Histological analysis and gene expression profile following augmentation of the anterior maxilla using rhBMP-2/ACS versus autogenous bone graft.

AIM: The objective of this report was to present histological characteristics and gene expression profile of newly formed bone following horizontal augmentation of the atrophic anterior maxilla using recombinant human bone morphogenetic protein-2 in an absorbable collagen sponge carrier (rhBMP-2/ACS) versus an autogenous bone graft (ABG). METHODS: Bone core biopsies from 24 subjects participating in a randomized clinical trial were obtained at dental implant placement, 6 months following alveolar ridge augmentation using rhBMP-2/ACS (rhBMP-2 at 1.5 mg/ml; total dose 4.2 mg) or a particulate ABG harvested from the mandibular retro-molar region. A titanium mesh was used to provide wound stability and space for bone formation. Analysis included histological/histometric observations and gene expression profile of the newly formed bone. RESULTS: rhBMP-2/ACS yielded bone marrow rich in capillaries, undifferentiated cells and bone lining cells compared with the ABG (p = 0.002). Whereas no significant differences were observed in total bone fraction (p = 0.53), non-vital bone particles trapped in lamellar vital bone were observed in the ABG group (p < 0.001). Real-time PCR showed greater BMP-2 and RUNX2 expression for rhBMP-2/ACS over the ABG (p = 0.001 and 0.0021, respectively), while the ABG exhibited greater expression of RANKL:OPG, BSP and OPN over rhBMP-2/ACS (p = 0.01, 0.005 and 0.0009, respectively). CONCLUSIONS: Our observations suggest that formative biological processes explain bone formation following implantation of rhBMP-2/ACS, whereas remodelling, resorptive/formative processes, characterizes sites receiving ABGs.

Inflammatory Bowel Disease: An Overview of Immune Mechanisms and Biological Treatments.

Inflammatory bowel diseases (IBD) are characterized by chronic inflammation of the intestinal tract associated with an imbalance of the intestinal microbiota. Crohn's disease (CD) and ulcerative colitis (UC) are the most widely known types of IBD and have been the focus of attention due to their increasing incidence. Recent studies have pointed out genes associated with IBD susceptibility that, together with environment factors, may contribute to the outcome of the disease. In ulcerative colitis, there are several therapies available, depending on the stage of the disease. Aminosalicylates, corticosteroids, and cyclosporine are used to treat mild, moderate, and severe disease, respectively. In Crohn's disease, drug choices are dependent on both location and behavior of the disease. Nowadays, advances in treatments for IBD have included biological therapies, based mainly on monoclonal antibodies or fusion proteins, such as anti-TNF drugs. Notwithstanding the high cost involved, these biological therapies show a high index of remission, enabling a significant reduction in cases of surgery and hospitalization. Furthermore, migration inhibitors and new cytokine blockers are also a promising alternative for treating patients with IBD. In this review, an analysis of literature data on biological treatments for IBD is approached, with the main focus on therapies based on emerging recombinant biomolecules.

Evaluation of in vitro anti-inflammatory effects of crude ginger and rosemary extracts obtained through supercritical CO2 extraction on macrophage and tumor cell line: the influence of vehicle type.

BACKGROUND: Numerous plants from have been investigated due to their anti-inflammatory activity and, among then, extracts or components of ginger (Zingiber officinale Roscoe) and rosemary (Rosmarinus officinalis L.), sources of polyphenolic compounds. 6-gingerol from ginger rhizome and carnosic acid and carnosol from rosemary leaves present anti-tumor, anti-inflammatory and antioxidant activities. However, the evaluation of the mechanisms of action of these and other plant extracts is limited due to their high hydrophobicity. Dimethylsulfoxide (DMSO) is commonly used as a vehicle of liposoluble materials to mammalian cells in vitro, presenting enhanced cell penetration. Liposomes are also able to efficiently deliver agents to mammalian cells, being capable to incorporate in their structure not only hydrophobic molecules, but also hydrophilic and amphiphilic compounds. Another strategy is based on the use of Pluronic F-68, a biocompatible low-foaming, non-ionic surfactant, to disperse hydrophobic components. Here, these three delivery approaches were compared to analyze their influence on the in vitro anti-inflammatory effects of ginger and rosemary extracts, at different concentrations, on primary mammalian cells and on a tumor cell line. METHODS: Ginger and rosemary extracts free of organic solvents were obtained by supercritical fluid extraction and dispersed in DMSO, Pluronic F-68 or liposomes, in variable concentrations. Cell viability, production of inflammatory mediators and nitric oxide (NO) release were measured in vitro on J774 cell line and murine macrophages primary culture stimulated with bacterial lipopolysaccharide and interferon-γ after being exposed or not to these extracts. RESULTS: Ginger and rosemary extracts obtained by supercritical CO2 extraction inhibited the production of pro-inflammatory cytokines and the release of NO by peritoneal macrophages and J774 cells. The delivery vehicles influenced the anti-inflammatory effects. Comparatively, the ginger extract showed the highest anti-inflammatory activity on the tumor cell line. Controversially, rosemary extract dispersed on DMSO induced a more significant IL-1 and TNF-α reduction than ginger extract in primary macrophages. CONCLUSIONS: Amongst the tested delivery vehicles, DMSO was the most suitable, presenting reduced cytotoxicity, followed by Pluronic F-68 and liposomes, provably due to differences in their form of absorption, distribution and cellular metabolism. Co-administration of liposomes and plant extracts may cause death of macrophages cells and induction of NO production. It can be concluded that some of the beneficial effects attributed to extracts of ginger and rosemary may be associated with the inhibition of inflammatory mediators due to their high antioxidant activity. However, these effects were influenced by the type of delivery vehicle.

