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Introduction: Post-translational modifications of proteins provide cellular physiology with a broad range of adaptability to the external environment flexibly and rapidly. In the case of the protozoan parasite Giardia lamblia , the study of these modifications has gained relevance in recent years, mainly focusing on methylation and deacetylation of proteins. This study investigates the significance of acetylation in this protozoan parasite., Methods: This study explores the role of acetylation in G. lamblia through a combination of immunofluorescence assays, manipulation of acetyltransferase enzymes, and the use of garcinol, an acetylation inhibitor, during the growth phase., Results: The acetylation of histone marks H3K9 and H3K27 occurs during growth and is followed by deacetylation during encystation. Transfections modifying acetyltransferase activity induced a latent cellular state, underscoring the importance of protein acetylation for parasite survival. Garcinol treatment during growth caused significant morphological changes, including plasma membrane blebbing and apoptotic-like bodies. Immunofluorescence revealed these bodies contained α-tubulin/acetylated α-tubulin and reactive oxygen species. Flow cytometry and Annexin V staining indicated early apoptosis within 24 hours of treatment. Additionally, garcinol led to the deacetylation of H3K9 and H3K27, with redistribution of tubulin and acetylated tubulin from microtubules to the cytosol. Significantly, garcinol prevented parasite recrudescence after treatment withdrawal., Discussion: These results demonstrate that acetylation is essential for trophozoite survival and highlight the natural histone acetyltransferase inhibitor garcinol as a potential candidate for drug development against giardiasis, considering its giardicidal activity., 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 © 2025 Patolsky, Laiolo, Díaz-Pérez, Luna Pizarro, Mayol, Touz, Feliziani and Rópolo.)

Introduction: Giardia lamblia is a flagellated protozoan parasite causing giardiasis, a common intestinal infection characterized by diarrhea, abdominal cramps, and nausea. Treatments employed to combat this parasitic infection have remained unchanged for the past 40 years, leading to the emergence of resistant strains and prompting the search for new therapeutic agents., Methods: This study investigated the cytotoxic effects of ivermectin (IVM) on G. lamblia trophozoites. We conducted dose-response experiments to assess IVM-induced cytotoxicity. We utilized various biochemical and ultrastructural analyses to explore the underlying mechanisms of cell death, including reactive oxygen species (ROS) production, DNA fragmentation, cell cycle arrest, and apoptosis markers., Results: Our findings demonstrate that IVM induces dose-dependent cytotoxicity and triggers cell death pathways. We found that IVM treatment generates elevated levels of reactive oxygen species (ROS), DNA fragmentation, and arrests of trophozoites in the cell cycle's S phase. Additionally, ultrastructural analysis reveals morphological alterations consistent with apoptosis, such as cytoplasmic vacuolization, chromatin condensation, and tubulin distribution., Discussion: The insights gained from this study may contribute to developing new therapeutic strategies against giardiasis, addressing the challenge posed by drug-resistant strains., 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 © 2024 Barzola, Laiolo, Cotelo, Joray, Volpini, Rivero, Rópolo, Touz and Feliziani.)

Histone post-translational modifications are extensively studied for their role in regulating gene transcription and cellular environmental adaptation. Research into these modifications has recently begun in the protozoan parasite Giardia lamblia, focusing on histone-modifying enzymes and specific post-translational changes. In the transformation from the trophozoite to the cyst form in the life cycle of this parasite, significant morphological and genetic alterations occur, culminating in the synthesis of cyst wall proteins responsible for forming the protective cyst wall. It has been previously demonstrated that histone deacetylation is required during encystation and that the enzyme lysine methyltransferase 1 is involved in the upregulation of encystation. Our study aims to extend the analysis to lysine methyltransferase 2 (GlKMT2) function. For this, two constructs were generated: one that downregulate the expression of GLKMT2 via antisense (glkmt2-as transgenic cells) and the other overexpressing GlKMT2 (glkmt2-ha transgenic cells). We found that the glktm2-as transgenic cells showed an arrest in progress at the late encystation stage. Consequently, the number of cysts produced was lower than that of the control cells. On the other hand, we found that the overexpression of GlKMT2 acts as a negative mutant of the enzyme. In this way, these glktm2-ha transgenic cells showed the same behavior during growth and encystation as glkmt2-as transgenic cells. This interplay between different enzymes acting during encystation reveals the complex process behind the differentiation of the parasite. Understanding how these enzymes play their role during the encystation of the parasite would allow the design of inhibitors to control the parasite., 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 © 2024 Elsevier B.V. All rights reserved.)

