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Junin virus (JV) infection of adult (resistant) BALB/c mice induces antigen non-specific delayed-type hypersensitivity (DTH) suppressor T cells, termed Tsv, bearing the Thy-1+, Ly-1+2- phenotype. These cells may be related to survival to infection since DTH reaction is associated with lethal meningoencephalitis. Employing several xenogeneic red blood cell (RBC) sensitization schedules to induce different cell subpopulations, we have attempted to establish the target of JV-induced suppressor cells (Tsv). The target of Tsv cells was actually included in the antigen-specific suppressor cell compartment, as demonstrated for the RBC system. Tsv cells were able to trigger suppressor cells to act without loss of their specificity. The presence of two sets of sheep RBC-induced DTH suppressor cells bearing the Ly-1+2- and Ly-1 2+ phenotypes was disclosed in low (106)-dose sensitized mice. Both sets were simultaneously required by Tsv to achieve DTH suppression. In contrast, in high (108) SRBC-dose sensitized animals treated with cyclophosphamide (doses of 50 mg/kg), a single Ly-1-2+ suppressor cell was required. [ABSTRACT FROM AUTHOR]

Intracerebral (i.c.) Junin virus (JV) infection of adult BALB/c mice is characterized by the absence of morbidity and a low mortality (barely 8-10%). In contrast, the suckling mouse model exhibits almost 100% mortality following central nervous system (CNS) alterations consistent with a delayed-type hypersensitivity (DTH)-like immune response. Besides, JV infection of adult (resistant) mice leads to immunosuppression of DTH to unrelated antigens. Here we present evidence demonstrating that such suppression is mediated by JV-induced cells present in spleen from 24 hr to 24 days post- infection, bearing the Thy-1+, Ly- 1 + 2- phenotype and reactive to an unrelated antigen such as sheep red blood cells (SRBC). No evidence of suppressor factors was found. A relatively low number of total splenic cells (5 × 106 cells/mouse) was enough to transfer suppression. Therefore, this cell population may be involved in adult mouse survival to JV infection. [ABSTRACT FROM AUTHOR]

Intracerebral inoculation of Junin virus (JV) in all susceptible mouse models available induces a lethal meningoencephalitis compatible with a delayed-type hypersensitivity (DTH) immune response. In contrast, adult BALB/c mice prove resistant to infection and no DTH response is seen. JV inoculation in adult BALB/c mice induces DTH suppression to unrelated antigens such as sheep red blood cells. (SRBC). This suppression is mediated by JV-induced T cells (Tsv), which are operative from 1 to 24 days post-infection (p.i.), and seems to be related to adult mouse survival. The presence of JV-induced contrasuppressor cells (CS) bearing the Thy-1+, Ly 1+2- phenotype, able to abrogate Tsv cells-mediated suppression, is described here. Thus, CS cells may be involved in the mechanism by which mice avoid over-exposure to Tsv-mediated DTH suppression. Such CS cells were found in the spleen of inoculated animals and may also be induced by transferring JV-free Tsv cells to ‘naive’ recipients, in which JV inoculation then induces morbidity. [ABSTRACT FROM AUTHOR]

