Improved biodistribution and enhanced immune response of subunit vaccine using a nanostructure formed by self-assembly of ascorbyl palmitate.

New adjuvant strategies are needed to improve protein-based subunit vaccine immunogenicity. We examined the potential to use nanostructure of 6-O-ascorbyl palmitate to formulate ovalbumin (OVA) protein and an oligodeoxynucleotide (CpG-ODN) (OCC). In mice immunized with a single dose, OCC elicited an OVA-specific immune response superior to OVA/CpG-ODN solution (OC). Rheological studies demonstrated OCC's self-assembling viscoelastic properties. Biodistribution studies indicated that OCC prolonged OVA and CpG-ODN retention at injection site and lymph nodes, reducing systemic spread. Flow-cytometry assays demonstrated that OCC promoted OVA and CpG-ODN co-uptake by Ly6ChiCD11bhiCD11c+ monocytes. OCC and OC induced early IFN-γ in lymph nodes, but OCC led to higher concentration. Conversely, mice immunized with OC showed higher serum IFN-γ concentration compared to those immunized with OCC. In mice immunized with OCC, NK1.1+ cells were the IFN-γ major producers, and IFN-γ was essential for OVA-specific IgG2c switching. These findings illustrate how this nanostructure improves vaccine's response.

COVID-19 patients display changes in lymphocyte subsets with a higher frequency of dysfunctional CD8lo T cells associated with disease severity.

This work examines cellular immunity against SARS-CoV-2 in patients from Córdoba, Argentina, during two major waves characterized by different circulating viral variants and different social behavior. Using flow cytometry, we evaluated the main lymphocyte populations of peripheral blood from hospitalized patients with moderate and severe COVID-19 disease. Our results show disturbances in the cellular immune compartment, as previously reported in different cohorts worldwide. We observed an increased frequency of B cells and a significant decrease in the frequency of CD3+ T cells in COVID-19 patients compared to healthy donors (HD). We also found a reduction in Tregs, which was more pronounced in severe patients. During the first wave, the frequency of GZMB, CD107a, CD39, and PD-1-expressing conventional CD4+ T (T conv) cells was significantly higher in moderate and severe patients than in HD. During the second wave, only the GZMB+ T conv cells of moderate and severe patients increased significantly. In addition, these patients showed a decreased frequency in IL-2-producing T conv cells. Interestingly, we identified two subsets of circulating CD8+ T cells with low and high CD8 surface expression in both HD and COVID-19 patients. While the percentages of CD8hi and CD8lo T cells within the CD8+ population in HD are similar, a significant increase was observed in CD8lo T cell frequency in COVID-19 patients. CD8lo T cell populations from HD as well as from SARS-CoV-2 infected patients exhibited lower frequencies of the effector cytokine-producing cells, TNF, IL-2, and IFN-γ, than CD8hi T cells. Interestingly, the frequency of CD8lo T cells increased with disease severity, suggesting that this parameter could be a potential marker for disease progression. Indeed, the CD8hi/CD8lo index helped to significantly improve the patient's clinical stratification and disease outcome prediction. Our data support the addition of, at least, a CD8hi/CD8lo index into the panel of biomarkers commonly used in clinical labs, since its determination may be a useful tool with impact on the therapeutic management of the patients.

High-dimensional analysis of 16 SARS-CoV-2 vaccine combinations reveals lymphocyte signatures correlating with immunogenicity.

The range of vaccines developed against severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) provides a unique opportunity to study immunization across different platforms. In a single-center cohort, we analyzed the humoral and cellular immune compartments following five coronavirus disease 2019 (COVID-19) vaccines spanning three technologies (adenoviral, mRNA and inactivated virus) administered in 16 combinations. For adenoviral and inactivated-virus vaccines, heterologous combinations were generally more immunogenic compared to homologous regimens. The mRNA vaccine as the second dose resulted in the strongest antibody response and induced the highest frequency of spike-binding memory B cells irrespective of the priming vaccine. Priming with the inactivated-virus vaccine increased the SARS-CoV-2-specific T cell response, whereas boosting did not. Distinct immune signatures were elicited by the different vaccine combinations, demonstrating that the immune response is shaped by the type of vaccines applied and the order in which they are delivered. These data provide a framework for improving future vaccine strategies against pathogens and cancer.

Different cytokine and chemokine profiles in hospitalized patients with COVID-19 during the first and second outbreaks from Argentina show no association with clinical comorbidities.