The genetically related assemblages of the intestinal protozoa parasite Giardia lamblia are morphologically indistinguishable and are often derived from specific hosts. The Giardia assemblages are separated by large genetic distances, which might account for their relevant biological and pathogenic differences. In this work, we analyzed the RNAs cargo released into exosomal-like vesicles (ElVs) by the assemblages A and B, which differentially infect humans, and the assemblage E, which infects hoofed animals. The RNA sequencing analysis revealed that the ElVs of each assemblage contained distinct small RNA (sRNA) biotypes, suggesting a preference for specific packaging in each assemblage. These sRNAs were classified into three categories, ribosomal-small RNAs (rsRNAs), messenger-small RNAs (msRNAs), and transfer-small RNAs (tsRNAs), which may play a regulatory role in parasite communication and contribute to host-specificity and pathogenesis. Uptake experiments showed, for the first time, that ElVs were successfully internalized by the parasite trophozoites. Furthermore, we observed that the sRNAs contained inside these ElVs were first located below the plasma membrane but then distributed along the cytoplasm. Overall, the study provides new insights into the molecular mechanisms underlying the host-specificity and pathogenesis of G. lamblia and highlights the potential role of sRNAs in parasite communication and regulation.

The protozoan parasite Giardia lamblia acquires cholesterol from the environment since it is unable to synthesise cholesterol de novo and this is vital for trophozoite growth. Conversely, the lack of cholesterol was described as an essential event to trigger encystation, the differentiation of trophozoites to mature cysts. During the G. lamblia cell cycle, cholesterol is acquired as a free molecule as well as through receptor-mediated endocytosis (RME) of lipoproteins. In this work, we describe the involvement of RME in the cell differentiation process of G. lamblia. We found that a reduction in the expression of the medium subunit (Glµ2) of the giardial adaptin protein GlAP2 impaired RME, triggering the process of encystation in growing cells. Contrary to expectations, decreasing Glµ2 expression produced a cohort of trophozoites that yielded significantly less mature cysts when cells were induced to encyst. Analysis of the subcellular localization of Glµ2 and the cyst wall protein 1 (CWP1) during encystation was later performed, to dissect the process. Our results showed, on one hand, that blocking RME by inhibiting Glµ2 expression, and probably cholesterol entry, is sufficient to induce cell differentiation but not to complete the process of encystation. On the other hand, we observed that GlAP2 is necessary to accomplish the final steps of encystation by sorting CWP1 to the plasma membrane for cyst wall formation. The understanding of the mechanisms involved in cyst formation should provide novel insights into the control of giardiasis, an endemic worldwide neglected disease., (Copyright © 2022 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Nuclear-cytoplasmic trafficking of proteins is a highly regulated process that modulates multiple biological processes in eukaryotic cells. In Giardia lamblia, shuttling has been described from the cytoplasm to nuclei of proteins during the biological cell cycle of the parasite. This suggests that a mechanism of nucleocytoplasmic transport is present and functional in G. lamblia. By means of computational biology analyses, we found that there are only two genes for nuclear transport in this parasite, named Importin α and Importin β. When these transporters were overexpressed, both localized close to the nuclear envelope, and no change was observed in trophozoite growth rate. However, during the encystation process, both transporters induced an increase in the number of cysts produced. Importazole and Ivermectin, two known specific inhibitors of importins, separately influenced the encysting process by inducing an arrest in the trophozoite stage that prevents the production of cysts. This effect was more noticeable when Ivermectin, an anti-parasitic drug, was used. Finally, we tested whether the enzyme arginine deiminase, which shuttles from the cytoplasm to the nuclei during encystation, was influenced by these transporters. We found that treatment with each of the inhibitors abrogates arginine deiminase nuclear translocation and favors perinuclear localization. This suggests that Importin α and Importin β are key transporters during the encystation process and are involved, at least, in the transport of arginine deiminase into the nuclei. Considering the effect produced by Ivermectin during growth and encystation, we postulate that this drug could be used to treat giardiasis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The capacity of the parasite Giardia duodenalis to perform complex functions with minimal amounts of proteins and organelles has attracted increasing numbers of scientists worldwide, trying to explain how this parasite adapts to internal and external changes to survive. One explanation could be that G. duodenalis evolved from a structurally complex ancestor by reductive evolution, resulting in adaptation to its parasitic lifestyle. Reductive evolution involves the loss of genes, organelles, and functions that commonly occur in many parasites, by which the host renders some structures and functions redundant. However, there is increasing data that Giardia possesses proteins able to perform more than one function. During recent decades, the concept of moonlighting was described for multitasking proteins, which involves only proteins with an extra independent function(s). In this chapter, we provide an overview of unusual proteins in Giardia that present multifunctional properties depending on the location and/or parasite requirement. We also discuss experimental evidence that may allow some giardial proteins to be classified as moonlighting proteins by examining the properties of moonlighting proteins in general. Up to date, Giardia does not seem to require the numerous redundant proteins present in other organisms to accomplish its normal functions, and thus this parasite may be an appropriate model for understanding different aspects of moonlighting proteins, which may be helpful in the design of drug targets., (© 2019 Elsevier Ltd All rights reserved.)

