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Thiel J.,University Hospital Freiburg | Kimmig L.,University Hospital Freiburg | Salzer U.,University Hospital Freiburg | Grudzien M.,University Hospital Freiburg | And 19 more authors.
Journal of Allergy and Clinical Immunology | Year: 2012

Background: Complement receptor 2 (CR2/CD21) is part of the B-cell coreceptor and expressed by mature B cells and follicular dendritic cells. CD21 is a receptor for C3d-opsonized immune complexes and enhances antigen-specific B-cell responses. Objective: Genetic inactivation of the murine CR2 locus results in impaired humoral immune responses. Here we report the first case of a genetic CD21 deficiency in human subjects. Methods: CD21 protein expression was analyzed by means of flow cytometry and Western blotting. CD21 transcripts were quantified by using real-time PCR. The CD21 gene was sequenced. Wild-type and mutant CD21 cDNA expression was studied after transfection of 293T cells. Binding of EBV-gp350 or C3d-containing immune complexes and induction of calcium flux in CD21-deficient B cells were analyzed by means of flow cytometry. Antibody responses to protein and polysaccharide vaccines were measured. Results: A 28-year-old man presented with recurrent infections, reduced class-switched memory B cells, and hypogammaglobulinemia. CD21 receptor expression was undetectable. Binding of C3d-containing immune complexes and EBV-gp350 to B cells was severely reduced. Sequence analysis revealed a compound heterozygous deleterious mutation in the CD21 gene. Functional studies with anti-immunoglobulin- and C3d-containing immune complexes showed a complete loss of costimulatory activity of C3d in enhancing suboptimal B-cell receptor stimulation. Vaccination responses to protein antigens were normal, but the response to pneumococcal polysaccharide vaccination was moderately impaired. Conclusions: Genetic CD21 deficiency adds to the molecular defects observed in human subjects with hypogammaglobulinemia. © 2011 American Academy of Allergy, Asthma & Immunology.

Silva J.A.,University of Campinas | Ferrucci D.L.,University of Campinas | Peroni L.A.,University of Campinas | Abrahao P.G.,University of Campinas | And 5 more authors.
Journal of Cellular Physiology | Year: 2012

Molecular mechanisms responsible for periodontal disease (PD) and its worsening in type 1 Diabetes Mellitus (DM1) remain unknown. Cytokine profile and expression levels of collagenases, Mmp14, and tissue inhibitors were determined, as were the numbers of neutrophils and macrophages in combined streptozotocin-induced DM1 and ligature-induced PD models. Increased IL-23 (80-fold) and Mmp8 expression (25-fold) was found in DM1. Ligature resulted in an IL-1β/IL-6 profile, increased expression of Mmp8, Mmp13, and Mmp14 (but not Mmp1), and transient expression of Timp1 and Reck in non-diabetics. PD in DM1 involved IL-1β (but not IL-6) and IL-23/IL-17, reduced IL-6 and IL-10, sustained Mmp8 and Mmp14, increased Mmp13 and reduced Reck expression in association with 20-fold higher counts of neutrophils and macrophages. IL-23 and Mmp8 expression are hallmarks of DM1. In association with the IL-1/IL-6 (Th1) response in PD, one found a secondary IL-17 (Th17) pathway in non-diabetic rats. Low IL-6/TNF-α suggest that the Th1 response was compromised in DM1, while IL-17 indicates a prevalence of the Th17 pathway, resulting in high neutrophil recruitment. Mmp8, Mmp13, and Mmp14 expression seems important in the tissue destruction during PD in DM1. PD-associated IL-1/IL-6 (Th1), IL-10, and Reck expression are associated with the acute-to-chronic inflammation transition, which is lost in DM1. In conclusion, IL-23/IL-17 are associated with the PD progression in DM1. © 2011 Wiley Periodicals, Inc.