Background: COVID-19 severity has been linked to an increased production of inflammatory mediators called "cytokine storm". Available data is mainly restricted to the first international outbreak and reports highly variable results. This study compares demographic and clinical features of patients with COVID-19 from Córdoba, Argentina, during the first two waves of the pandemic and analyzes association between comorbidities and disease outcome with the "cytokine storm", offering added value to the field. Methods: We investigated serum concentration of thirteen soluble mediators, including cytokines and chemokines, in hospitalized patients with moderate and severe COVID-19, without previous rheumatic and autoimmune diseases, from the central region of Argentina during the first and second infection waves. Samples from healthy controls were also assayed. Clinical and biochemical parameters were collected. Results: Comparison between the two first COVID-19 waves in Argentina highlighted that patients recruited during the second wave were younger and showed less concurrent comorbidities than those from the first outbreak. We also recognized particularities in the signatures of systemic cytokines and chemokines in patients from both infection waves. We determined that concurrent pre-existing comorbidities did not have contribution to serum concentration of systemic cytokines and chemokines in COVID-19 patients. We also identified immunological and biochemical parameters associated to inflammation which can be used as prognostic markers. Thus, IL-6 concentration, C reactive protein level and platelet count allowed to discriminate between death and discharge in patients hospitalized with severe COVID-19 only during the first but not the second wave. Conclusions: Our data provide information that deepens our understanding of COVID-19 pathogenesis linking demographic features of a COVID-19 cohort with cytokines and chemokines systemic concentration, presence of comorbidities and different disease outcomes. Altogether, our findings provide information not only at local level by delineating inflammatory/anti-inflammatory response of patients but also at international level addressing the impact of comorbidities and the infection wave in the variability of cytokine and chemokine production upon SARS-CoV-2 infection.

TLR9 activation is required for cytotoxic response elicited by baculovirus capsid display.

Although the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects lepidopteran invertebrates as natural hosts, represents an efficient vector for vaccine development. Baculovirus surface display induces strong humoral responses against viruses and parasites. A novel strategy based on capsid display carrying foreign antigens in the AcMNPV particle further improved the immune response by eliciting CD8+ T cell activation. In this study, we analyze the intracellular mechanisms and signalling pathways involved in CD8+ T cell activation by capsid display. Our results show that baculovirus can attach to the cell surface, enter dendritic cells (DCs), transit within endocytic vesicles and escape to the cytosol for further degradation by the proteasome. We found that the availability of viral proteins, endosomal acidification, and proteasome activity are needed for efficient Major Histocompatibility Complex class-I presentation by baculovirus carrying Ovalbumin in the viral capsid. Importantly, we demonstrated with this strategy that the induction of cytotoxic T cells and IL-12 production by DCs are TLR9-dependent and STING-independent. Finally, our study shows differential intracellular processing for capsid and surface baculovirus proteins in DCs and highlights the role of different danger receptors during cytotoxic T cell priming through the capsid display delivery system, which could lead to improved baculovirus-based vaccines development.

Spray-drying-microencapsulated Minthostachys verticillata essential oil and limonene as innovative adjuvant strategy to bovine mastitis vaccines.

Design of innovative adjuvant strategies with an appropriate safety profile is relevant to developed subunit or inactivated microorganism vaccines for bovine mastitis. Minthostachys verticillata essential oil (EO) has demonstrated ability to stimulate the innate immune response and adjuvant effect similar to Al(OH)3. Here we evaluated the adjuvant effect of EO and its metabolite, limonene (L) alone and microencapsulated by spray-drying, using an inactivated Enterococcus faecium strain bovine-mastitis inducer. The gas chromatography-mass spectrometry analysis showed that microencapsulation process did not alter the EO or L chemistry. Microencapsulated EO (McEO) or L (McL) (2.0, 2.5 and 5.0 mg/ml) decreased the viability of bovine mammary gland epithelial cells in a dose-dependent way. Balb/c mice (n = 32) were subcutaneously inoculated (day 0) and revaccinated (day 14 and 28) with saline solution, inactivated bacteria alone or combined with Incomplete Freund's Adjuvant; EO or L (2.5 mg/ml); McEO or McL (5.0 mg/ml); or microcapsule wall material (Mc) alone (2.5 mg/ml). EO, L, McEO and McL stimulated E. faecium-specific IgG (IgG1 or IgG2a) with opsonizing capacity and increased the proportion of CD4+ and CD8+ T cells producers of IFN-γ. Microencapsulation was an effective strategy to increase the adjuvant potential of EO or L. These new adjuvants deserve further study to evaluate their incorporation into vaccines for bovine mastitis.