Lactoferrin (LF) is an 80 KDa iron-binding glycoprotein that plays a significant role in the innate immune system and is considered to be an important microbicide molecule. It has been suggested to be effective in the treatment of giardiasis, an intestinal disease caused by the protozoan parasite G. lamblia. However, the molecular mechanisms by which LF exerts its effect on this parasite are unknown. Most of the microbicidal activity of human or bovine LF (hLF or bLF) has been associated with the N-terminal region of the mature LF - lactoferricin (LFcin). LFcin is produced by pepsin cleavage of the native protein in vitro and likely in vivo. In this work, we analyse the participation of the endocytic machinery of G. lamblia in the internalization of bLF and bLFcin and their effects on cell homeostasis. Our results show that, when bLF or bLFcin are internalized by receptor-mediated endocytosis, cell growth stops, and morphological changes are produced in the trophozoites, which ultimately will produce immature cysts. Our findings contribute to disclose the fine mechanism by which bLF and bLFcin may function as an antigiardial molecule and why they have therapeutic potential to eradicate giardiasis.

The manner in which membrane-associated proteins interact with the membrane defines their subcellular fate and function. This interaction relies on the characteristics of the proteins, their journey after synthesis, and their interaction with other proteins or enzymes. Understanding these properties may help to define the function of a protein and also the role of an organelle. In the case of microorganisms like protozoa parasites, it may help to understand singular features that will eventually lead to the design of parasite-specific drugs. The protozoa parasite Giardia lamblia is an example of a widespread parasite that has been infecting humans and animals from ancestral times, adjusting itself to the changes of the environment inside and outside the host. Several membrane-associated proteins have been posted in the genome database GiardiaDB, although only a few of them have been characterized. This review discusses the data regarding membrane-associated proteins in relationship with lipids and specific organelles and their implication in the discovery of anti-giardial therapies.

In eukaryotes, histone lysine methylation is associated with either active or repressed chromatin states, depending on the status of methylation. Even when the amino-terminus of Giardia lamblia histones diverges from other organisms, these regions contain lysine residues that are potential targets for methylation. When we examined the role of the histone methyltransferase 1 (HMT1) in the regulation of the encystation process by giardial histone methyltransferase 1 (GlHMT1) overexpression or downregulation, we observed an increase or a decrease in cyst production, respectively, compared to wild-type trophozoites. A time-lapse analysis of encystation showed that overexpression of GlHMT1 induced an earlier and faster process than in wild-type cells together with an upregulation of mRNA expression of cyst wall proteins. Subcellular localization studies indicated that GlHMT1-hemaglutinin was mainly associated with the nuclear and perinuclear region in both growing and encysting parasites, in agreement with bioinformatics analyses showing that GlHMT-1 possesses nuclear localization signals in addition to the classical SU(var)3-9, Enhancer-of-Zeste, Trithorax (SET), and post-SET domains. Altogether, these findings suggest that the function of HMT1 is critical for the success and timing of the encystation process, and reinforce the idea that epigenetic marks are critical for cyst formation in G. lamblia., (© 2017 Federation of European Biochemical Societies.)