Silva J.A.F.,University of Campinas | Lopes Ferrucci D.,University of Campinas | Peroni L.A.,University of Campinas | Carvalho H.F.,University of Campinas | And 2 more authors.
Cells Tissues Organs | Year: 2012

Alveolar bone resorption results from the inflammatory response to periodontal pathogens. Systemic diseases that affect the host response, such as type 1 diabetes mellitus (DM1), can potentiate the severity of periodontal disease (PD) and accelerate bone resorption. However, the biological mechanisms by which DM1 modulates PD are not fully understood. The aim of this study was to determine the influence of DM1 on alveolar bone resorption and to evaluate the role of receptor activator of nuclear factor-kappaB ligand (RANKL)/ osteoprotegerin (OPG) in osteoclastogenesis in rats. PD was induced by means of ligature in nondiabetic and in streptozotocyn-induced DM1 rats. Morphological and morphometric analyses, stereology and osteoclast counting were performed. RANKL and OPG mRNA levels, protein content, and location were determined. PD caused alveolar bone resorption, increased the number of osteoclasts in the alveolar bone crest and also promoted changes in RANKL/OPG mRNA expression. DM1 alone showed alveolar bone destruction and an increased number of osteoclasts at the periapical and furcal regions. DM1 exacerbated these characteristics, with a greater impact on bone structure, resulting in a low OPG content and a higher RANKL/OPG ratio, which correlated with prominent osteoclastogenesis. This work demonstrates that the effects of PD and DM1 enhance bone destruction, confirms the importance of the RANKL signaling pathway in bone destruction in DM1 in animal models and suggests the existence of alternative mechanisms potentiating bone degradation in PD. © 2012 S. Karger AG, Basel.

Machado L.C.,University of Sao Paulo | Pelegati V.B.,Applied Cell Sciences | Oliveira A.L.,University of Sao Paulo
Journal of Supercritical Fluids | Year: 2016

Supercritical CO2 was studied for impregnation or encapsulation of essential oils in modified starches via Particle from Gas Saturated Solutions or Suspensions (PGSS). Modified starches were choose as function of its low cost. The advantage of this method over conventional encapsulation that use modified starch via spray drier refers to the low temperatures used and absence of water in the process. Modified starch presents hydrophobic elements and this molecules present amphiphilic character. Usually it is employed in the encapsulation of essences as wall material with excellent volatiles retention due to its polar and nonpolar interface. Considering its hydrophobic characteristics, interactions between the modified starch and supercritical CO2 occurred, resulting in two different structural interactions of the limonene and modified starch in the PGSS (50 °C and 60 °C at 100 bar and 120 bar). When hydrous ethanol was used in the suspension, impregnation occurred and, when anhydrous ethanol was used, encapsulation occurred. Analysis of particle morphology via scanning electron and confocal microscopy, thermo-oxidative characterization by differential scanning calorimetry and determination of microencapsulated limonene via gas chromatography coupled to mass spectrometry indicated limonene microencapsulation and impregnation occurred despite the highly solubility of limonene in supercritical CO2. The retention of limonene by Purity Gum Ultra® was 86% when encapsulated and, 53% when impregnated, similar values to those obtained in conventional microencapsulation methods via a spray drier or via PGSS-drying. © 2015 Elsevier B.V. All rights reserved.

Singh A.,Applied Cell Sciences | Leng L.,Yale University | Fan J.,Yale University | Gajda M.,University Hospital | And 4 more authors.
Rheumatology International | Year: 2013

Rheumatoid arthritis (RA) is characterized by the interaction of multiple mediators, among the most important of which are cytokines. In recent years, extensive studies demonstrate a pivotal role for one cytokine, macrophage migration inhibitory factor (MIF), in fundamental events in innate and adaptive immunity. MIF has now been demonstrated to be involved in the pathogenesis of many diseases, but in the case of RA the evidence for a role of MIF is very strong. MIF is abundantly expressed in the sera of RA patients and in RA synovial tissue correlating with disease activity. MIF-deficient mice were used to induce arthritis by serum transfer from K/BxN mice. K/BxN serum transfer arthritis was markedly attenuated in MIF- mice, with reduction in clinical index and histological severity as well as decrease in synovial cytokines. Macrophage transfers were done to investigate the specific role of macrophage-derived MIF. We show that adoptive transfer of wild-type macrophages into MIF- mice restores the sensitivity of MIF- mice to arthritis development, and this affect was associated with a restoration in serum IL-1β and IL-6 production. These results indicate that MIF plays a critical role in inflammation and joint destruction in K/BxN serum-induced arthritis and that the systemic expression of MIF by a subpopulation of macrophages is necessary and sufficient for the full development of arthritis. © 2013 Springer-Verlag Berlin Heidelberg.

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