Bovine alphaherpesvirus-1 (BoAHV-1) causes several symptoms in cattle, leading to significant costs for the livestock industry. In this study, we used a plasmid encoding a secreted form of BoAHV-1 glycoprotein D (pCIgD) as a DNA vaccine. To enhance the potency of the pCIgD vaccine, we used Montanide™ GEL01 PR (GEL01) and introduced Galectin-8 (Gal-8), a lectin considered a novel adjuvant due to its immunostimulatory effects, into the formulation. Animals were vaccinated with pCIgD, pCIgD with Gal-8 (pCIgD-Gal-8), pCIgD with Gal-8 and GEL01 (pCIgD-Gal-8-GEL01), or the control plasmid pCIneo. The immune response was first assessed in a mouse model and then in bovines. The results showed that combining Gal-8 and GEL01 with pCIgD modulated immune responses at both the humoral and cellular levels in both animal models. This study evaluates the efficacy of a DNA vaccine with Gal-8 and GEL01 as potential adjuvants to enhance immune protection against BoAHV-1., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Some of the authors of this paper are authors of a relevant patent; see ref (Campetella and Tribulatti, 2023)., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Galectins constitute a family of soluble lectins with unique capacity to induce macroscale rearrangements upon interacting with cell membrane glycoconjugates. Galectin-8 (Gal-8) is acknowledged for its role in facilitating antigen uptake and processing upon engaging with cell surface glycoconjugates on antigen-presenting cells (APCs). Gal-8 consists of two covalently fused N- and C-terminal carbohydrate recognition domains (N- and C-CRD), each exhibiting distinct glycan specificity. In this study, we utilized single N- and C-CRD recombinant proteins to dissect the nature of Gal-8-glycan interactions during antigen internalization enhancement. Single C-CRD was able to replicate the effect of full-length Gal-8 (FLGal-8) on antigen internalization in BMDCs. Antigen uptake enhancement was diminished in the presence of lactose or when N-glycosylation-deficient macrophages served as APCs, underscoring the significance of glycan recognition. Measurement of the elastic modulus using Atomic Force Microscopy unveiled that FLGal-8- and C-CRD-stimulated macrophages exhibited heightened membrane stiffness compared to untreated cells, providing a plausible mechanism for their involvement in endocytosis. C-CRD proved to be as efficient as FLGal-8 in promoting antigen degradation, suggesting its implication in antigen-processing induction. Lastly, C-CRD was able to replicate FLGal-8-induced antigen presentation in the MHC-II context both in vitro and in vivo. Our findings support the notion that Gal-8 binds through its C-CRD to cell surface N-glycans, thereby altering membrane mechanical forces conducive to soluble antigen endocytosis, processing, and presentation to cognate CD4 T-cells. These findings contribute to a deeper comprehension of Gal-8 and its mechanisms of action, paving the way for the development of more efficacious immunotherapies., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology.)

Human African trypanosomiasis or sleeping sickness, caused by the protozoan parasite Trypanosoma brucei, is characterized by the manipulation of the host's immune response to ensure parasite invasion and persistence. Uncovering key molecules that support parasite establishment is a prerequisite to interfere with this process. We identified Q586B2 as a T. brucei protein that induces IL-10 in myeloid cells, which promotes parasite infection invasiveness. Q586B2 is expressed during all T. brucei life stages and is conserved in all Trypanosomatidae. Deleting the Q586B2-encoding Tb927.6.4140 gene in T. brucei results in a decreased peak parasitemia and prolonged survival, without affecting parasite fitness in vitro, yet promoting short stumpy differentiation in vivo. Accordingly, neutralization of Q586B2 with newly generated nanobodies could hamper myeloid-derived IL-10 production and reduce parasitemia. In addition, immunization with Q586B2 delays mortality upon a challenge with various trypanosomes, including Trypanosoma cruzi. Collectively, we uncovered a conserved protein playing an important regulatory role in Trypanosomatid infection establishment., (© 2024. The Author(s).)

