An in vitro model mimicking the complement system to favor directed phagocytosis of unwanted cells

BACKGROUND: Opsonization, is the molecular mechanism by which target molecules promote interactions with phagocyte cell surface receptors to remove unwanted cells by induced phagocytosis. We designed an in vitro system to demonstrate that this procedure could be driven to eliminate adipocytes, using peptides mimicking regions of the complement protein C3b to promote opsonization and enhance phagocytosis. Two cell lines were used: (1) THP-1 monocytes differentiated to macrophages, expressing the C3b opsonin receptor CR1 in charge of the removal of unwanted coated complexes; (2) 3T3-L1 fibroblasts differentiated to adipocytes, expressing AQP7, to evaluate the potential of peptides to stimulate opsonization. (3) A co-culture of the two cell lines to demonstrate that phagocytosis could be driven to cell withdrawal with high efficiency and specificity. RESULTS: An array of peptides were designed and chemically synthesized p3691 and p3931 joined bound to the CR1 receptor activating phagocytosis (p < 0.033) while p3727 joined the AQP7 protein (p < 0.001) suggesting that opsonization of adipocytes could occur. In the co-culture system p3980 and p3981 increased lipid uptake to 91.2% and 89.0%, respectively, as an indicator of potential adipocyte phagocytosis. CONCLUSIONS: This in vitro model could help understand the receptor­ligand interaction in the withdrawal of unwanted macromolecules in vivo. The adipocyte-phagocytosis discussed may help to control obesity, since peptides of C3b stimulated the CR1 receptor, promoting opsonisation and phagocytosis of lipidcontaining structures, and recognition of AQP7 in the differentiated adipocytes, favored the phagocytic activity of macrophages, robustly supported by the co-culture strategy.

Protein corona meets freeze-drying: overcoming the challenges of colloidal stability, toxicity, and opsonin adsorption.

Freeze-drying of nanoparticle suspensions is capable of generating stable nanoformulations with improved storage times and easier transportation. Nonetheless, nanoparticle aggregation is likely induced during freeze-drying, which reduces its redispersibility upon reconstitution and leads to undesirable effects such as non-specific toxicity and impaired efficacy. In this work, bovine serum albumin (BSA) is described as a suitable protectant for silica nanoparticles (SNPs), which result in solid structures with excellent redispersibility and negligible signs of aggregation even when longer storage times are considered. We experimentally demonstrated that massive system aggregation can be prevented when a saturated BSA corona around the nanoparticle is formed before the lyophilization process. Furthermore, the BSA corona is able to suppress non-specific interactions between these nanoparticles and biological systems, as evidenced by the lack of residual cytotoxicity, hemolytic activity and opsonin adsorption. Hence, BSA can be seriously considered for industry as an additive for nanoparticle freeze-drying since it generates solid and redispersible nanoformulations with improved biocompatibility.

IL-17A and complement contribute to killing of pneumococci following immunization with a pneumococcal whole cell vaccine.

The pneumococcal whole cell vaccine (PWCV) has been investigated as an alternative to polysaccharide-based vaccines currently in use. It is a non-encapsulated killed vaccine preparation that induces non-capsular antibodies protecting mice against invasive pneumococcal disease (IPD) and reducing nasopharyngeal (NP) carriage via IL-17A activation of mouse phagocytes. Here, we show that PWCV induces antibody and IL-17A production to protect mice against challenge in a fatal aspiration-sepsis model after only one dose. We observed protection even with a boiled preparation, attesting to the stability and robustness of the vaccine. PWCV antibodies were shown to bind to different encapsulated strains, but complement deposition on the pneumococcal surface was observed only on serotype 3 strains; using flow cytometer methodology, variations in PWCV quality, as in the boiled vaccine, were detected. Moreover, anti-PWCV induces phagocytosis of different pneumococcal serotypes by murine peritoneal cells in the presence of complement or IL-17A. These findings suggest that complement and IL-17A may participate in the process of phagocytosis induced by PWCV antibodies. IL-17A can stimulate phagocytic cells to kill pneumococcus and this is enhanced in the presence of PWCV antibodies bound to the bacterial cell surface. Our results provide further support for the PWCV as a broad-range vaccine against all existing serotypes, potentially providing protection for humans against NP colonization and IPD. Additionally, we suggest complement deposition assay as a tool to detect subtle differences between PWCV lots.