Corrigendum to "CpG-ODN formulated with a nanostructure as adjuvant for anticrotalic serum production. Studies in mice" [Toxicon 215 (2022) 28-36].

Antivenom is the only safe and effective treatment to neutralize snake venom. Specific anti-venom used to treat snake bite is usually obtained from horses after hyperimmunization with crude snake venom in combination with Freund's Adjuvant. Freund's complete and incomplete adjuvant can cause severe local and systemic acute and chronic inflammation, its potentially severe inflammatory effects have led many researchers to seek alternative immunological adjuvants. CpG-ODN formulated in a 6-O-ascorbyl palmitate nanostructure (Coa-ASC16) was more efficient as adjuvant than CpG-ODN alone using ovalbumin (OVA) as an antigen model. Particularly, immunization of mice with OVA/CpG-ODN/Coa-ASC16 resulted in high OVA specific antibody titers and IFN-γ and IL-17 secretion compared to immunization with OVA/CpG-ODN. First of all, we estimated the effect of Coa-ASC16 nanostructure preparation on venom activity. Additionally, in order to evaluate the immune response induced by this adjuvant strategy using Crotalus durissus terrificus (C. d. terrificus) venom (CdtV), we determined the titer of antibodies (IgG, IgG1 and IgG2) and their specificity. BALB/c mice were subcutaneously immunizated on days 0, 15 and 30 with CdtV/CpG-ODN/Coa-ASC16 or CdtV/Freund's Adyuvant (complete first and incomplete-booster) (dose/mice: CdtV: 6-10 µg, CpG-ODN: 30 µg). On day 45 mice were sacrificed. The neutralizing ability of serum from animals immunized with CdtV/CpG-ODN/Coa-ASC16 or CdtV/Freund's adjuvant was tested against PLA2 activity and lethality. In both immunized group mice, the antibody titers in plasma were high (1 × 105), with a similar IgG1/IgG2a ratio. The antibodies recognized phospholipase A2 and thrombin-like proteins, the main toxins from C. d. terrificus venom. Macroscopic and microscopic analysis at the site of injection of mice injected with Freund's adjuvant showed local damage (with non-infectious abscesses) and hypertrophy of inguinal lymph nodes, whereas mice injected with CpG-ODN/Coa/ASC16 did not. Our results show that CpG-ODN/Coa-ASC16 produces a humoral response as strong and specific as Freund's adjuvant, with minor or null local deleterious effect, demonstrating the potentiality and advisability of an alternative formulation as a new adjuvant option for future immunizations to produce C. d. terrificus antivenom.

Polymeric antigen BLSOmp31 formulated with class B CpG-ODN in a nanostructure (BLSOmp31/CpG-ODN/Coa-ASC16) administered by parenteral or mucosal routes confers protection against Brucella ovis in Balb/c mice.

Previously, we demonstrated that the chimera BLSOmp31 formulated in chitosan microspheres or Poloxamer407-Chitosan administered via the nasal and the ocular mucosa conferred partial protection in sheep against B. ovis. In this work, we tested a new delivery system for mucosal immunization with BLSOmp31 in the murine model to improve the efficacy of previously used formulations. First, we evaluated the protective efficacy against B. ovis induced by BLSOmp31 administered by the subcutaneous route using either BLSOmp31 alone, co-administered with immunostimulatory synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG-ODN) or with CpG-ODN in a nanostructure called Coa-ASC16 compared with BLSOmp31 emulsified in Incomplete Freund Adjuvant. Then, we evaluated the protection conferred by the best performing formulation (BLSOmp31/CpG-ODN/Coa-ASC16) administered by both subcutaneous and ocular routes. BLSOmp31/CpG-ODN/Coa-ASC16 injected subcutaneously did not induce higher IgG antibody levels compared to BLSOmp31 alone or BLSOmp31/CpG-ODN but it did stimulate a mixed immune Th1-Th2 response with the highest levels of IFN-É£ and conferred significant protection against the B. ovis challenge. Although ocular instillation of BLSOmp31/CpG-ODN/Coa-ASC16 showed a similar degree of protection compared to the parenteral route (3.66 and 3.60 logs of protection, respectively), it induced lower levels in serum of specific IgG (with mixed IgG1/IgG2a) and IgA antibodies and, less IFN-É£ and IL-4 than the subcutaneous route. No antibodies were detected in vaginal lavages or saliva. Fecal antigen-specific IgA was slightly higher in mice immunized with BLSOmp31/CpG-ODN/Coa-ASC16 subcutaneously compared with the ocular route. These results indicate that BLSOmp31/CpG-ODN/Coa-ASC16 was a safe and effective vaccine against B. ovis in mice.