Nearly all expression vectors currently available for Trypanosoma cruzi were conceived to produce a single primary transcript containing the genes of interest along with those that confer antibiotic resistance. However, since each messenger RNA (mRNA) matures separately, drug selection will only guarantee the expression of those derived from the selectable marker. Therefore, commonly a considerable fraction of the cells recovered after selection with these expression vectors, although resistant do not express the protein of interest. Consequently, in order to counteract this disadvantage, we developed vectors with an alternative arrangement in which the gene of interest and antibiotic resistance are fused sharing the same mRNA. To test this configuration, we included the coding sequence for the green fluorescent protein (mEGFP) linked to the one conferring neomycin resistance (Neo). Additionally, to allow for the production of two independent proteins the sequence for a Thosea asigna virus self-cleaving 2A peptide (T2A) was inserted in-between. Cells obtained with these vectors displayed higher mEGFP expression levels with more homogeneous transgenic parasite populations than those transfected with more conventional independent mRNA-based alternatives. Moreover, as determined by Western blot, 2A mediated fusion protein dissociation occurred with high efficiency in all parasite stages. In addition, these vectors could easily be transformed into endogenous tagging constructs that allowed the insertion, by ends-in homologous recombination, of a hemagglutinin tag (HA) fused to the actin gene. The use of 2A self-cleaving peptides in the context of single mRNA vectors represents an interesting strategy capable of improving ectopic transgene expression in T. cruzi as well as providing a simple alternative to more sophisticated methods, such as the one based on CRISPR/Cas9, for the endogenous labeling of genes., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Trypanosoma cruzi infection induces a polyclonal B cell proliferative response characterized by maturation to plasma cells, excessive generation of germinal centers, and secretion of parasite-unrelated antibodies. Although traditionally reduced to the humoral response, several infectious and non-infectious models revealed that B lymphocytes could regulate and play crucial roles in cellular responses. Here, we analyze the trypomastigote-induced effect on B cells, their effects on CD4 + T cells, and their correlation with in vivo findings. The trypomastigotes were able to induce the proliferation and the production of IL-10 or IL-6 of naïve B cells in co-culture experiments. Also, we found that IL-10-producing B220 lo cells were elicited in vivo . We also found up-regulated expression of FasL and PD-L1, proteins involved in apoptosis induction and inhibition of TCR signaling, and of BAFF and APRIL mRNAs, two B-cell growth factors. Interestingly, it was observed that IL-21, which plays a critical role in regulatory B cell differentiation, was significantly increased in B220 + /IL-21 + in in vivo infections. This is striking since the secretion of IL-21 is associated with T helper follicular cells. Furthermore, trypomastigote-stimulated B-cell conditioned medium dramatically reduced the proliferation and increased the apoptotic rate on CD3/CD28 activated CD4 + T cells, suggesting the development of effective regulatory B cells. In this condition, CD4 + T cells showed a marked decrease in proliferation and viability with marginal IL-2 or IFNγ secretion, which is counterproductive with an efficient immune response against T. cruzi . Altogether, our results show that B lymphocytes stimulated with trypomastigotes adopt a particular phenotype that exerts a strong regulation of this T cell compartment by inducing apoptosis, arresting cell division, and affecting the developing of a proinflammatory response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Somoza, Bertelli, Pratto, Verdun, Campetella and Mucci.)

Matrix metalloproteinases (MMPs) not only play a relevant role in homeostatic processes but are also involved in several pathological mechanisms associated with infectious diseases. As their clinical relevance in Chagas disease has recently been highlighted, we studied the modulation of circulating MMPs by Trypanosoma cruzi infection. We found that virulent parasites from Discrete Typing Units (DTU) VI induced higher proMMP-2 and MMP-2 activity in blood, whereas both low (DTU I) and high virulence parasites induced a significant decrease in proMMP-9 plasma activity. Moreover, trans-sialidase, a relevant T. cruzi virulence factor, is involved in MMP-2 activity modulation both in vivo and in vitro. It removes α2,3-linked sialyl residues from cell surface glycoconjugates, which then triggers the PKC/MEK/ERK signaling pathway. Additionally, bacterial