Adjuvant ArtinM favored the host immunity against Cryptococcus gattii infection in C57BL/6 mice.

Aim: Cryptococcus gattii causes a severe fungal infection with high mortality rate among immunosuppressed and immunocompetent individuals. Due to limitation of current antifungal treatment, new immunotherapeutic approaches are explored. Methods: This study investigated an immunization strategy utilizing heat-inactivated C. gattii with ArtinM as an adjuvant. C57BL/6 mice were intranasally immunized with heat-killed C. gattii and ArtinM was administrated either before immunization or along with HK-C. gattii. Mice were infected with C. gattii and the efficacy of the immunization protocol was evaluated. Results: Mice that received ArtinM exhibited increased levels of IL-10 and relative expression of IL-23 in the lungs, reduced fungal burden and preserved tissue integrity post-infection. Conclusion: Adjuvant ArtinM improved immunization against C. gattii infection in C57BL/6 mice.

Cryptococcus gattii is a fungus that can make lungs sick. Right now, there are no good treatments for it, so scientists are trying to find new ways to fight it. In a recent study, they tested a type of immunotherapy called ArtinM to see if it could help. When they gave ArtinM to mice, the mice got healthier and had less fungus in their lungs. This means ArtinM might be able to help fight this fungus.

Eventos gerenciais e assistenciais como interferências na carga de trabalho / Managerial and assistance events such as workload interference / Eventos gerenciales y de asistencia como interferencia en la carga de trabajo

Objetivo: Analisar eventos no processo de trabalho gerencial e assistencial da enfermagem, como interferências na carga de trabalho de enfermagem em Unidade de Terapia Intensiva. Métodos: Estudo epidemiológico, transversal, de série histórica na análise da carga de trabalho. Foram realizadas aferições do Nursing Activities Score entre 2007 a 2014. Os eventos foram divididos em ações gerenciais e assistenciais. Resultados: O Nursing Activities Score foi aplicado 35262 vezes em 4731 pacientes. Entre as intervenções gerenciais, a carga de trabalho foi maior na vigência da Resolução da Diretoria Colegiada nº. 26 da Agência Nacional de Vigilância Sanitária concomitante ao aumento da proporção de pacientes por enfermeiro (p<0,001), na implantação dos sítios assistenciais (p<0,001) e na expansão da unidade (p<0,001). Entre as intervenções assistenciais, houve aumento da carga de trabalho no período de incorporação tecnológica (p<0,001), credenciamento de transplante hepático (p<0,001) e implantação de bundle de controle de infecção (p<0,001). Conclusão: Os eventos no processo de trabalho gerencial e assistencial da enfermagem que tiveram como objetivo melhorar a qualidade da assistência, constituem fatores que interferem na carga de trabalho da enfermagem em UTI, uma vez que esta tem aumentado ao longo dos anos. (AU)

Objective: To analyze events in the managerial and care work process, such as interferences in the nursing workload in the Intensive Care Unit. Methods: Epidemiological, cross-sectional, historical series study on workload analysis. Measurements of the Nursing Activities Score were carried out between 2007 and 2014. The events were divided into managerial and assistance actions. Results: The Nursing Activities Score was applied 35262 times to 4731 patients. Among management interventions, the workload was greater under the Directors' Collegiate Resolution no. 26 of the National Health Surveillance Agency concomitant to the increase in the proportion of patients per nurse (p<0.001), in the implantation of assistance sites (p<0.001) and in the expansion of the unit (p<0.001). Among assistance interventions, there was an increase in the workload in the period of technological incorporation (p<0.001), accreditation of liver transplantation (p<0.001) and implantation of an infection control bundle (p<0.001). Conclusion: Interventions that aimed to improve the quality of care are factors that interfere with the nursing workload in the ICU, whereas it has increased over the years. (AU)

