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Several biosimilar versions of enoxaparin are already approved and in use globally. Analytical characterization can establish good quality control in manufacturing, but they may not assure similarity in clinical outcomes between biosimilar and branded enoxaparin. This study evaluated the efficacy and safety of biosimilar Cristália versus branded Sanofi enoxaparin in venous thromboembolism (VTE) prevention in patients undergoing major abdominal surgery at risk for VTE. In this randomized, prospective single-blind study, we compared Cristália enoxaparin (Ce), a biosimilar version, versus branded Sanofi enoxaparin (Se; at a dose of 40 mg subcutaneously per day postoperatively from 7 to 10 days) in 243 patients submitted to major abdominal surgery at risk for VTE for VTE prevention. The primary efficacy outcome was occurrence of VTE or death related to VTE. The principal safety outcomes were a combination of major bleeding and clinically relevant non-major bleeding. Bilateral duplex scanning of the legs was performed from days 10 to 14, and follow-ups were performed up to 60 days after surgery. The incidence of VTE was 4.9% in the Cristália group and 1.1% in the Sanofi group (absolute risk difference = 3.80%, 95% confidence interval [CI]: -1.4%-9.0%) yielding noninferiority since the 95% CI does not reach the prespecified value Δ = 20%. Clinically significant bleeding occurred in 9.9% in the Cristália group and in 5.5% in the Sanofi group (n.s. ). In conclusion, this study suggests that 40 mg once daily of Ce, a biosimilar enoxaparin, is as effective and safe as the branded Sanofi enoxaparin in the prophylaxis of VTE in patients submitted to major abdominal surgery at risk for VTE.

Due to its physicochemical properties, nanostructured mesoporous SBA-15 silica shows great potential as a vaccine adjuvant. This study evaluated the capacity of SBA-15 to encapsulate/adsorb the recombinant purified HBsAg from the Hepatitis B virus and the immunoresponsiveness of mice orally immunized with HBsAg inside SBA-15. A simulation of small angle X-ray scattering experimental results, together with the nitrogen adsorption isotherms data, allowed to determine the appropriate mass ratio of HBsAg:SBA-15, indicating antigen encapsulation into SBA-15 macroporosity. This was also evaluated by bicinchoninic acid assay and gel electrophoresis. The recruitment of inflammatory cells, an increase in production of specific antibodies, and the non-influence of silica on T H 1 or T H 2 polarization were observed after oral immunization. Besides, SBA-15 enhanced the phagocytosis of ovalbumin by dendritic cells, an important key to prove how this adjuvant works. Thus, it seems clear that the nanostructured SBA-15 is an effective and safe adjuvant for oral immunizations., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Propionibacterium acnes (P. acnes) is a gram-positive anaerobic bacillus present in normal human skin microbiota, which exerts important immunomodulatory effects, when used as heat- or phenol-killed suspensions. We previously demonstrated that heat-killed P. acnes or its soluble polysaccharide (PS), extracted from the bacterium cell wall, suppressed or potentiated the Th2 response to ovalbumin (OVA) in an immediate hypersensitivity model, depending on the treatment protocol. Herein, we investigated the mechanisms responsible for these effects, using the same model and focusing on the activation status of antigen-presenting cells (APCs). We verified that higher numbers of APCs expressing costimulatory molecules and higher expression levels of these molecules are probably related to potentiation of the Th2 response to OVA induced by P. acnes or PS, while higher expression of toll-like receptors (TLRs) seems to be related to Th2 suppression. In vitro cytokines production in cocultures of dendritic cells and T lymphocytes indicated that P. acnes and PS seem to perform their effects by acting directly on APCs. Our data suggest that P. acnes and PS directly act on APCs, modulating the expression of costimulatory molecules and TLRs, and these differently activated APCs drive distinct T helper patterns to OVA in our model.