In the protozoa parasite Giardia lamblia, endocytosis and lysosomal protein trafficking are vital parasite-specific processes that involve the action of the adaptor complexes AP-1 and AP-2 and clathrin. In this work, we have identified a single gene in Giardia encoding a protein containing an ENTH domain that defines monomeric adaptor proteins of the epsin family. This domain is present in the epsin or epsin-related (epsinR) adaptor proteins, which are implicated in endocytosis and Golgi-to-endosome protein trafficking, respectively, in other eukaryotic cells. We found that GlENTHp (for G. lamblia ENTH protein) localized in the cytosol, strongly interacted with PI3,4,5P3, was associated with the alpha subunit of AP-2, clathrin and ubiquitin and was involved in receptor-mediated endocytosis. It also bonded PI4P, the gamma subunit of AP-1 and was implicated in ER-to-PV trafficking. Alteration of the GlENTHp function severely affected trophozoite growth showing an unusual accumulation of dense material in the lysosome-like peripheral vacuoles (PVs), indicating that GlENTHp might be implicated in the maintenance of PV homeostasis. In this study, we showed evidence suggesting that GlENTHp might function as a monomeric adaptor protein supporting the findings of other group indicating that GlENTHp might be placed at the beginning of the ENTH family., (Copyright © 2015 Elsevier B.V. All rights reserved.)

SUMOylation, a posttranslational modification of proteins, has been recently described as vital in eukaryotic cells. In a previous work, we analyzed the role of SUMO protein and the genes encoding the putative enzymes of the SUMOylation pathway in the parasite Giardia lamblia. Although we observed several SUMOylated proteins, only the enzyme Arginine Deiminase (ADI) was confirmed as a SUMOylated substrate. ADI is involved in the survival of the parasite and, besides its role in ATP production, it also catalyzes the modification of arginine residues to citrulline in the cytoplasmic tail of surface proteins. During encystation, however, ADI translocates to the nuclei and downregulates the expression of the Cyst Wall Protein 2 (CWP2). In this work, we made site-specific mutation of the ADI SUMOylation site (Lys101) and observed that transgenic trophozoites did not translocate to the nuclei at the first steps of encystation but shuttled in the nuclei late during this process through classic nuclear localization signals. Inside the nuclei, ADI acts as a peptidyl arginine deiminase, being probably involved in the downregulation of CWPs expression and cyst wall formation. Our results strongly indicate that ADI plays a regulatory role during encystation in which posttranslational modifications of proteins are key players., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.

The retromer is a pentameric protein complex that mediates the retrograde transport of acid hydrolase receptors between endosomes and the trans-Golgi network and is conserved across all eukaryotes. Unlike other eukaryotes, the endomembrane system of Giardia trophozoite is simple and is composed only of the endoplasmic reticulum and peripheral vesicles (PVs), which may represent an ancient organellar system converging compartments such as early and late endosomes and lysosomes. Sorting and trafficking of membrane proteins and soluble hydrolases from the endoplasmic reticulum to the PVs have been described as specific and conserved but whether the giardial retromer participates in receptor recycling remains elusive. Homologs of the retromer Vacuolar Protein Sorting (Vps35p, Vps26p, and Vps29p) have been identified in this parasite. Cloning the GlVPS35 subunit and antisera production enabled the localization of this protein in the PVs as well as in the cytosol. Tagged expression of the subunits was used to demonstrate their association with membranes, and immunofluorescence confocal laser scanning revealed high degrees of colabeling between the retromer subunits and also with the endoplasmic reticulum and PV compartment markers. Protein-protein interaction data revealed interaction between the subunits of GlVPS35 and the cytosolic domain of the hydrolase receptor GlVps. Altogether our data provide original information on the molecular interactions that mediate assembly of the cargo-selective retromer subcomplex and its involvement in the recycling of the acid hydrolase receptor in this parasite., (© 2013.)

Post-translational modifications are able to regulate protein function and cellular processes in a rapid and reversible way. SUMOylation, the post-translational modification of proteins by the addition of SUMO, is a highly conserved process that seems to be present in modern cells. However, the mechanism of protein SUMOylation in earlier divergent eukaryotes, such as Giardia lamblia, is only starting to become apparent. In this work, we report the presence of a single SUMO gene encoding to SUMO protein in Giardia. Monoclonal antibodies against recombinant Giardia SUMO protein revealed the cytoplasmic localization of native SUMO in wild-type trophozoites. Moreover, the over-expression of SUMO protein showed a mainly cytoplasmic localization, though also neighboring the plasma membrane, flagella, and around and even inside the nuclei. Western blot assays revealed a number of SUMOylated proteins in a range between 20 and 120 kDa. The genes corresponding to putative enzymes involved in the SUMOylation pathway were also explored. Our results as a whole suggest that SUMOylation is a process conserved in the eukaryotic lineage, and that its study is significant for understanding the biology of this interesting parasite and the role of post-translational modification in its evolution.