sialidases specific for this sialyl residue linkage displayed similar MMP modulation profiles and triggered the same signaling pathways. This novel pathogenic mechanism, dependent on sialic acid removal by the neuraminidase activity of trans-sialidase, can be exploited by different pathogens expressing sialidases with similar specificity. Thus, here we present a new pathogen strategy through the regulation of the MMP network., Competing Interests: Declaration of competing interest All authors declare no conflicts of interest and have approved the final article., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Galectins are soluble lectins that participate in many physiological and pathological functions. Since they can act extracellularly, the use of the recombinant protein is a recurrent strategy for studying their biological functions. Here, we provide a general protocol for the production of Galectins and their isolated or chimeric domains. We take advantage of their lectin activity and the 6xHis-tag addition for purification, thus obtaining a highly pure and active Galectin to use in both in vitro and in vivo assays. For complete details on the use and execution of this protocol, please refer to Cattaneo et al. (2011), Tribulatti et al. (2012), and Prato et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Galectin-8 (Gal-8) is a mammalian lectin endowed with immunostimulatory ability. In the present work, we demonstrate that Gal-8-glycan interactions on the surface of antigen-presenting cells (APCs) promote antigen binding and internalization, independently from antigen nature. Both Gal-8 and antigen were together internalized and localized in early endosomes. Interestingly, antigen processing by APCs was also accelerated in the presence of Gal-8 as a separate mechanism, distinct from the increased antigen internalization. Moreover, APCs pulsed together with antigen and Gal-8 were able to activate cognate CD4 + T cells more efficiently than those pulsed with antigen alone. This enhanced antigen presentation was still evident in the absence of costimulatory signals and APCs-derived soluble mediators. Therefore, our results provide evidence for as yet unrecognized mechanism by which Gal-8 stimulates the elicitation of the immune response in a lectin-dependent manner, by inducing antigen uptake and processing upon lattice formation at APCs surface., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Many important pathogen-host interactions rely on highly specific carbohydrate binding events. In the case of the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, glycointeractions involving sialic acid (SA) residues are pivotal for parasite infectivity, escape from immune surveillance and pathogenesis. Though unable to synthesize SA de novo, T. cruzi displays a unique trans-Sialidase (TS) enzyme, which is able to cleave terminal SA residues from host donor glycoconjugates and transfer them onto parasite surface mucins, thus generating protective/adhesive structures. In addition, this parasite sheds TS into the bloodstream, as a way of modifying the surface SA signature, and thereby the signaling/functional properties of mammalian host target cells on its own advantage. Here, we discuss the pathogenic aspects of T. cruzi TS: its molecular adaptations, the multiplicity of interactions in which it is involved during infections, and the array of novel and appealing targets for intervention in Chagas disease provided by TS-remodeled sialoglycophenotypes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Galectins (Gals), a family of mammalian lectins, have emerged as key regulators of the immune response, being implicated in several physiologic and pathologic conditions. Lately, there is increasing data regarding the participation of Galectin-8 (Gal-8) in both the adaptive and innate immune responses, as well as its high expression in inflammatory disorders. Here, we focus on the pro- and anti-inflammatory properties of Gal-8 and discuss the potential use of this lectin in order to shape the immune response, according to the context., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Among mammary tumor-infiltrating immune cells, the highest expression of podoplanin (PDPN) is found in a subset of tumor-associated macrophages (TAMs). We hereby demonstrate that PDPN is involved in the attachment of this TAM subset to lymphatic endothelial cells (LECs). Mechanistically, the binding of PDPN to LEC-derived galectin 8 (GAL8) in a glycosylation-dependent manner promotes the activation of pro-migratory integrin β1. When proximal to lymphatics, PDPN-expressing macrophages (PoEMs) stimulate local matrix remodeling and promote vessel growth and lymphoinvasion. Anti-integrin β1 blockade, macrophage-specific Pdpn knockout, or GAL8 inhibition impairs TAM adhesion to LECs, restraining lymphangiogenesis and reducing