Objetivo: Analizar eventos en el proceso de trabajo gerencial y asistencial, como interferencias en la carga de trabajo de enfermería en la Unidad de Cuidados Intensivos. Métodos: Estudio epidemiológico, transversal, de serie histórica sobre análisis de carga de trabajo. Las mediciones del Puntaje de Actividades de Enfermería se realizaron entre 2007 y 2014. Los eventos se dividieron en acciones gerenciales y asistenciales. Resultados: El puntaje de actividades de enfermería se aplicó 35262 veces a 4731 pacientes. Entre las intervenciones de gestión, la carga de trabajo fue mayor bajo la Resolución del Consejo Colegiado no. 26 de la Agencia Nacional de Vigilancia Sanitaria concomitante al aumento de la proporción de pacientes por enfermera (p <0,001), en la implantación de sitios asistenciales (p <0,001) y en la ampliación de la unidad (p <0,001). Entre las intervenciones asistenciales, hubo un aumento de la carga de trabajo en el período de incorporación tecnológica (p <0,001), acreditación de trasplante hepático (p <0,001) e implantación de un paquete de control de infecciones (p <0,001). Conclusion: Las intervenciones dirigidas a mejorar la calidad de la atención son factores que interfieren con la carga de trabajo de enfermería en la UCI, mientras que se ha incrementado con los años. (AU)

Role of paracoccin on Paracoccidioides brasiliensis virulence and susceptibility to antifungal drugs in the Galleria mellonella larvae model.

Paracoccin (PCN), a Paracoccidioides brasiliensis glycoprotein, has been reported to play roles in fungal biology and paracoccidioidomycosis pathogenesis. Lectin and chitinase domains account for the PCN's dual roles as an immunomodulatory agent and virulence factor. Soluble PCN injected in P. brasiliensis infected mice, by interacting with TLRs' N-glycans, drives the host immune response toward a protective Th1 axis. Otherwise, mice infection with yeasts overexpressing PCN (ov-PCN) revealed that PCN acts as a fungal virulence factor, thanks to its chitinase activity on the cell wall, resulting in resistance to phagocytes' fungicidal activity and development of severe paracoccidioidomycosis. Because antifungal drug administration follows the disease diagnosis, we studied the PCN effect on yeast resistance or susceptibility to antifungal agents. Using a paracoccidioidomycosis model developed in Galleria mellonella larvae, we confirmed the observation, in the murine host, that ov-PCN yeasts display maximum virulence compared to wild-type (wt-PCN) or PCN-silenced (kd-PCN) yeasts. PCN overexpression accounted for the highest susceptibility of P. brasiliensis to antifungal and reduced relative mRNA expression of genes encoding proteins related to cell wall remodeling. The lowest virulence, detected in infection with kd-PCN yeasts, correlated with the lowest susceptibility to antifungals and impact on genes for cell wall remodeling. So, we defined that the grade of endogenous PCN production influences the P. brasiliensis virulence and susceptibility to antifungal drugs, as well as the expression of genes related to cell wall remodeling. We postulate that this variable gene expression is mechanistically associated with P. brasiliensis virulence changes.

ArtinM Cytotoxicity in B Cells Derived from Non-Hodgkin's Lymphoma Depends on Syk and Src Family Kinases.

Receptors on the immune cell surface have a variety of glycans that may account for the immunomodulation induced by lectins, which have a carbohydrate recognition domain (CRD) that binds to monosaccharides or oligosaccharides in a specific manner. ArtinM, a D-mannose-binding lectin obtained from Artocarpus heterophyllus, has affinity for the N-glycans core. Immunomodulation by ArtinM toward the Th1 phenotype occurs via its interaction with TLR2/CD14 N-glycans on antigen-presenting cells, as well as recognition of CD3γ N-glycans on murine CD4+ and CD8+ T cells. ArtinM exerts a cytotoxic effect on Jurkat human leukemic T-cell line and human myeloid leukemia cell line (NB4). The current study evaluated the effects of ArtinM on murine and human B cells derived from non-Hodgkin's lymphoma. We found that murine B cells are recognized by ArtinM via the CRD, and the ArtinM stimulus did not augment the proliferation rate or production of IL-2. However, murine B cell incubation with ArtinM augmented the rate of apoptosis, and this cytotoxic effect of ArtinM was also seen in human B cell-lines sourced from non-Hodgkin's lymphoma Raji cell line. This cytotoxic effect was inhibited by the phosphatase activity of CD45 on Lck, and the protein kinases of the Src family contribute to cell death triggered by ArtinM.

Enzymatically amplified linear dbDNATM as a rapid and scalable solution to industrial lentiviral vector manufacturing.