In Giardia, lysosome-like peripheral vacuoles (PVs) need to specifically coordinate their endosomal and lysosomal functions to be able to successfully perform endocytosis, protein degradation and protein delivery, but how cargo, ligands and molecular components generate specific routes to the PVs remains poorly understood. Recently, we found that delivering membrane Cathepsin C and the soluble acid phosphatase (AcPh) to the PVs is adaptin (AP1)-dependent. However, the receptor that links AcPh and AP1 was never described. We have studied protein-binding to AcPh by using H6-tagged AcPh, and found that a membrane protein interacted with AcPh. This protein, named GlVps (for Giardia lamblia Vacuolar protein sorting), mainly localized to the ER-nuclear envelope and in some PVs, probably functioning as the sorting receptor for AcPh. The tyrosine-binding motif found in the C-terminal cytoplasmic tail domain of GlVps was essential for its exit from the endoplasmic reticulum and transport to the vacuoles, with this motif being necessary for the interaction with the medium subunit of AP1. Thus, the mechanism by which soluble proteins, such as AcPh, reach the peripheral vacuoles in Giardia appears to be very similar to the mechanism of lysosomal protein-sorting in more evolved eukaryotic cells.

Background: To date, eight assemblages of Giardia lamblia have been described, but only assemblages A and B are known to infect humans. Despite the fact that the genomic, biological, and clinical differences found between these two assemblages has raised the possibility that they may be considered different species, there is relatively limited information on their phenotypic differences. In the present study, we developed monoclonal antibodies against alpha-1 and beta giardin, two immunodominant proteins produced during G. lamblia infection, and studied their expression and localization in WB (assemblage A) and GS trophozoites (assemblage B)., Results: The polyclonal antibodies generated against WB trophozoites, particularly those recognizing intracellular proteins as well as the proteins present at the plasma membrane (variable-specific surface proteins), showed cross-reactivity with intracellular proteins in GS trophozoites. The use of monoclonal antibodies against beta giardin indicated ventral disc localization, particularly at the periphery in WB trophozoites. Interestingly, although beta giardin was also restricted to the ventral disc in GS trophozoites, the pattern of localization clearly differed in this assemblage. On the other hand, monoclonal antibodies against alpha-1 giardin showed plasma membrane localization in both assemblages with the bare area of GS trophozoites also being distinguished. Moreover, the same localization at the plasma membrane was observed in Portland-1 (Assemblage A) and in P15 (Assemblage E) trophozoites., Conclusions: We found differences in localization of the beta giardin protein between assemblages A and B, but the same pattern of localization of alpha-1 giardin in strains from Assemblages A, B and E. These findings reinforce the need for more studies based on phenotypic characteristics in order to disclose how far one assemblage is from the other.

As Giardia lamblia is unable to synthesize cholesterol de novo, this steroid might be obtained from the host's intestinal milieu by endocytosis of lipoproteins. In this work, we identified a putative Giardia lamblia low-density lipoprotein receptor-related proteins (GlLRP), a type I membrane protein, which shares the substrate N-terminal binding domain and a FXNPXY-type endocytic motif with human LRPs. Expression of tagged GlLRP showed that it was localized predominantly in the endoplasmic reticulum, lysosomal-like peripheral vacuoles and plasma membrane. However, the FXNPXY-deleted GlLRP was retained at the plasma membrane suggesting that it is abnormally transported and processed. The low-density lipoprotein and chylomicrons interacted with GlLRP, with this interaction being necessary for lipoprotein internalization and cell proliferation. Finally, we show that GlLRP binds directly to the medium subunit of Giardia adaptor protein 2, indicating that receptor-mediated internalization occurs through an adaptin mechanism., (© 2011 Blackwell Publishing Ltd.)