Association of serum bactericidal antibody and opsonic antibody levels after Neisseria meningitidis serogroup C conjugate vaccine in Brazilian children and adolescents infected or not infected with HIV.

Neisseria meningitidis serogroup C (MenC) is the main causative agent of meningitis in Brazil. HIV infection affects the quality of the immune system. HIV+ children have an increased risk of infection to encapsulated bacteria such as N. meningitidis. We evaluated the opsonic antibody (OPA) levels and its correlation with serum bactericidal antibody (SBA) levels induced by one and two doses of a MenC conjugate vaccine in children and adolescents HIV+ and HIV-exposed but uninfected children (HEU) group. Overall the data show the importance of two doses of vaccine for HIV+ individuals. About 79% and 58% of HIV+ patients showed SBA and OPA positive response after two doses of vaccine, respectively. For HEU group, 62% and 41% of patients showed SBA and OPA positive response after one dose of vaccine, respectively. A positive and significant association between SBA and OPA levels was seen after two doses of vaccine in HIV+ patients.

Anti-erythrocyte antibodies may contribute to anaemia in Plasmodium vivax malaria by decreasing red blood cell deformability and increasing erythrophagocytosis.

BACKGROUND: Plasmodium vivax accounts for the majority of human malaria infections outside Africa and is being increasingly associated in fatal outcomes with anaemia as one of the major complications. One of the causes of malarial anaemia is the augmented removal of circulating non-infected red blood cells (nRBCs), an issue not yet fully understood. High levels of auto-antibodies against RBCs have been associated with severe anaemia and reduced survival of nRBCs in patients with falciparum malaria. Since there are no substantial data about the role of those antibodies in vivax malaria, this study was designed to determine whether or not auto-antibodies against erythrocytes are involved in nRBC clearance. Moreover, the possible immune mechanisms elicited by them that may be associated to induce anaemia in P. vivax infection was investigated. METHODS: Concentrations of total IgG were determined by sandwich ELISA in sera from clinically well-defined groups of P. vivax-infected patients with or without anaemia and in healthy controls never exposed to malaria, whereas the levels of specific IgG to nRBCs were determined by cell-ELISA. Erythrophagocytosis assay was used to investigate the ability of IgGs purified from each studied pooled sera in enhancing nRBC in vitro clearance by THP-1 macrophages. Defocusing microscopy was employed to measure the biomechanical modifications of individual nRBCs opsonized by IgGs purified from each group. RESULTS: Anaemic patients had higher levels of total and specific anti-RBC antibodies in comparison to the non-anaemic ones. Opsonization with purified IgG from anaemic patients significantly enhanced RBCs in vitro phagocytosis by THP-1 macrophages. Auto-antibodies purified from anaemic patients decreased the nRBC dynamic membrane fluctuations suggesting a possible participation of such antibodies in the perturbation of erythrocyte flexibility and morphology integrity maintenance. CONCLUSIONS: These findings revealed that vivax-infected patients with anaemia have increased levels of IgG auto-antibodies against nRBCs and that their deposition on the surface of non-infected erythrocytes decreases their deformability, which, in turn, may enhance nRBC clearance by phagocytes, contributing to the anaemic outcome. These data provide insights into the immune mechanisms associated with vivax malaria anaemia and may be important to the development of new therapy and vaccine strategies.

N-Formyl-Perosamine Surface Homopolysaccharides Hinder the Recognition of Brucella abortus by Mouse Neutrophils.

Brucella abortus is an intracellular pathogen of monocytes, macrophages, dendritic cells, and placental trophoblasts. This bacterium causes a chronic disease in bovines and in humans. In these hosts, the bacterium also invades neutrophils; however, it fails to replicate and just resists the killing action of these leukocytes without inducing significant activation or neutrophilia. Moreover, B. abortus causes the premature cell death of human neutrophils. In the murine model, the bacterium is found within macrophages and dendritic cells at early times of infection but seldom in neutrophils. Based on this observation, we explored the interaction of mouse neutrophils with B. abortus In contrast to human, dog, and bovine neutrophils, naive mouse neutrophils fail to recognize smooth B. abortus bacteria at early stages of infection. Murine normal serum components do not opsonize smooth Brucella strains, and neutrophil phagocytosis is achieved only after the appearance of antibodies. Alternatively, mouse normal serum is capable of opsonizing rough Brucella mutants. Despite this, neutrophils still fail to kill Brucella, and the bacterium induces cell death of murine leukocytes. In addition, mouse serum does not opsonize Yersinia enterocolitica O:9, a bacterium displaying the same surface polysaccharide antigen as smooth B. abortus Therefore, the lack of murine serum opsonization and absence of murine neutrophil recognition are specific, and the molecules responsible for the Brucella camouflage are N-formyl-perosamine surface homopolysaccharides. Although the mouse is a valuable model for understanding the immunobiology of brucellosis, direct extrapolation from one animal system to another has to be undertaken with caution.