Traditional bacterial fermentation techniques used to manufacture plasmid are time-consuming, expensive, and inherently unstable. The production of sufficient GMP grade material thus imposes a major bottleneck on industrial-scale manufacturing of lentiviral vectors (LVV). Touchlight's linear doggybone DNA (dbDNATM) is an enzymatically amplified DNA vector produced with exceptional speed through an in vitro dual enzyme process, enabling industrial-scale manufacturing of GMP material in a fraction of the time required for plasmid. We have previously shown that dbDNATM can be used to produce functional LVV; however, obtaining high LVV titres remained a challenge. Here, we aimed to demonstrate that dbDNATM could be optimised for the manufacture of high titre LVV. We found that dbDNATM displayed a unique transfection and expression profile in the context of LVV production, which necessitated the optimisation of DNA input and construct ratios. Furthermore, we demonstrate that efficient 3' end processing of viral genomic RNA (vgRNA) derived from linear dbDNATM transfer vectors required the addition of a strong 3' termination signal and downstream spacer sequence to enable efficient vgRNA packaging. Using these improved vector architectures along with optimised transfection conditions, we were able to produce a CAR19h28z LVV with equivalent infectious titres as achieved using plasmid, demonstrating that dbDNATM technology can provide a highly effective solution to the plasmid bottleneck.

Adjuvant Curdlan Contributes to Immunization against Cryptococcus gattii Infection in a Mouse Strain-Specific Manner.

The low efficacy and side effects associated with antifungal agents have highlighted the importance of developing immunotherapeutic approaches to treat Cryptococcus gattii infection. We developed an immunization strategy that uses selective Dectin-1 agonist as an adjuvant. BALB/c or C57BL/6 mice received curdlan or ß-glucan peptide (BGP) before immunization with heat-killed C. gattii, and the mice were infected with viable C. gattii on day 14 post immunization and euthanized 14 days after infection. Adjuvant curdlan restored pulmonary tumor necrosis factor- α (TNF-α) levels, as induced by immunization with heat-killed C. gattii. The average area and relative frequency of C. gattii titan cells in the lungs of curdlan-treated BALB/c mice were reduced. However, this did not reduce the pulmonary fungal burden or decrease the i0,nflammatory infiltrate in the pulmonary parenchyma of BALB/c mice. Conversely, adjuvant curdlan induced high levels of interferon-γ (IFN-γ) and interleukin (IL)-10 and decreased the C. gattii burden in the lungs of C57BL/6 mice, which was not replicated in ß-glucan peptide-treated mice. The adjuvant curdlan favors the control of C. gattii infection depending on the immune response profile of the mouse strain. This study will have implications for developing new immunotherapeutic approaches to treat C. gattii infection.

Virulence Vs. Immunomodulation: Roles of the Paracoccin Chitinase and Carbohydrate-Binding Sites in Paracoccidioides brasiliensis Infection.

Paracoccin (PCN) is a bifunctional protein primarily present in the cell wall of Paracoccidioides brasiliensis, a human pathogenic dimorphic fungus. PCN has one chitinase region and four potential lectin sites and acts as both a fungal virulence factor and an immunomodulator of the host response. The PCN activity on fungal virulence, mediated by the chitinase site, was discovered by infecting mice with yeast overexpressing PCN (PCN-ov). PCN-ov are characterized by increased chitin hydrolysis, a narrow cell wall, and augmented resistance to phagocytes' fungicidal activity. Compared to wild-type (wt) yeast, infection with PCN-ov yeast causes a more severe disease, which is attributed to the increased PCN chitinase activity. In turn, immunomodulation of the host response was demonstrated by injecting, subcutaneously, recombinant PCN in mice infected with wt-P. brasiliensis. Through its carbohydrate binding site, the injected recombinant PCN interacts with Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) N-glycans on macrophages, triggers M1 polarization, and stimulates protective Th1 immunity against the fungus. The PCN-treatment of wt yeast-infected mice results in mild paracoccidioidomycosis. Therefore, PCN paradoxically influences the course of murine paracoccidioidomycosis. The disease is severe when caused by yeast that overexpress endogenous PCN, which exerts a robust local chitinase activity, followed by architectural changes of the cell wall and release of low size chito-oligomers. However, the disease is mild when exogenous PCN is injected, which recognizes N-glycans on systemic macrophages resulting in immunomodulation.

Transforming growth factor (TGF)-ß1 and interferon (IFN)-γ differentially regulate ICAM-1 expression and adhesion of Toxoplasma gondii to human trophoblast (BeWo) and uterine cervical (HeLa) cells.