A natural oil increases specific anti-OVA IgG levels and induces a cellular immune response combined with aluminum hydroxide.

The efficacy of certain vaccines is improved by the use of adjuvants. Nowadays, the development of new, effective, and safe adjuvants that stimulate the innate immune response is researched. In this context, medicinal plants appear as a suitable alternative. Minthostachys verticillata essential oil (EO) has demonstrated the ability to modulate mechanisms of the innate immune response. Thus, the present work aimed to evaluate the EO adjuvant effect on humoral and cellular immunity, coadministered with OVA as antigen. The chemical analysis of EO by gas chromatography-mass spectrometry revealed a predominant pulegone-menthone chemotype. EO (1.25, 2.5, or 5.0 mg/ml) did not alter the viability of murine fibroblasts (3T3 cell line) neither showed signs of toxicity in Balb/c mice inoculated subcutaneously. The serum of mice immunized with OVA + EO showed increased levels of anti-OVA-specific antibodies of IgG1 subclass compared with the mice immunized with OVA alone revealing an adjuvant effect of EO. The delayed type hypersensitivity showed that the combination OVA + Al(OH)3  + EO was the best to induce a cellular immune response that extended until 48 h postinjection of OVA. M. verticillata EO appears as a new, safe, and effective adjuvant, which should continue to be studied for their possible future incorporation into vaccine formulations.

A Vaccine Based on Kunitz-Type Molecule Confers Protection Against Fasciola hepatica Challenge by Inducing IFN-γ and Antibody Immune Responses Through IL-17A Production.

Fasciola hepatica is helminth parasite found around the world that causes fasciolosis, a chronic disease affecting mainly cattle, sheep, and occasionally humans. Triclabendazole is the drug of choice to treat this parasite. However, the continuous use of this drug has led to the development of parasite resistance and, consequently, the limitation of its effectiveness. Hence, vaccination appears as an attractive option to develop. In this work, we evaluated the potential of F. hepatica Kunitz-type molecule (FhKTM) as an antigen formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate ester (Coa-ASC16) and the synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) during an experimental model of fasciolosis in mice, and we further dissected the immune response associated with host protection. Our results showed that immunization of mice with FhKTM/CpG-ODN/Coa-ASC16 induces protection against F. hepatica challenge by preventing liver damage and improving survival after F. hepatica infection. FhKTM/CpG-ODN/Coa-ASC16-immunized mice elicited potent IFN-γ and IL-17A with high levels of antigen-specific IgG1, IgG2a, and IgA serum antibodies. Strikingly, IL-17A blockade during infection decreased IgG2a and IgA antibody levels as well as IFN-γ production, leading to an increase in mortality of vaccinated mice. The present study highlights the potential of a new vaccine formulation to improve control and help the eradication of F. hepatica infection, with potential applications for natural hosts such as cattle and sheep.

Neutrophils Which Migrate to Lymph Nodes Modulate CD4+ T Cell Response by a PD-L1 Dependent Mechanism.

It is well known that neutrophils are rapidly recruited to a site of injury or infection and perform a critical role in pathogen clearance and inflammation. However, they are also able to interact with and regulate innate and adaptive immune cells and some stimuli induce the migration of neutrophils to lymph nodes (LNs). Previously, we demonstrated that the immune complex (IC) generated by injecting OVA into the footpad of OVA/CFA immunized mice induced the migration of OVA+ neutrophils to draining LNs (dLNs). Here we investigate the effects of these neutrophils which reach dLNs on CD4+ T cell response. Our findings here strongly support a dual role for neutrophils in dLNs regarding CD4+ T cell response modulation. On the one hand, the CD4+ T cell population expands after the influx of OVA+ neutrophils to dLNs. These CD4+ T cells enlarge their proliferative response, activation markers and IL-17 and IFN-γ cytokine production. On the other hand, these neutrophils also restrict CD4+ T cell expansion. The neutrophils in the dLNs upregulate PD-L1 molecules and are capable of suppressing CD4+ T cell proliferation. These results indicate that neutrophils migration to dLNs have an important role in the homeostasis of adaptive immunity. This report describes for the first time that the influx of neutrophils to dLNs dependent on IC presence improves CD4+ T cell response, at the same time controlling CD4+ T cell proliferation through a PD-L1 dependent mechanism.