Safety, tolerability and immunogenicity of 15-valent pneumococcal conjugate vaccine in toddlers previously vaccinated with 7-valent pneumococcal conjugate vaccine.

BACKGROUND: Widespread use of 7-valent pneumococcal conjugate vaccine (PCV7) in children has led to significant reduction in pneumococcal disease in children and adults. However, diseases caused by serotypes not included in PCV7 have increased. A 15-valent pneumococcal conjugate vaccine (PCV15) containing serotypes in PCV7 and 8 additional serotypes (1, 3, 5, 6A, 7F, 19A, 22F, 33F) was developed and evaluated in toddlers 12 to 15 months of age. METHODS: Ninety toddlers who completed an infant series with PCV7 received a single dose of either aluminum-adjuvanted PCV15, nonadjuvanted PCV15, or PCV7. Injection-site and systemic adverse events (AEs) were collected for 14 days postvaccination and serious AEs (SAEs) were collected for 30 days postvaccination. Solicited AEs included local (pain/tenderness, swelling, nodule and redness) and systemic (fatigue, arthralgia and myalgia) AEs. Serotype-specific immunoglobulin G (IgG) and opsonophagocytic (OPA) responses were measured immediately prior and 30 days postvaccination. RESULTS: Incidences of local and systemic AEs were comparable across vaccine groups. The majority of reported events, regardless of vaccine received, were transient and of mild to moderate intensity. No clinically significant differences were observed when comparing duration and severity of AEs. No vaccine-related SAEs or discontinuations from the study due to AEs were reported. Pneumococcal IgG concentrations and OPA titers increased postvaccination, with appreciable fold rises for all serotypes. Antibody levels were comparable between both PCV15 formulations and generally comparable to PCV7 for the shared serotypes. CONCLUSION: Both formulations of PCV15 display acceptable safety profiles and induce IgG and OPA responses to all vaccine serotypes.

Open-label trial of immunogenicity and safety of a 13-valent pneumococcal conjugate vaccine in adults ≥ 50 years of age in Mexico.

This open-label multicenter clinical trial conducted in Mexico assessed the immunogenicity and safety of a 13-valent pneumococcal conjugate vaccine (PCV13) in adults ≥ 50 years of age not previously vaccinated with the 23-valent pneumococcal polysaccharide vaccine (PPSV23). The PCV13 elicited a robust immune response in this study population, as reflected by the magnitude of fold rises in functional antibody levels measured by serotype-specific opsonophagocytic activity (OPA) assays before and 1 month after vaccination. Although the prevaccination OPA geometric mean titers (GMTs) for the majority of the serotypes were significantly lower in the 50- to 64-year age group than those in the ≥ 65-year age group, the postvaccination immune responses were generally similar. The overall immune responses were higher for the majority of the serotypes in the Mexican study population than those in similar adult study populations who received the PCV13 in Europe and the United States. PCV13 was well tolerated, and there were no vaccine-related serious adverse events. In conclusion, PCV13 is safe and immunogenic when administered to adults ≥ 50 years of age in Mexico and has the potential to protect against vaccine-type pneumococcal disease. (This study has been registered at ClinicalTrials.gov under registration no. NCT01432262.).

Bordetella parapertussis survives inside human macrophages in lipid raft-enriched phagosomes.