Yacon (Smallanthus sonchifolius): a functional food.

Yacon (Smallanthus sonchifolius) is an Andean tuberous root that is regarded as a functional food given that it contains fructooligosaccharides (FOS), inulin and phenolic compounds. The consumption of FOS and inulin improves the growth of bifidobacteria in the colon, enhances mineral absorption and gastrointestinal metabolism and plays a role in the regulation of serum cholesterol. Furthermore, the literature reports that the consumption of these prebiotics promotes a positive modulation of the immune system, improving resistance to infections and allergic reactions. Certain studies have demonstrated the potential of yacon as an alternative food source for those patients with conditions that require dietary changes. This review intends to describe the potential of yacon as a prebiotic and its cultivation and industrial processing for human consumption.

Oral tolerance and OVA-induced tolerogenic dendritic cells reduce the severity of collagen/ovalbumin-induced arthritis in mice.

Dietary proteins play an important role in the regulation of systemic immune response, in a phenomenon known as oral tolerance (OT). To evaluate the effects of OT on a murine model of type II collagen (CII) plus ovalbumin (OVA)-induced arthritis (CIA), mice were fed with OVA either before or after CIA induction. OT significantly reduced the paw edema and synovial inflammation, as well as serum levels of anti-CII, the ex vivo proliferation and inflammatory cytokine production by spleen cells from CIA mice. The frequencies of Foxp3(+) and IL-10(+) cells were higher, whereas IFNγ(+) cells and IL-17(+) cells were lower, among gated CD4(+) spleen T cells from tolerized CIA mice than in those from non-tolerized CIA mice. Adoptive transfer of tolerogenic dendritic cells (DCs) before CIA induction mimics the effects observed in the OT. We demonstrate here that bystander suppression induced by OT can modify the course of CIA and tolerogenic DCs play a role this phenomenon.

Phenotypical and functional analysis of intraepithelial lymphocytes from small intestine of mice in oral tolerance.

In this work, we evaluated the effects of administration of OVA on phenotype and function of intraepithelial lymphocytes (IELs) from small intestine of transgenic (TGN) DO11.10 and wild-type BALB/c mice. While the small intestines from BALB/c presented a well preserved structure, those from TGN showed an inflamed aspect. The ingestion of OVA induced a reduction in the number of IELs in small intestines of TGN, but it did not change the frequencies of CD8(+) and CD4(+) T-cell subsets. Administration of OVA via oral + ip increased the frequency of CD103(+) cells in CD4(+) T-cell subset in IELs of both BALB/c and TGN mice and elevated its expression in CD8ß(+) T-cell subset in IELs of TGN. The frequency of Foxp3(+) cells increased in all subsets in IELs of BALB/c treated with OVA; in IELs of TGN, it increased only in CD25(+) subset. IELs from BALB/c tolerant mice had lower expression of all cytokines studied, whereas those from TGN showed high expression of inflammatory cytokines, especially of IFN-γ, TGF-ß, and TNF-α. Overall, our results suggest that the inability of TGN to become tolerant may be related to disorganization and altered proportions of inflammatory/regulatory T cells in its intestinal mucosa.

Role of prebiotics in regulation of microbiota and prevention of obesity.

There is an increasing incidence in non-communicable diseases (NCD) such as obesity, diabetes and metabolic syndrome all over the world, despite of the governmental investments in healthy policies. Some of these diseases represent a high economic burden to individuals and society, demanding urgency in the identification of risk factors and appropriate solutions. Inadequate nutrition habits are an important risk factor for developing these diseases, and the consumption of functional foods such as prebiotics, probiotics, and phenolic compounds may contribute to mitigate them. It has been reported that the consumption of prebiotics can cause positive changes in the intestinal microbiota. The healthy microbiota, consisting mainly of Bifidobacterium and Lactobacillus, ferment prebiotics producing compounds that appear to play a protective role against metabolic diseases. Despite the growing number of publications on the effects of regular consumption of prebiotics in experimental models, additional studies on their effects on humans are needed to bring more subsidies to public health programs. This manuscript aims to review the literature on the possible benefits of ingestion of prebiotics in the control of obesity.

Tolerogenic Dendritic Cells on Transplantation: Immunotherapy Based on Second Signal Blockage.