Toxoplasma gondii is a parasite able to infect various cell types, including trophoblast cells. Studies have demonstrated that interleukin (IL)-10, transforming growth factor (TGF)-ß1 and interferon (IFN)-γ are involved in the susceptibility of BeWo trophoblast cells to T. gondii infection. Furthermore, T. gondii is able to adhere to the plasma membrane of host cells through intercellular adhesion molecule (ICAM)-1. Thus, the present study aimed to assess the role of IL-10, TGF-ß1 and IFN-γ in the expression of ICAM-1 in BeWo and HeLa cells and to analyze the role of ICAM-1 in the adhesion and invasion of T. gondii to these cells under the influence of these cytokines. For this purpose, BeWo and HeLa cells were treated or not, before and after T. gondii infection, with rIL-10, rTGF-ß1 or rIFN-γ. For the BeWo cells, rIL-10 and rTGF-ß1 favored susceptibility to infection, but only rTGF-ß1 and rIFN-γ increased ICAM-1 expression, and TNF-α release. On the other hand, rIFN-γ downregulated the expression of ICAM-1 triggered by T. gondii in HeLa cells, leading to control of the infection. Moreover, we observed that upregulation of ICAM-1, mediated by cytokine's stimulation, in BeWo and HeLa cells resulted in a high number rate of both parasite adhesion and invasion to these cells, which were strongly reduced after ICAM-1 neutralization. Likewise, the blockage of ICAM-1 molecule also impaired T. gondii infection in human villous explants. Taken together, these findings demonstrate that TGF-ß1 and IFN-γ differentially regulate ICAM-1 expression, which may interfere in the adhesion/invasion of T. gondii to BeWo and HeLa cells for modulating susceptibility to infection.

Administration of artinm lectin reduces the severity of the acute phase infection with Trypanosoma cruzi.

The acute phase of experimental Trypanosoma cruzi infection is associated with a strong inflammatory reaction, physiological changes, amastigote nests in tissues, and hematological alterations. ArtinM, a lectin extracted from Artocarpus heterophyllus seeds, is a homotetramer exhibiting immunomodulatory properties that promotes Th1 immune responses against intracellular pathogens, including the induction of neutrophil migration and increase in IL-12 production. This study aimed to evaluate the effects of ArtinM on experimental Chagas disease in mice. We evaluated mouse survival curves, parasitemia, hematological parameters including quantification of inflammatory infiltrates, and amastigote nests in cardiac tissue during infection. The results showed a reduced number of parasites in the blood, an increase in animal survival, improvements in hematological parameters, and decrease in inflammatory infiltrates and amastigote nests in the group treated with ArtinM. Collectively, these data suggest that the administration of ArtinM can lower the number of parasites in peak parasitemia caused by the Colombian strain of T. cruzi and can increase survival of infected mice. The observed reduction in cardiac tissue injury may be due to fewer T. cruzi amastigote nests and lower levels of inflammatory infiltrates. This study highlights the need for further investigation into the use of ArtinM as a potential alternative therapeutic for treating Chagas disease.

Microneme Proteins 1 and 4 From Toxoplasma gondii Induce IL-10 Production by Macrophages Through TLR4 Endocytosis.

The protozoan parasite Toxoplasma gondii modulates host cell responses to favor its success in the early stage of infections by secreting proteins from its apical organelles. Some of these proteins, including microneme proteins (MICs) 1 and 4, trigger pro-inflammatory host cell responses. The lectins MIC1 and MIC4 interact with N-linked glycans on TLR2 and TLR4, activating NF-κB and producing IL-12, TNF-α, and IL-6. Interestingly, MIC1 and MIC4 also trigger secretion of the anti-inflammatory cytokine IL-10 through mechanisms as yet unknown. Herein, we show that the ability of these MICs to induce macrophages to produce IL-10 depends on TLR4 internalization from the cell surface. Macrophages subjected to blockade of endocytosis by Dynasore continued to release TNF-α, but failed to produce IL-10, in response to MIC1 or MIC4 exposure. Similarly, IL-10 was not produced by Dynasore-conditioned T. gondii-infected macrophages. Furthermore, MIC1- or MIC4-stimulated macrophages gained transient tolerance to LPS. We report a previously undiscovered mechanism by which well-defined T. gondii components inhibit a host inflammatory response.

Paracoccin Overexpression in Paracoccidioides brasiliensis Enhances Fungal Virulence by Remodeling Chitin Properties of the Cell Wall.