Bordetella parapertussis is a human pathogen that causes whooping cough. The increasing incidence of B. parapertussis has been attributed to the lack of cross protection induced by pertussis vaccines. It was previously shown that B. parapertussis is able to avoid bacterial killing by polymorphonuclear leukocytes (PMN) if specific opsonic antibodies are not present at the site of interaction. Here, we evaluated the outcome of B. parapertussis innate interaction with human macrophages, a less aggressive type of cell and a known reservoir of many persistent pathogens. The results showed that in the absence of opsonins, O antigen allows B. parapertussis to inhibit phagolysosomal fusion and to remain alive inside macrophages. The O antigen targets B. parapertussis to lipid rafts that are retained in the membrane of phagosomes that do not undergo lysosomal maturation. Forty-eight hours after infection, wild-type B. parapertussis bacteria but not the O antigen-deficient mutants were found colocalizing with lipid rafts and alive in nonacidic compartments. Taken together, our data suggest that in the absence of opsonic antibodies, B. parapertussis survives inside macrophages by preventing phagolysosomal maturation in a lipid raft- and O antigen-dependent manner. Two days after infection, about 15% of macrophages were found loaded with live bacteria inside flotillin-enriched phagosomes that had access to nutrients provided by the host cell recycling pathway, suggesting the development of an intracellular infection. IgG opsonization drastically changed this interaction, inducing efficient bacterial killing. These results highlight the need for B. parapertussis opsonic antibodies to induce bacterial clearance and prevent the eventual establishment of cellular reservoirs of this pathogen.

Long-term immune responses to pneumococcal conjugate vaccines in children previously vaccinated with 7-valent pneumococcal conjugate vaccine.

BACKGROUND: Seven-valent pneumococcal conjugate vaccine (PCV7) has reduced incidence of vaccine-serotype pneumococcal diseases. Using a single dose of 13-valent pneumoccal conjugate vaccine (PCV13), we evaluated late immune responses 10 years after vaccination with PCV7 in infancy, compared with a PCV7-naïve cohort. METHODS: In this open-label study, we administered 1 dose of PCV13 to children aged 11-14 years who had previously received PCV7 (PCV7/PCV13) or meningococcal group C conjugate vaccine (MnCC/PCV13) during infancy. We evaluated serotype-specific immunoglobulin G concentrations and opsonophagocytic activity prevaccination and 1 week and 1 month postvaccination. We recorded local reactions and systemic events for 4 days postvaccination and adverse events for 6 months. RESULTS: Seventy-four subjects received PCV13 (PCV7/PCV13, n = 38; MnCC/PCV13, n = 36). Prevaccination with PCV13, >62.9% of subjects had immunoglobulin G concentrations ≥0.35 µg/mL for all serotypes except serotype 4 (28-29%); proportions increased at 1 month postvaccination to 100% for all serotypes except serotypes 3 (PCV7/PCV13, 94.7%; MnCC/PCV13, 97.0%) and 14 (MnCC/PCV13, 97.1%). Immunoglobulin G and opsonophagocytic activity concentrations for the 7 common and 6 additional serotypes were similar in both groups prevaccination and increased in both groups from prevaccination to 1 week and 1 month postvaccination. Local reactions and fever were mild or moderate; no serious adverse events were reported. CONCLUSION: Late immune responses after a single dose of PCV13 were similar in children aged 11-14 years regardless of previous vaccination with PCV7 or MnCC. PCV13 was immunogenic, safe and well tolerated.

Bordetella pertussis iron regulated proteins as potential vaccine components.

Bordetella pertussis is the etiologic agent of whooping cough, an illness whose incidence has been increasing over the last decades. Pertussis reemergence despite high vaccination coverage, together with the recent isolation of circulating strains deficient in some of the vaccine antigens, highlight the need for new vaccines. Proteins induced under physiological conditions, such as those required for nutrient acquisition during infection, might represent good targets for better preventive strategies. By mean of serological proteome analysis we identified two novel antigens of B. pertussis potentially involved in iron acquisition during host colonization. We had previously demonstrated that one of them, designated IRP1-3, is protective against pertussis infection in mice. In the present study, we show that the other antigen, named AfuA (BP1605), is a highly antigenic protein, exposed on the bacterial surface, conserved among clinical isolates and expressed during infection. Immunization of mice with the recombinant AfuA induced opsonophagocytic antibodies which could explain the protection against B. pertussis infection conferred by mice immunization with rAfuA. Importantly, we found that the addition of rAfuA and rIRP1-3 proteins to the commercial three pertussis components acellular vaccine significantly increased its protective activity. Taken together, our results point at these two antigens as potential components of a new generation of acellular vaccines.