Dendritic cells (DCs), the most important professional antigen-presenting cells (APC), play crucial role in both immunity and tolerance. It is well known that DCs are able to mount immune responses against foreign antigens and simultaneously tolerate self-antigens. Since DCs can be modulated depending on the surrounding microenvironment, they can act as a bridge between innate and adaptive immunity. However, the mechanisms that support this dual role are not entirely clear. Recent studies have shown that DCs can be manipulated ex vivo in order to trigger their tolerogenic profile, what can be a tool to be used in clinical trials aiming the treatment of various diseases and the prevention of transplant rejection. In this sense, the blockage of costimulatory molecules on DC, in the attempt of inhibiting the second signal in the immunological synapse, can be considered as one of the main strategies under development. This review brings an update on current therapies using tolerogenic dendritic cells modulated with costimulatory blockers with the aim of reducing transplant rejection. However, although there are current clinical trials using tolerogenic DC to treat allograft rejection, the actual challenge is to modulate these cells in order to maintain a permanent tolerogenic profile.

Role of the Mac-1 and VLA-4 integrins, and concomitant Th2-cytokine production, in nitric oxide modulated eosinophil migration from bone marrow to lungs in allergic mice.

Although numerous studies demonstrate the participation of nitric oxide (NO) in various inflammatory diseases, the precise function of NO in allergic asthma remains unclear. We investigated whether iNOS inhibition could interfere with the kinetics of VLA-4 and Mac-1 expression and adhesion properties of bone marrow and peripheral blood eosinophils of sensitized mice after antigen exposure. Treatment of allergic mice with 1400 W (iNOS inhibitor) increased the adhesion of bone marrow eosinophils to ICAM-1, but not blood eosinophils, at 24h and 48 h after OVA-challenge. Conversely, adhesion of blood eosinophils from 1400 W-treated mice to VCAM-1 diminished at 24h and was almost completely blocked at 48 h. 1400 W did not induce any change in the adhesion of bone marrow eosinophils to VCAM-1, at 24h, but cells collected 48 h after challenge showed significantly lower adherence. Flow cytometry demonstrated that 1400 W resulted in a significantly increased Mac-1 expression on bone marrow eosinophils at 24h, as compared to control mice. However, at 24h, 1400 W significantly decreased Mac-1 and VLA-4 expressions on blood eosinophils. At 48 h, the expressions of both Mac-1 and VLA-4 returned to previous levels. Results show a temporal effect of iNOS upon Mac-1 expression and function, the chief adhesion molecule involved in the eosinophil efflux from the bone marrow at 24h. In contrast, Mac-1 and VLA-4 were involved in eosinophil mobilization from blood to lungs at 48 h after antigen challenge. Data suggest an important role of the Mac-1 and VLA-4 in the iNOS-modulated migration of eosinophils to the lungs of allergic mice.

Production of monoclonal antibodies for detection of a secreted aspartyl proteinase from Candida spp. in biologic specimens.

Secreted acid proteinases (SAP) constitute an important group of virulence factors in Candida albicans. In the present work, an acid proteinase from C. albicans was sequentially purified from the supernatant of a yeast culture by precipitation with ammonium sulfate, ion exchange chromatography, and molecular exclusion chromatography, yielding a specific enzymatic activity of 204.1 IU/mg on bovine serum albumin (BSA). The molecular mass of the purified proteinase was estimated at 43 kd after exclusion chromatography and at 41 kd by nondenaturating sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified proteinase was able to degrade BSA at pH 2.5, but was not active on collagen, and it was significantly inhibited by pepstatin A. The immunization of BALB/c mice with the purified proteinase and later fusion of their spleen cells with myeloma cells resulted in 19 monoclonal antibody secreting hybridomas (MAbs) capable of detecting SAP in enzyme-linked immunosorbent assay (ELISA) assays. All MAbs obtained are isotype IgG1 kappa (kappa) immunoglobulins and develop a 41 kd protein band by Western blot (WB) in samples of SAP obtained from C. albicans (12-A) and C. dubliniensis (strain 778) crude extracts. The anti-SAP MAbs were used in capture ELISA and two combinations of these antibodies proved suitable for SAP detection, that is, MAP1 (1B1B3) or MAP2 (2D2C10) as coat antibodies, and biotinylated MAP3 (2A6E8) as detect antibody. Capture ELISA using these sets of MAbs detected over 32 ng/mL protein in purified SAP samples as well as in crude C. albicans and C. dubliniensis extracts. The results herein obtained allow for the prediction of how this set of antibodies can be useful for SAP detection in biologic specimens.

Detecçäo de VERO citotoxina (VT) em amostras de escherichia coli isoladas de bezerros com diarréia / Detection of VERO cytotoxin (VT) among strains of escherichia coli isolated from calves with diarrhea

Entre 104 amostras de Escherichia coli isoladas de bezerros com diarréia, 70 produziram citoxina ativa em células VERO.Nenhuma das amostras produziu enteroxina termoestável (STa) detectável pelo teste do camundongo recém-nascido ou enterotoxina termolábel ensaiada pelo teste de imunohemólise radial. Os autores fazem consideraçöes sobre o papel que essa citotoxina pode desempenhar na etiologia da diarréia neonatal em bezerros.