BACKGROUND: The thermodimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis. Although poorly studied, paracoccin (PCN) from Paracoccidioides brasiliensis has been shown to harbor lectinic, enzymatic, and immunomodulatory properties that affect disease development. METHODS: Mutants of P. brasiliensis overexpressing PCN (ov-PCN) were constructed by Agrobacterium tumefaciens-mediated transformation. ov-PCN strains were analyzed and inoculated intranasally or intravenously to mice. Fungal burden, lung pathology, and survival were monitored to evaluate virulence. Electron microscopy was used to evaluate the size of chito-oligomer particles released by ov-PCN or wild-type strains to growth media. RESULTS: ov-PCN strains revealed no differences in cell growth and viability, although PCN overexpression favored cell separation, chitin processing that results in the release of smaller chito-oligomer particles, and enhanced virulence. Our data show that PCN triggers a critical effect in the cell wall biogenesis through the chitinase activity resulting from overexpression of PCN. As such, PCN overexpression aggravates the disease caused by P. brasiliensis. CONCLUSIONS: Our data are consistent with a model in which PCN modulates the cell wall architecture via its chitinase activity. These findings highlight the potential for exploiting PCN function in future therapeutic approaches.

Pro-inflammatory response ensured by LPS and Pam3CSK4 in RAW 264.7 cells did not improve a fungistatic effect on Cryptococcus gattii infection.

BACKGROUND: The macrophage lineage is characterized by plasticity due to the acquisition of distinct functional phenotypes, and two major subsets are evaluated; classical M1 activation (strong microbicidal activity) and alternative M2 activation (immunoregulatory functions). The M1 subset expresses inducible nitric oxide synthase (iNOS), which is a primary marker to identify these cells, whereas M2 macrophages are characterized by expression of Arginase-1, found in inflammatory zone 1 (Fizz1), chitinase-like molecule (Ym-1), and CD206. The micro-environmental stimuli and signals in tissues are critical in the macrophage polarization. Toll-like receptors (TLR) ligands, such as lipopolysaccharide (LPS), palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4), and ArtinM (mannose-binding lectin) are inductors of M1 subset. The impact of TLR2 and TLR4 signals to fight against Cryptococcus gattii infection is unknown, which is a fungal pathogen that preferentially infects the lung of immunocompetent individuals. The macrophages initiate an immune response to combat the C. gattii, then we evaluated in RAW 264.7 cell the effect of TLR2 and TLR4 agonists on the macrophage polarization dynamic and the impact on the growth of C. gattii. METHODS AND RESULTS: We demonstrated that P3C4, LPS, and ArtinM induced an increase in the levels of iNOS transcripts in RAW 264.7 cells, whereas the relative expression of arginase-1, Ym-1, and Fizz1 was significantly increased in the presence of IL-4 alone. The effects of TLR2 and TLR4 agonists on repolarization from the M2 to M1 subset was evaluated, and the first stimulus was composed of IL-4 and, after 24 h of incubation, the cells were submitted to a second stimulus of P3C4, LPS, ArtinM, or Medium. These TLR agonists induced the production of TNF-α in polarized RAW 264.7 cells to the M2 subset, moreover the measurement of M1/M2 markers using qRT-PCR demonstrated that a second stimulus with LPS for 24 h induced a significant augmentation of levels of iNOS mRNA. This impact of TLR2 and TLR4 agonists in the activation of the RAW 264.7 macrophage was assayed in the presence of C. gattii, the macrophages stimulated with TLR2 and TLR4 agonists for 24 h and co-cultured with C. gattii, as a second stimulus, reached high levels of TNF-α even after incubation with different concentrations of C. gattii. The activation of RAW 264.7 cells induced by TLR2 and TLR4 agonists favored the phagocytosis of C. gattii and inhibited the growth of yeast in the early period of infection. However, RAW 264.7 cells incubated with C. gattii in the presence of TLR2 and TLR4 agonists did not result a significant difference in the colony forming unit (CFU) assay in the early period of C. gattii infection, compared to negative control. CONCLUSION: Polarized RAW 264.7 cells to the M1 subset with TLR2 and TLR4 agonists did not inhibit the growth of C. gattii, whereas robust immunity was identified that could dysregulate host tolerance to this pathogen.

Brain-Targeted Delivery of Pre-miR-29b Using Lactoferrin-Stearic Acid-Modified-Chitosan/Polyethyleneimine Polyplexes.