In the relationships between host and parasites, there is a cross-talk that involves diverse mechanisms developed by two different genetic systems during years of evolution. On the one hand, immunocompetent hosts have developed effective innate and acquired immune responses that are used to restrict or avoid parasitism. On the other hand, parasites evade the immune response, expressing different antigens on their surface or by using other specific mechanisms, such as nutrient depletion. In this review, we analyze the survival mechanisms used by the protozoan parasite Giardia lamblia during infection. In particular, we examine the multiple roles played by the enzyme arginine deiminase during colonization of the gut, also involving the parasite's mechanism of antigenic variation. Potential drug targets for the treatment of giardiasis are also discussed.

The protozoan parasite Giardia lamblia uses arginine deiminase (ADI) to produce energy from free L-arginine under anaerobic conditions. In this work, we demonstrate that, in addition to its known role as a metabolic enzyme, it also functions as a peptidylarginine deiminase, converting protein-bound arginine into citrulline. G. lamblia ADI specifically binds to and citrullinates the arginine in the conserved CRGKA tail of variant-specific surface proteins (VSPs), affecting both antigenic switching and antibody-mediated cell death. During encystation, ADI translocates from the cytoplasm to the nuclei and appears to play a regulatory role in the expression of encystation-specific genes. ADI is also sumoylated, which might modulate its activity. Our findings reveal a dual role played by ADI and define novel regulatory pathways used by Giardia for survival.

During aging, there is an increased rise in susceptibility to infectious diseases. However, it is still unresolved whether standard vaccine adjuvants are efficient in the elderly. We report that immunization with OVA plus synthetic oligodeoxinucleotides containing immunostimulatory CpG motifs (CpG-ODN) stimulates specific Th1 response in aged mice. The immunization with OVA/CpG-ODN induced an increase in CD19+ cells. These cells from aged mice in vivo captured OVA-FITC as efficiently as those from young mice. Interestingly, naïve aged mice, which showed Th2 polarization and up-regulation of the expression of GATA-3, with immunization with OVA/CpG-ODN inducing Th1-specific response but maintaining a Th2 pattern in response to a non-specific stimulator of T cells. Our data suggest that the response elicited by CpG-ODN in aged mice have similar properties to the response developed in young mice, emphasizing the importance of the use of this adjuvant during aging.

We have previously demonstrated that subcutaneously administered ovalbumin (OVA) plus synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG-ODN) as adjuvant stimulate cellular and humoral immunity and promote T helper cell type 1 differentiation in aged mice. The present study assessed the ability of CpG-ODN to induce an OVA-specific immune response after oral immunization in young (3-month-old) and aged (18-month-old) BALB/c mice. Oral OVA/CpG-ODN immunization induces a similar OVA-specific T cell-proliferative response (in mucosal and systemic tissues), immunoglobulin G (IgG) in plasma, and IgA in intestinal washes in both groups of ages. The OVA-specific humoral immune response observed in aged mice was similar to the one observed in young mice, peaking at day 7 after the last oral immunization and was present over 40 days after the last oral immunization. The pattern of cytokines released in culture supernatants in both groups of mice was similar, with specific interferon-gamma secretion in the absence of interleukin-5 responses. These results provide evidence that orally administered OVA/CpG-ODN induces a young-like, specific, immune response against OVA in aged mice, showing that CpG-ODN might be used as a mucosal adjuvant during aging.

We examined whether CpG-DNA could be used as adjuvant to induce a T helper cell type-1 (Th1) immunity in aged BALB/c mice that showed a Th2 polarization. Bordetella pertussis and complete Freund's adjuvant (CFA) were used as well. Immunization with ovalbumin (OVA)/CpG-DNA showed that the immunoglobulin G (IgG)2a/IgG1 ratio and OVA-specific T cell response were similar in young and aged mice. OVA/CpG-DNA induced the secretion of interferon-gamma (IFN-gamma) and absence of interleukin (IL)-5. Similar results were found in mice immunized with OVA/CFA. When mice were immunized with OVA/B. pertussis, we found that the IgG2a/IgG1 ratio and OVA-specific T cell response were lower in aged mice and elicited IFN-gamma and IL-5. In vitro CpG-DNA stimulated antigen-presenting cells to display IL-12 and up-regulate the expression of major histocompatibility complex class II and B7-2 on B cells as efficiently in aged as in young mice, but the up-regulation of B7-1 was stronger in aged mice. The findings demonstrate that CpG-DNA is able to induce a young-like Th1 specific immune response in aged mice.