B-1 lymphocytes are the predominant cells in mouse peritoneal cavity. They express macrophage and lymphocyte markers and are divided into B-1a, B-1b and B-1c subtypes. The role of B-1 cells is not completely clear, but they are responsible for natural IgM production and seem to play a regulatory role. An enriched B-1b cell population can be obtained from non-adherent peritoneal cell cultures, and we have previously demonstrated that these cells undergo differentiation to acquire a mononuclear phagocyte phenotype upon attachment to the substrate in vitro. Nevertheless, the B-1 cell response to antigens or adjuvants has been poorly investigated. Because killed Propionibacterium acnes exhibits immunomodulatory effects on both macrophages and B-2 lymphocytes, we analyzed whether a killed bacterial suspension or its soluble polysaccharide (PS) could modulate the absolute number of peritoneal B-1 cells in BALB/c mice, the activation status of these cells and their ability to differentiate into phagocytes in vitro. In vivo, P. acnes treatment elevated the absolute number of all B-1 subsets, whereas PS only increased B-1c. Moreover, the bacterium increased the number of B-1b cells that were positive for MHC II, TLR2, TLR4, TLR9, IL-4, IL-5 and IL-12, in addition to up-regulating TLR9, CD80 and CD86 expression. PS increased B-1b cell expression of TLR4, TLR9, CD40 and CD86, as well as IL-10 and IL-12 synthesis. Both of the treatments decreased the absolute number of B-1b cells in vitro, suggesting their early differentiation into B-1 cell-derived phagocytes (B-1CDP). We also observed a higher phagocytic activity from the phagocytes that were derived from B-1b cells after P. acnes and PS treatment. The adjuvant effect that P. acnes has on B-1 cells, mainly the B-1b subtype, reinforces the importance of B-1 cells in the innate and adaptive immune responses.

A 500 bp fragment encoding an isoform of cysteine proteinase from Leishmania (Leishmania) amazonensis was subcloned and expressed in the pHis vector, resulting in a recombinant protein of 24 kDa, rLacys24. In Western blots of L. (L.) amazonensis extracts, antibodies directed to rLacys24 recognized a cysteine proteinase isoform of 30 kDa. Analysis by fluorescence-activated cell sorter showed a significantly higher expression of CD8(+) lymphocytes in animals immunized with rLacys24 plus CFA, whereas a low expression of CD4(+) lymphocytes was observed in these animals. The cytotoxicity of lymphocytes isolated from mice immunized with rLacys24 plus CFA on L. (L.) amazonensis-infected macrophages was significantly higher than that observed in the presence of lymphocytes from control animals. Immunization of BALB/c mice with rLacys24 plus CFA resulted in a low but significant decrease of foot lesions after challenge with L. (L.) amazonensis compared to those exhibited by control mice., (Copyright 2009 Elsevier Inc. All rights reserved.)

Gastrointestinal helminthic infection is an important worldwide sheep disease. The emergence of anthelminthic resistance has led to drives to seek new means of therapeutic control of helminthiasis in sheep. Several data demonstrated the adjuvant effect of Propionibacterium acnes on resistance to infection. Herein, we evaluate the adjuvant effect of the commercial suspension containing LPS and P. acnes on experimental helminthiasis. Sheep received three doses of LPS and P. acnes commercial suspension or saline 0.9% (control group). Both groups received orally Haemonchus contortus infective larvae on day 0. Parasitological, haematological, lymphoproliferation analysis, IL-5 and IgE determination were made once a week until 35th day after infection. Our results revealed increase on packed cell volumes on day 14, in LPS + P. acnes treated group. On 21st and 35th days after infection in the same group occurred increase on circulating eosinophils and lymphocytes, and also in the lymphoproliferative response to mitogen. On 35th day, the faecal eggs peak in LPS + P. acnes treated group was significantly lower than control. A negative correlation between faecal eggs counts and circulating eosinophils in the immunostimulant treated group was also observed. Our findings suggest that LPS + P. acnes suspension can be used as a strategy to control helminthiasis in sheep.