Bordetella parapertussis survives the innate interaction with human neutrophils by impairing bactericidal trafficking inside the cell through a lipid raft-dependent mechanism mediated by the lipopolysaccharide O antigen.

Whooping cough is a reemerging disease caused by two closely related pathogens, Bordetella pertussis and Bordetella parapertussis. The incidence of B. parapertussis in whooping cough cases has been increasing since the introduction of acellular pertussis vaccines containing purified antigens that are common to both strains. Recently published results demonstrated that these vaccines do not protect against B. parapertussis due to the presence of the O antigen on the bacterial surface that impairs antibody access to shared antigens. We have investigated the effect of the lack of opsonization of B. parapertussis on the outcome of its interaction with human neutrophils (polymorphonuclear leukocytes [PMNs]). In the absence of opsonic antibodies, PMN interaction with B. parapertussis resulted in nonbactericidal trafficking upon phagocytosis. A high percentage of nonopsonized B. parapertussis was found in nonacidic lysosome marker (lysosome-associated membrane protein [LAMP])-negative phagosomes with access to the host cell-recycling pathway of external nutrients, allowing bacterial survival as determined by intracellular CFU counts. The lipopolysaccharide (LPS) O antigen was found to be involved in directing B. parapertussis to PMN lipid rafts, eventually determining the nonbactericidal fate inside the PMN. IgG opsonization of B. parapertussis drastically changed this interaction by not only inducing efficient PMN phagocytosis but also promoting PMN bacterial killing. These data provide new insights into the immune mechanisms of hosts against B. parapertussis and document the crucial importance of opsonic antibodies in immunity to this pathogen.

Impaired function of antibodies to pneumococcal surface protein A but not to capsular polysaccharide in Mexican American adults with type 2 diabetes mellitus.

The goal of the study was to determine baseline protective titers of antibodies to Streptococcus pneumoniae surface protein A (PspA) and capsular polysaccharide in individuals with and individuals without type 2 diabetes mellitus. A total of 561 individuals (131 individuals with diabetes and 491 without) were screened for antibodies to PspA using a standard enzyme-linked immunosorbent assay (ELISA). A subset of participants with antibodies to PspA were retested using a WHO ELISA to determine titers of antibodies to capsular polysaccharide (CPS) (serotypes 4, 6B, 9V, 14, 18C, 19A, 19F, and 23F). Functional activity of antibodies was measured by assessing their ability to enhance complement (C3) deposition on pneumococci and promote killing of opsonized pneumococci. Titers of antibodies to protein antigens (PspA) were significantly lower in individuals with diabetes than controls without diabetes (P = 0.01), and antibodies showed a significantly reduced complement deposition ability (P = 0.02). Both antibody titers and complement deposition were negatively associated with hyperglycemia. Conversely, titers of antibodies to capsular polysaccharides were either comparable between the two groups or were significantly higher in individuals with diabetes, as was observed for CPS 14 (P = 0.05). The plasma specimens from individuals with diabetes also demonstrated a higher opsonophagocytic index against CPS serotype 14. Although we demonstrate comparable protective titers of antibodies to CPS in individuals with and individuals without diabetes, those with diabetes had lower PspA titers and poor opsonic activity strongly associated with hyperglycemia. These results suggest a link between diabetes and impairment of antibody response.

Phagocytic receptors on macrophages distinguish between different Sporothrix schenckii morphotypes.

Sporothrix schenckii is a human pathogen that causes sporotrichosis, a cutaneous subacute or chronic mycosis. Little is known about the innate immune response and the receptors involved in host recognition and phagocytosis of S. schenckii. Here, we demonstrate that optimal phagocytosis of conidia and yeast is dependent on preimmune human serum opsonisation. THP-1 macrophages efficiently ingested opsonised conidia. Competition with D-mannose, methyl α-D-mannopyranoside, D-fucose, and N-acetyl glucosamine blocked this process, suggesting the involvement of the mannose receptor in binding and phagocytosis of opsonised conidia. Release of TNF-α was not stimulated by opsonised or non-opsonised conidia, although reactive oxygen species (ROS) were produced, resulting in the killing of conidia by THP-1 macrophages. Heat inactivation of the serum did not affect conidia internalization, which was markedly decreased for yeast cells, suggesting the role of complement components in yeast uptake. Conversely, release of TNF-α and production of ROS were induced by opsonised and non-opsonised yeast. These data demonstrate that THP-1 macrophages respond to opsonised conidia and yeast through different phagocytic receptors, inducing a differential cellular response. Conidia induces a poor pro-inflammatory response and lower rate of ROS-induced cell death, thereby enhancing the pathogen's survival.