The efficacy of brain therapeutics is largely hampered by the presence of the blood-brain barrier (BBB), mainly due to the failure of most (bio) pharmaceuticals to cross it. Accordingly, this study aims to develop nanocarriers for targeted delivery of recombinant precursor microRNA (pre-miR-29b), foreseeing a decrease in the expression of the BACE1 protein, with potential implications in Alzheimer's disease (AD) treatment. Stearic acid (SA) and lactoferrin (Lf) were successfully exploited as brain-targeting ligands to modify cationic polymers (chitosan (CS) or polyethyleneimine (PEI)), and its BBB penetration behavior was evaluated. The intracellular uptake of the dual-targeting drug delivery systems by neuronal cell models, as well as the gene silencing efficiency of recombinant pre-miR-29b, was analyzed in vitro. Labeled pre-miR-29b-CS/PEI-SA-Lf systems showed very strong fluorescence in the cytoplasm and nucleus of RBE4 cells, being verified the delivery of pre-miR-29b to neuronal cells after 1 h transfection. The experiment of transport across the BBB showed that CS-SA-Lf delivered 65% of recombinant pre-miR-29b in a period of 4 h, a significantly higher transport ratio than the 42% found for PEI-SA-Lf in the same time frame. Overall, a novel procedure for the dual targeting of DDS is disclosed, opening new perspectives in nanomedicines delivery, whereby a novel drug delivery system harvests the merits and properties of the different immobilized ligands.

Expression of Hsp60 and its cell location in Paracoccidioides brasiliensis.

Paracoccidioides species cause paracoccidioidomycosis (PCM), a systemic mycosis highly prevalent in Brazil. Therapy of PCM has some issues that make studies for new therapeutic and vaccine targets relevant, such as the P. brasiliensis 60-kDa-heat-shock protein (PbHsp60), an immunogenic antigen that induces protection in experimental mice infection. Here, we investigated the relative expression of mRNA for PbHsp60 in P. brasiliensis in the different morphotypes of P. brasiliensis and in morphological transition phases. In addition, antibodies to rPbHsp60 were produced and used to analyze the location of PbHsp60 in yeast and hyphae by electron microscopy. The analyses showed a substantial increase in the relative amounts of HSP60 mRNA in yeast when compared to mycelium and an intermediate expression in transitional forms. Regarding the cell location, immunoelectron microscopy analysis revealed that PbHsp60 is within the cell wall. These observations suggest that this protein may be involved in the maintenance of the cell wall integrity and the interaction with the host for colonization, infection and pathogenesis.

Paracoccin: Purification and Validation of Its Lectin and Enzymatic Properties.

Studies on the effects of components derived from the human pathogenic fungi Paracoccidioides brasiliensis have identified paracoccin (PCN), as a bifunctional protein with lectin (GlcNAc-binding) and enzymatic (chitinase) activities, able to induce modulation of host immune response. Endogenous PCN acts as a fungal virulence factor, whereas exogenous purified PCN, administered to the host, confers protective immunity in a murine model of paracoccidioidomycosis. The immunomodulation induced by purified-PCN injection has characterized it as an agent applicable in the therapy and vaccine against paracoccidioidomycosis. This section describes methods for PCN purification and validation of its lectin and enzymatic activities. It includes detailed protocols to obtain homogeneous PCN from P. brasiliensis yeasts, as well as to purify recombinant PCN from transformed heterologous microorganisms.

ArtinM: Purification and Evaluation of Biological Activities.

The immunomodulatory activity of plant lectins has been evaluated because of their high selectivity for glycans linked to receptors on innate and adaptative immune cells. ArtinM is a mannosyl-binding lectin, obtained from the seeds of Artocarpus heterophyllus, that induces the differentiation of CD4+ T cells and macrophages by interacting with CD3 and TLR2/CD14, respectively. This ArtinM property ultimately favors the combat of intracellular pathogens, opening new perspectives on the lectins application as immunomodulatory agents. The current section describes protocols for purification and evaluation of ArtinM biological activity. The purification is based on the ArtinM-D-mannose affinity. The effect of inducing IL-12 production by murine macrophages cell line is adopted to evaluate the ArtinM biological activity.

MIC4 from Toxoplasma gondii: A Lectin Acting as a Toll-Like Receptor Agonist.