Late phase reaction (LPR) of immediate hypersensitivity is a Th2 response characterized by eosinophil recruitment and related to allergic asthma pathogenesis. Several strategies were developed trying to control the tissue damage observed in this reaction. Recently, we verified that killed Propionibacterium acnes (P. acnes), a Gram-positive bacillus, immunomodulated LPR in a murine model, potentiating or suppressing it depending on the treatment protocol used. However, the bacterium compounds responsible for this effect are not known, leading us to investigate if P. acnes purified soluble polysaccharide (PS) could be a major component involved on the modulation induced by the bacterium. Recently, we demonstrated that PS, like P. acnes, induces adjuvant effect on DNA vaccine, increases bone marrow dendritic cell precursors in vivo and its maturation in vitro, and modulates in vitro macrophage tumoricidal activity. Herein, we determined the chemical PS composition, which is mainly constituted by galactopyranose, ribopyranose, arabinopyranose, glucopyranose, ribofuranose and mannopyranose, and analyzed its capacity to modulate the immediate hypersensitivity in mice. Animals were subcutaneously implanted with coagulated hen's egg white (HEW) and 14 days later challenged with ovalbumin (OVA) in the footpad, developing a typical LPR after 24h. Similarly to the whole bacterium, Th2 response to OVA was potentiated when PS was administered concomitantly to HEW implantation, by increase in footpad eosinophilia and IL-4-producing spleen cells, and decrease in anti-OVA IgG2a titers and IL-12- or IFN-gamma-producing cells. On the other hand, the reaction was abrogated when HEW implantation was performed 1 week after PS-treatment, by decrease in footpad swelling, eosinophilia and anti-OVA IgG1 levels, and increase in IgG2a titers and IL-12-producing cells. These data suggest that PS seems to be the major P. acnes compound responsible for its effects on the modulation of immediate hypersensitivity reaction in mice.

Purpose: To evaluate subretinal detection of bevacizumab 2 hours after intravitreous injection of 1.25 mg in rabbit eyes., Methods: Anterior chamber paracentesis using a 30-gauge needle was performed in nine female Dutch-belted rabbits by removal of 0.05 mL of aqueous humor. Transscleral retinal detachment was performed with a modified 25-gauge infusion cannula connected to a bottle of physiologic saline solution (PSS). The animals were divided into experimental group 1, with intravitreous injection of 0.05 mL of (1.25 mg) with a 30-gauge needle (n = 6) and the control group 2, with intravitreous injection of 0.05 mL of PSS with a 30-gauge needle (n = 3). Two hours after the intravitreous bevacizumab or PSS injection, subretinal fluid was aspirated and immunoassayed to measure the level of bevacizumab. The rabbits were killed by intravenous pentobarbital injection. The eyes were enucleated and fixed in 10% formaldehyde. The pars plana site at which the transscleral cannula was introduced was analyzed by light microscopy, to exclude iatrogenic retinal tears. Eyes with accidental retinal tears were excluded., Results: Subretinal bevacizumab molecules were detected in the six eyes that received an intravitreous bevacizumab injection. No subretinal bevacizumab was detected in the control eyes. Light microscopy showed no evidence of retinal tears or holes in any rabbits used for the bevacizumab detection and control group., Conclusions: Bevacizumab molecules were detected in the subretinal space after intravitreous injection of 1.25 mg of bevacizumab, possibly as the result of diffusion through the retina in a rabbit model.

Propionibacterium acnes has been described as a potent adjuvant to immune responses in vitro and in vivo. Presently, we analysed the modulation of peritoneal exudate cells (PEC) by heat-killed P. acnes or its purified soluble polysaccharide (PS), both injected intraperitoneally in C57Bl/6 mice, aiming at their recruitment and cytotoxicity. Both treatments induced an increase in macrophages, immature dendritic cells, B1a lymphocytes and NK1.1(+) CD3(+) cells. The bacterium caused a remarkable increase in a NK1.1(+) CD3(+) CD4(-) CD8(-) cells subpopulation, whereas the PS component seemed responsible for the recruitment of mainly macrophage cells. To assess P. acnes and PS adjuvant effect on PEC cytotoxicity we evaluated their in vitro effect on murine B16F10 melanoma cells. The effector cells from the heat-killed bacteria and PS-treated groups lysed melanoma cells in co-cultures with PEC. Mice genetically deficient in IFN-gamma, when stimulated with P. acnes or PS, had reduced PEC cytotoxicity, and the cytotoxic effect was completely abrogated in PEC from iNOS(-/-) mice. The tumoricidal activity of PEC from P. acnes-treated mice was mediated by macrophages and NKT cells stimulated with IL-12. In PS-treated mice the cytotoxicity was mediated mainly by macrophages. Moreover, both treatments increased IL-4 and IFN-gamma production by NKT cells. In conclusion, we show that P. acnes act mainly by recruiting and activating NKT double-negative cells in PEC, which were shown to be tumoricidal in vitro when induced by IL-12. Macrophages induced by both P. acnes and PS have their antitumour effect dependent on NO production.