Immune evasion and recognition of the syphilis spirochete in blood and skin of secondary syphilis patients: two immunologically distinct compartments.

BACKGROUND: The clinical syndrome associated with secondary syphilis (SS) reflects the propensity of Treponema pallidum (Tp) to escape immune recognition while simultaneously inducing inflammation. METHODS: To better understand the duality of immune evasion and immune recognition in human syphilis, herein we used a combination of flow cytometry, immunohistochemistry (IHC), and transcriptional profiling to study the immune response in the blood and skin of 27 HIV(-) SS patients in relation to spirochetal burdens. Ex vivo opsonophagocytosis assays using human syphilitic sera (HSS) were performed to model spirochete-monocyte/macrophage interactions in vivo. RESULTS: Despite the presence of low-level spirochetemia, as well as immunophenotypic changes suggestive of monocyte activation, we did not detect systemic cytokine production. SS subjects had substantial decreases in circulating DCs and in IFNγ-producing and cytotoxic NK-cells, along with an emergent CD56-/CD16+ NK-cell subset in blood. Skin lesions, which had visible Tp by IHC and substantial amounts of Tp-DNA, had large numbers of macrophages (CD68+), a relative increase in CD8+ T-cells over CD4+ T-cells and were enriched for CD56+ NK-cells. Skin lesions contained transcripts for cytokines (IFN-γ, TNF-α), chemokines (CCL2, CXCL10), macrophage and DC activation markers (CD40, CD86), Fc-mediated phagocytosis receptors (FcγRI, FcγR3), IFN-ß and effector molecules associated with CD8 and NK-cell cytotoxic responses. While HSS promoted uptake of Tp in conjunction with monocyte activation, most spirochetes were not internalized. CONCLUSIONS: Our findings support the importance of macrophage driven opsonophagocytosis and cell mediated immunity in treponemal clearance, while suggesting that the balance between phagocytic uptake and evasion is influenced by the relative burdens of bacteria in blood and skin and the presence of Tp subpopulations with differential capacities for binding opsonic antibodies. They also bring to light the extent of the systemic innate and adaptive immunologic abnormalities that define the secondary stage of the disease, which in the skin of patients trends towards a T-cell cytolytic response.

IgG keeps virulent Salmonella from evading dendritic cell uptake.

Dendritic cells (DCs) are phagocytic professional antigen-presenting cells that can prime naive T cells and initiate anti-bacterial immunity. However, several pathogenic bacteria have developed virulence mechanisms to impair DC function. For instance, Salmonella enterica serovar Typhimurium can prevent DCs from activating antigen-specific T cells. In addition, it has been described that the Salmonella Pathogenicity Island 1 (SPI-1), which promotes phagocytosis of bacteria in non-phagocytic cells, can suppress this process in DCs in a phosphatidylinositol 3-kinase (PI3K) -dependent manner. Both mechanisms allow Salmonella to evade host adaptive immunity. Recent studies have shown that IgG-opsonization of Salmonella can restore the capacity of DCs to present antigenic peptide-MHC complexes and prime T cells. Interestingly, T-cell activation requires Fcγ receptor III (FcγRIII) expression over the DC surface, suggesting that this receptor could counteract both antigen presentation and phagocytosis evasion by bacteria. We show that, despite IgG-coated Salmonella retaining its capacity to secrete anti-capture proteins, DCs are efficiently capable of engulfing a large number of IgG-coated bacteria. These results suggest that DCs employ another mechanism to engulf IgG-coated Salmonella, different from that used for free bacteria. In this context, we noted that DCs do not employ PI3K, actin cytoskeleton or dynamin to capture IgG-coated bacteria. Likewise, we observed that the capture is an FcγR-independent mechanism. Interestingly, these internalized bacteria were rapidly targeted for degradation within lysosomal compartments. Hence, our results suggest a novel mechanism in DCs that does not employ PI3K/actin cytoskeleton/dynamin/FcγRs to engulf IgG-coated Salmonella, is not affected by anti-capture SPI-1-derived effectors and enhances DC immunogenicity, bacterial degradation and antigen presentation.