Class-B CpG-ODN Formulated With a Nanostructure Induces Type I Interferons-Dependent and CD4+ T Cell-Independent CD8+ T-Cell Response Against Unconjugated Protein Antigen.

There is a need for new vaccine adjuvant strategies that offer both vigorous antibody and T-cell mediated protection to combat difficult intracellular pathogens and cancer. To this aim, we formulated class-B synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) with a nanostructure (Coa-ASC16 or coagel) formed by self-assembly of 6-0-ascorbyl palmitate ester. Our previous results demonstrated that mice immunized with ovalbumin (OVA) and CpG-ODN formulated with Coa-ASC16 (OVA/CpG-ODN/Coa-ASC16) elicited strong antibodies (IgG1 and IgG2a) and Th1/Th17 cellular responses without toxic systemic effects. These responses were superior to those induced by a solution of OVA with CpG-ODN or OVA/CpG-ODN formulated with aluminum salts. In this study, we investigated the capacity of this adjuvant strategy (CpG-ODN/Coa-ASC16) to elicit CD8+ T-cell response and some of the underlying cellular and molecular mechanisms involved in adaptive response. We also analyzed whether this adjuvant strategy allows a switch from an immunization scheme of three-doses to one of single-dose. Our results demonstrated that vaccination with OVA/CpG-ODN/Coa-ASC16 elicited an antigen-specific long-lasting humoral response and importantly-high quality CD8+ T-cell immunity with a single-dose immunization. Moreover, Coa-ASC16 promoted co-uptake of OVA and CpG-ODN by dendritic cells. The CD8+ T-cell response induced by OVA/CpG-ODN/Coa-ASC16 was dependent of type I interferons and independent of CD4+ T-cells, and showed polyfunctionality and efficiency against an intracellular pathogen. Furthermore, the cellular and humoral responses elicited by the nanostructured formulation were IL-6-independent. This system provides a simple and inexpensive adjuvant strategy with great potential for future rationally designed vaccines.

Evaluation of the Performance of an Ophthalmic Thermosensitive Hydrogel Containing Combination of Suramin and Bevacizumab.

Suramab (SUM) is a new pharmaceutical combination made up of suramine (SUR) and bevacizumab (BVM), which showed a high synergistic effect when administered jointly. As the pharmaceutical vehicle, poloxamer aqueous dispersions were used since this system is able to maintain their fluidity at low temperatures (<15ºC) but which become gel in the corporal environment (>35ºC). In the present study we aimed at evaluating the effect of Poloxamer to prolong the effect of SUM. These formulations were characterized using rheological, biopharmaceutical (drug release) and morphological (SEM) technique. Corneal NV was induced in Sprague Dawley rats Corneal. At 15 days of follow up animals were sacrificed and perfused with black drawing ink. Digital photographs were taken and the area of neovascularisation (ANV) was calculated using the image programmed. The rheological behavior was influenced by the addition of drugs, resulting in a decrease in the gelation temperature (Tsol/gel). Both drugs were released from poloxamer gels by means of an anomalous mechanism. However, BVM was released faster than SUR, with their combination (SUM) to appearing to reduce delivery, probably due to interactions between the drugs or with the polymeric matrix. The in vivo studies showed that SUM-poloxamer gel was able to increase the corneal antiangiogenic effect compared to the SUM solution and BVM alone at 15 days of follow-up. Furthermore no injurious effects were observed in the histological tissue examination after drug administration. The presence of Poloxamer, known to modulate control release of biological agents, seems to have a favorable effect on SUM subconjunctival administered.

Aging Impairs the Ability of Conventional Dendritic Cells to Cross-Prime CD8+ T Cells upon Stimulation with a TLR7 Ligand.

The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs) in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8+ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α+ cDCs from old mice have an impaired ability to activate naïve CD8+ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.

A liquid crystal of ascorbyl palmitate, used as vaccine platform, provides sustained release of antigen and has intrinsic pro-inflammatory and adjuvant activities which are dependent on MyD88 adaptor protein.