lymphatic cancer spread. In breast cancer patients, association of PoEMs with tumor lymphatic vessels correlates with incidences of lymph node and distant organ metastasis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: TolT was originally described as a Trypanosoma cruzi molecule that accumulated on the trypomastigote flagellum bearing similarity to bacterial TolA colicins receptors. Preliminary biochemical studies indicated that TolT resolved in SDS-PAGE as ~3-5 different bands with sizes between 34 and 45 kDa, and that this heterogeneity could be ascribed to differences in polypeptide glycosylation. However, the recurrent identification of TolT-deduced peptides, and variations thereof, in trypomastigote proteomic surveys suggested an intrinsic TolT complexity, and prompted us to undertake a thorough reassessment of this antigen., Methods/principle Findings: Genome mining exercises showed that TolT constitutes a larger-than-expected family of genes, with at least 12 polymorphic members in the T. cruzi CL Brener reference strain and homologs in different trypanosomes. According to structural features, TolT deduced proteins could be split into three robust groups, termed TolT-A, TolT-B, and TolT-C, all of them showing marginal sequence similarity to bacterial TolA proteins and canonical signatures of surface localization/membrane association, most of which were herein experimentally validated. Further biochemical and microscopy-based characterizations indicated that this grouping may have a functional correlate, as TolT-A, TolT-B and TolT-C molecules showed differences in their expression profile, sub-cellular distribution, post-translational modification(s) and antigenic structure. We finally used a recently developed fluorescence magnetic beads immunoassay to validate a recombinant protein spanning the central and mature region of a TolT-B deduced molecule for Chagas disease serodiagnosis., Conclusion/significance: This study unveiled an unexpected genetic and biochemical complexity within the TolT family, which could be exploited for the development of novel T. cruzi biomarkers with diagnostic/therapeutic applications., Competing Interests: The authors have declared that no competing interests exist.

Trypanosoma cruzi, the protozoan agent of Chagas disease, has evolved an innovative metabolic pathway by which protective sialic acid (SA) residues are scavenged from host sialylglycoconjugates and transferred onto parasite surface mucin-like molecules (or surface glycoconjugates from host target cells) by means of a unique trans-sialidase (TS) enzyme. TS-induced changes in the glycoprotein sialylation profile of both parasite and host cells are crucial for the establishment of a persistent T. cruzi infection and for the development of Chagas disease-associated pathogenesis. In this chapter, we describe a novel metabolic labeling method developed in our labs that enables straightforward identification and molecular characterization of SA acceptors of the TS-catalyzed reaction.

Galectin-8 (Gal-8) is a mammalian lectin endowed with the ability to co-stimulate antigen-specific immune responses. We have previously demonstrated that bone-marrow-derived dendritic cells produce high levels of interleukin-6 (IL-6) in response to Gal-8 stimulation. As IL-6 is a pleiotropic cytokine that has a broad effect on cells of the immune system, we aimed to elucidate whether IL-6 was involved in Gal-8-dependent co-stimulatory signals during antigen recognition by specific CD4 T cells. With this aim, splenocytes from DO11.10 mice were incubated with a low dose of the cognate ovalbumin peptide in combination with Gal-8. Interleukin-6 was found significantly increased in cultures stimulated with Gal-8 alone or Gal-8 plus cognate peptide. Moreover, IL-6 signalling was triggered during Gal-8-induced co-stimulation, as determined by phosphorylation of signal transducer and activator of transcription 3. Interleukin-6 blockade by neutralizing monoclonal antibody precluded Gal-8 co-stimulatory activity but did not affect the antigen-specific T-cell receptor activation. Different subsets of dendritic cells, as well as macrophages and B cells, were identified as the cellular source of IL-6 during Gal-8-induced co-stimulation. To confirm that IL-6 mediated the Gal-8 co-stimulatory effect, antigen-presenting cells from IL-6-deficient or wild-type mice were co-cultured with purified CD4 T cells from OTII mice in the presence of cognate peptide and Gal-8. Notably, Gal-8-induced co-stimulation, but not the antigen-specific response, was significantly impaired in the presence of IL-6-deficient antigen-presenting cells. In addition, exogenous IL-6 fully restored Gal-8-induced co-stimulation. Taken together, our results demonstrate that IL-6 signalling mediates the Gal-8 immune-stimulatory effect., (© 2018 John Wiley & Sons Ltd.)