Interferon gamma induces actin polymerization, Rac1 activation and down regulates phagocytosis in human monocytic cells.

IFNγ is a potent activator and IL-10 a powerful inhibitor of macrophage functions. However, neither all cellular functions are enhanced by IFNγ nor IL-10 inhibits all cellular responses. Thus, FcγRs-mediated phagocytosis in monocyte-derived macrophages (MDM) increases after IL-10 treatment, and decreases after treatment with IFNγ, although both IL-10 and IFNγ up regulate FcγRI expression. In this work we investigated the effect of IFNγ and IL-10 on phagocytic signaling by FcγRs in MDM. Treatment with IFNγ diminished phagocytosis of IgG-opsonized SRBC (IgG-SRBC) while treatment with IL-10 increased it. These opposite effects cannot be attributed to changes in FcγR expression induced by each cytokine. Early biochemical responses mediated by FcγRs were distinctly affected by cytokine treatment. Syk phosphorylation and the rise in [Ca(2+)](i) were higher after IL-10 treatment, whereas IFNγ treatment also increased Syk phosphorylation but had no effect on the rise in [Ca(2+)](i). IFNγ treatment led to increased basal levels of F-actin and this effect correlated with the decrease in phagocytosis of both IgG-SRBC and non-opsonized Escherichia coli. IL-10 did not alter F-actin basal levels, and enhanced the phagocytosis of E. coli and IgG-SRBC. The level of F-actin reached after IFNγ treatment was not further increased after stimulation with IgG-SRBC or CCL5, whereas MDM treated with IL-10 showed a slightly higher response than control cells to CCL5. IFNγ increased Rac1-GTP levels. Inhibition of PI3K with LY294002 prevented IFNγ-mediated actin polymerization. Our data suggest that IFNγ induces a higher basal level of F-actin and activation of Rac1, affecting the response to stimuli that induce cytoskeleton rearrangement such as phagocytic or chemotactic stimuli.

Immunogenicity, reactogenicity and safety of the 10-valent pneumococcal nontypeable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) in Mexican infants.

The immunogenicity and safety of the 10-valent pneumococcal conjugate vaccine, PHiD-CV, have been documented in European and Asian studies. In this open study conducted in Mexico (NCT00489554), 230 healthy infants received three doses of PHiD-CV and DTPa-HBV-IPV/Hib vaccines at 2, 4 and 6 months of age and two doses of oral human rotavirus vaccine at 2 and 4 months. Serotype-specific pneumococcal responses and opsonophagocytic activity (OPA) were measured one month post-dose 3. PHiD-CV's primary vaccination course was highly immunogenic against each of the 10 pneumococcal vaccine serotypes and carrier protein D. Antibody responses against pneumococcal serotypes and protein D were generally higher in Mexican infants compared with European antibody responses, and functional OPA responses were also higher or in the same range. The most frequent solicited local symptom was pain, with high but similar incidences of grade 3 pain reported at both injection sites (up to 15% of all doses). PHiD-CV was well tolerated, with no serious adverse events considered as causally related to vaccination. Most solicited symptoms were mild and there was no increase in incidence of solicited symptoms with successive vaccine doses.

Role of macrophages in early protective immune responses induced by two vaccines against foot and mouth disease.

Foot and Mouth Disease (FMD) is an acute disease of cloven-hoofed species. We studied the protection and early immune response induced in the murine model by vaccines formulated with inactivated virus and two different adjuvants. The presence of IMS12802PR or ISA206VG adjuvants yielded protection against viral challenge at early times post vaccination and induced FMDV-specific, but non neutralizing, antibody titers. In vivo macrophage depletion in vaccinated mice severely decreased the protection levels after virus challenge, indicating a central role of this cell population in the response elicited by the vaccines. Accordingly, opsonophagocytosis of FITC-labelled virus was augmented in 802-FMDVi and 206-FMDVi vaccinated mice. These results demonstrate the ability of the studied adjuvants to enhance the protective responses of these inactivated vaccines without the increase in seroneutralizing antibodies and the main role of opsonization and phagocytosis in the early protective immune responses against FMD infection in the murine model.