the rKDDR-plus protein improves the diagnostic performance of serological tests by increasing the specificity and accuracy of the diagnosis.

T follicular helper cells: Their development and importance in the context of helminthiasis.

The development of T follicular helper cells (Tfh) is a multifactorial process that occurs in multiple stages. After their activation the Tfh cells interact with the B cells to complete their differentiation. During this process, the Tfh cells begin to express canonical molecules such as the transcription factor B-cell lymphoma 6 protein, the CXC chemokine receptors type 5, and the inducible T-cell costimulator, as well as secreting other molecules such as IL-21. This whole process is regulated positively and negatively by several factors so that the best response is offered in the face of diseases of various origins, among them helminthiasis. In this context, the role of circulating Tfh, IL-4 and IgG subtypes is essential for an effective response against these pathogens. In this review, the migration process and the differentiation of Tfh, the regulation, their cell subtypes and the role of Tfh in the context of helminth infections will be addressed.

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.