In the present work we investigated the role of killed Propionibacterium acnes or a soluble polysaccharide extracted from bacterium cell wall in modulated experimental immunization with plasmidial DNA. We used a plasmid, p154/13, containing a gene-encoding catalytic domain of Trypanosoma cruzi (T. cruzi) trans-sialidase. As previously described, immunization of BALB/c mice with p154/13 elicited humoral, cell-mediated and protective immune responses against T. cruzi infection. In this study we describe that both P. acnes and its soluble polysaccharide fraction have the ability to modulate the immune response elicited by p154/13. Treatment with these adjuvants enhanced specific trans-sialidase Th1 immune response, as revealed by a lower IgG1/IgG2a ratio and stronger in vitro IFN-gamma synthesis by CD4+ T cells. The most important fact was that treatment with P. acnes or its soluble polysaccharide fraction in the presence of p154/13 significantly reduced the peak of parasitemia observed 7 to 8 days after T. cruzi challenge. These data suggest that P. acnes or its soluble polysaccharide fraction may improve the protective potential of a DNA vaccine against experimental T. cruzi infection.

Among the effects exerted by Propionibacterium acnes, a most relevant one is its capacity to modulate the Th1/Th2 cellular immune response. This effect depends on the induction and activation of antigen presenting cells, mainly dendritic cells (DCs), whose number is increased in the peripheral blood of animals treated with this bacterium. A soluble P. acnes polysaccharide (PS) extract also acts on DCs, modulating a Th1 immune response. These data led us to investigate the role of P. acnes and its soluble PS on murine bone marrow (BM) DCs. Bone marrow cells were analyzed by flow cytometry, showing an increase of stem cells and DCs in P. acnes- or PS-treated animals. Culturing in the presence of granulocyte monocyte colony stimulating factor (GM-CSF) increased the in vitro differentiation and maturation of these cells into BM-derived DCs (CD11c+ and MHC class II+). Maturation of DCs was determined by increased CD80 and CD86 expression, IL-4 and IL-12 production, reduction in phagocytic capacity and increase in the antigen presenting ability to primed or naïve T lymphocytes. These data indicate that P. acnes as well as its PS can modulate BM stem cells, originating mature DCs, which are important mainly at the initial antigen contact.

The administration of killed Propionibacterium acnes suspension to mice enhances macrophage phagocytic and tumoricidal activities, have an adjuvant effect to antibody response and increases resistance to infection. Recent reports demonstrated that P. acnes treatment promotes IL-12 and IL-18 synthesis in mice inducing IFN-gamma release, enhancement of IgG2a switch and inhibition of Th2 cell expansion. These findings led us to investigate whether P. acnes could modulate hypersensitivity type I reaction observed in a murine model. Animals were implanted with heat coagulated hen's egg white (HEW) into the subcutaneous tissue, followed by OVA-challenge in the footpad. The observed reaction was characterized by elevated Th2 cytokine levels, especially IL-4 and increase in eosinophil infiltration as occurs in the late phase reaction (LPR) of type I hypersensitivity, a pattern observed in allergic asthma in human. Two different biological effects were induced by killed P. acnes depending on the experimental protocol used. When mice were treated with one dose of P. acnes per week during 3 weeks and the last dose administrated at the same time of HEW implantation, a strong adjuvant effect on type I hypersensitivity reaction with intense eosinophilic infiltration was observed. On the other hand, when the HEW implant was made 1 week after the administration of the last dose of P. acnes, animals developed a typical delayed type hypersensitivity reaction, and a cytokines pattern characteristic of the Th1 immune response.