Tachyzoites, which are infective forms of Toxoplasma gondii, use their actinomyosin system to move over surfaces and invade host cells. Central to this process is the regulated release of micronemes organelles contents. The microneme protein 4 (MIC4) has the property to recognize galactosides residues linked to glycoproteins on the host cell surface. This property allows that MIC4 binds to TLR2- and TLR4 N-linked glycans and promote the activation of cell innate immune cells and secretion of inflammatory cytokines, acting on resistance against the parasite. Obtention of MIC4 from T. gondii requires several purification steps, is time-consuming and provides low yield. Therefore, this section details the protocol for prokaryotic expression, production, and purification of recombinant MIC4 (rMIC4) and for experimental assays to confirm its biological activity.

Production and Characterization of MIC1: A Lectin from Toxoplasma gondii.

Some lectins of pathogens interact with host cells through the recognition of specific carbohydrates displayed on the mammals' cell surface. The microneme protein 1 (MIC1) from Toxoplasma gondii has a lectin domain that specifically binds sialic acid residues, often found in the terminal positions of N-glycans of mammalian cells. The necessary studies on the MIC1 biological roles have been limited initially by the laborious purification of the protein from T. gondii tachyzoites and the low yields verified. Then Escherichia coli has been transformed with a construct containing the MIC1 gene, and the obtained recombinant MIC1 (rMIC1) has been purified from the inclusion bodies. Herein, we detail the methodology of heterologous production and purification of rMIC1 and protocols to assay the rMIC1 lectin ability.

The lectin ArtinM activates RBL-2H3 mast cells without inducing degranulation.

Mast cells are connective tissue resident cells with morphological and functional characteristics that contribute to their role in allergic and inflammatory processes, host defense and maintenance of tissue homeostasis. Mast cell activation results in the release of pro-inflammatory mediators which are largely responsible for the physiological functions of mast cells. The lectin ArtinM, extracted from Artocarpus heterophyllus (jackfruit), binds to D-manose, thus inducing degranulation of mast cells. ArtinM has several immunomodulatory properties including acceleration of wound healing, and induction of cytokine release. The aim of the present study was to investigate the role of ArtinM in the activation and proliferation of mast cells. The rat mast cell line RBL-2H3 was used throughout this study. At a low concentration (0.25µg/mL), ArtinM induced mast cell activation and the release of IL-6 without stimulating the release of pre-formed or newly formed mediators. Additionally, when the cells were activated by ArtinM protein tyrosine phosphorylation was stimulated. The low concentration of ArtinM also activated the transcription factor NFkB, but not NFAT. ArtinM also affected the cell cycle and stimulated cell proliferation. Therefore, ArtinM may have therapeutic applications by modulating immune responses due to its ability to activate mast cells and promote the release of newly synthesized mediators. Additionally, ArtinM could have beneficial effects at low concentrations without degranulating mast cells and inducing allergic reactions.

Topical application of lectin Artin M improves wound healing in defects created in the palatal mucosa: an in vivo study in dogs.

Previous studies have shown that topical application of lectin Artin-M accelerates wound healing in the rat oral mucosa. The aim of this study was to evaluate, by means of histology and immunohistochemistry (IHC) the effects of Artin-M on wound healing in the palatal mucosa in dogs. Three full thickness wounds of 6 mm diameter were surgically created in the palatal mucosa of twenty dogs and randomly divided into three groups according to one of the treatment assigned: Group C-Control (coagulum); Group A-Artin-M gel; Group V-Vehicle (carboxymethylcellulose 3%). Each animal received all the three experimental treatments. Afterwards, four animals were killed at 2, 4, 7, 14 and 21 days post-surgery. Wounded areas were photographed and scored for macroscopic evaluation. Biopsies were harvested and used for descriptive histological analysis, proliferating cell nuclear antigen IHC and measurement of myeloperoxidase activity. The results demonstrated faster wound closure in group A in comparison to the other groups in all the periods evaluated. Histological analyses exhibited improved re-epithelialization and collagen fiber formation resulting in faster maturation of granulation tissue in group A compared to the other groups by day 14. Treatment with Artin-M gel significantly induced cell proliferation and increased volumetric density of fibroblasts at day 2 and 4 (p < 0.05). Neutrophil infiltration in group A was significantly higher than the other groups (p < 0.05) at the same time points. Collectively, our findings demonstrated that Artin-M may potentially favor wound healing on palatal mucosa lesions via recruitment of neutrophils and promotion of cell proliferation.