Modern subunit vaccines require the development of new adjuvant strategies. Recently, we showed that CpG-ODN formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate (Coa-ASC16) is an attractive system for promoting an antigen-specific immune response to weak antigens. Here, we showed that after subcutaneous injection of mice with near-infrared fluorescent dye-labeled OVA antigen formulated with Coa-ASC16, the dye-OVA was retained at the injection site for a longer period than when soluble dye-OVA was administered. Coa-ASC16 alone elicited a local inflammation, but how this material triggers this response has not been described yet. Although it is known that some materials used as a platform are not immunologically inert, very few studies have directly focused on this topic. In this study, we explored the underlying mechanisms concerning the interaction between Coa-ASC16 and the immune system and we found that the whole inflammatory response elicited by Coa-ASC16 (leukocyte recruitment and IL-1ß, IL-6 and IL-12 production) was dependent on the MyD88 protein. TLR2, TLR4, TLR7 and NLRP3-inflammasome signaling were not required for induction of this inflammatory response. Coa-ASC16 induced local release of self-DNA, and in TLR9-deficient mice IL-6 production was absent. In addition, Coa-ASC16 revealed an intrinsic adjuvant activity which was affected by MyD88 and IL-6 absence. Taken together these results indicate that Coa-ASC16 used as a vaccine platform is effective due to the combination of the controlled release of antigen and its intrinsic pro-inflammatory activity. Understanding how Coa-ASC16 works might have significant implications for rational vaccine design.

Expansion of myeloid-derived suppressor cells with arginase activity lasts longer in aged than in young mice after CpG-ODN plus IFA treatment.

As we age, the homeostatic function of many systems in the body, such as the immune function declines, which in turn contributes to augment susceptibility to disease. Here we describe that challenging aged mice with synthetic oligodeoxynucleotides containing unmethylated cytosine guanine motifs (CpG-ODN) emulsified in incomplete Freund's adjuvant (IFA), (CpG-ODN+IFA) an inflammatory stimulus, led to the expansion of CD11b+Gr1+ myeloid cells with augmented expression of CD124 and CD31. These myeloid cells lasted longer in the spleen of aged mice than in their younger counterparts after CpG-ODN+IFA treatment and were capable of suppressing T cell proliferative response by arginase induction. Myeloid cells from aged CpG-ODN+IFA-treated mice presented increased arginase-1 expression and enzyme activity. In addition, we found a different requirement of cytokines for arginase induction according to mice age. In myeloid cells from young treated mice, arginase-1 expression and activity is induced by the presence of each IL-4 or IL-6 in their extracellular medium, unlike myeloid cells from aged treated mice which need the presence of both IL-4 and IL-6 together for arginase induction and suppressor function.

Neutrophils exhibit differential requirements for homing molecules in their lymphatic and blood trafficking into draining lymph nodes.

Although much is described about the molecules involved in neutrophil migration from circulation into tissues, less is known about the molecular mechanisms that regulate neutrophil entry into lymph nodes (LNs) draining a local inflammatory site. In this study, we investigated neutrophil migration toward LNs in a context of inflammation induced by immunization of BALB/c mice with OVA emulsified in CFA. We demonstrated that neutrophils can enter LNs of OVA/CFA-immunized mice not only via lymphatic vessels but also from blood, across high endothelial venules. By adoptive transfer experiments, we showed that this influx was dependent on an inflammatory-state condition and previous neutrophil stimulation with OVA/anti-OVA immune complexes. Importantly, we have demonstrated that, in the migratory pattern to LNs, neutrophils used L-selectin and P-selectin glycoprotein ligand-1, macrophage-1 Ag and LFA-1 integrins, and CXCR4 to get access across high endothelial venules, whereas macrophage-1 Ag, LFA-1, and CXCR4 were involved in their trafficking through afferent lymphatics. Strikingly, we found that stimulation with immune complexes significantly upregulated the expression of sphingosine-1-phosphate receptor 4 on neutrophils, and that treatment with the sphingosine-1-phosphate agonist FTY720 altered neutrophil LN-homing ability. These findings summarized in this article disclose the molecular pattern that controls neutrophil recruitment to LNs.

Novel poly(NIPA-co-AAc) functional hydrogels with potential application in drug controlled release.

The synthesis, characterization and properties of pH/thermosensitive hydrogels based on acrylic acid (AAc) and N-isopropylacrylamide (NIPA) using (+)-N,N'-diallyltartramide (DAT) as cross-linking agent and water as solvent, are presented in this article. Subsequently, the incorporation of ofloxacin (OFL) as model drug to evaluate the drug load capacity of hydrogels and the in vitro release from OFL-polymer conjugate are presented in order to define potential pharmaceutical applications. Interestingly, the incorporation of AAc diversified the properties of NIPA-based hydrogels allowing ionic interaction of these new materials with drugs of opposite charge and produced different release profiles at pH 1.2 and 6.8 simulated physiological media.

SUMOylation and deimination of proteins: two epigenetic modifications involved in Giardia encystation.

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.