Resistance of melanized yeast cells of Paracoccidioides brasiliensis to antimicrobial oxidants and inhibition of phagocytosis using carbohydrates and monoclonal antibody to CD18

Paracoccidioides brasiliensis, a thermal dimorphic fungal pathogen, produces a melanin-like pigment in vitro and in vivo. We investigated the involvement of carbohydrates and monoclonal antibody to CD18, on phagocytosis inhibition, involving macrophage receptors and the resistance of melanized fungal cells to chemically generated nitric oxide (NO), reactive oxygen species (ROS), hypochlorite and H2O2. Our results demonstrate that melanized yeast cells were more resistant than nonmelanized yeast cells to chemically generated NO, ROS, hypochlorite and H2O2, in vitro. Phagocytosis of melanized yeast cells was virtually abolished when mannan, N-acetyl glucosamine and anti-CD18 antibody were added together in this system. Intratracheal infection of BALB/c mice, with melanized yeast cells, resulted in higher lung colony forming units, when compared to nonmelanized yeast cells. Therefore, melanin is a virulence factor of P. brasiliensis.

Negative Anti-C1q Antibody Titers May Influence Therapeutic Decisions and Reduce the Number of Renal Biopsies in Systemic Lupus Erythematosus

In a cross-sectional study involving 62 patients with systemic lupus erythematosus (SLE), we found that patients with biopsy-proven lupus nephritis (LN) had higher titers of anti-C1q antibodies than active SLE without nephritis patients. Anti-C1q was associated with a negative predictive value of 94.59%, a positive predictive value of 52%, a sensitivity of 86.66% and a specificity of 74.47% for the diagnosis of LN. We conclude that high titers of anti-C1q antibodies are strongly associated with the presence of active LN, and the negative predictive value of this test for diagnosing LN is very high; therefore, it can influence therapeutic decisions and reduce the number of renal biopsies in patients with SLE. Copyright (C) 2011 S. Karger AG, Basel

Anti-Ro antibody and cutaneous vasculitis in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease with various clinical and serological manifestations. Previous studies have shown the association of SLE and anti-Ro antibody with a series of clinical manifestations. We investigated this association in Brazilian patients with SLE. Five hundred and nine consecutive patients who fulfilled the revised American College of Rheumatology criteria for the SLE were enrolled in the study from June to December 2007. All patients were from our Service of Rheumatology, School of Medicine, University of Sao Paulo, Brazil. Frequencies of a series of laboratorial and clinical manifestations were calculated. Anti-Ro antibody was associated to anti-La antibody, female, and cutaneous vasculitis. In multivariate analysis, patients with anti-Ro antibody has 1.63 (95% CI 1.07-2.50) more risk to develop cutaneous vasculitis than patients without this antibody. Our data have demonstrated that anti-Ro antibody is an independent useful serologic marker for cutaneous vasculitis.

Hepatoportal Sclerosis and Extrahepatic Portal Venous Obstruction Associated with Anti-phospholipid Antibody Syndrome in Child

We report a 2-year-old child with extrahepatic portal venous obstruction, hepatoportal sclerosis and pulmonary thromboembolism whose sole hypercoagulability factor was the presence of anti-phospholipid antibodies.

CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display.

Replacement of the hyperimmune anti-Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti-RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high-affinity anti-RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin-treated red blood cells enabled the isolation of two high-affinity Fab-expressing phage clones. LD-6-3 and LD-6-33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants D(III), D(IVa), D(IVb), D(Va), D(VI) types I and II. D(VII), Rh33 and DFR. Full-length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth-adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti-RhD antibody adapted to pilot-scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody-dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti-RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.

Anti-flagellin antibody responses elicited in mice orally immunized with attenuated Salmonella enterica serovar Typhimurium vaccine strains

In the present study we investigated the flagellin-specific serum (IgG) and fecal (IgA) antibody responses elicited in BALB/c mice immunized with isogenic mutant derivatives of the attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) SL3261 strain expressing phase 1 (FliCi), phase 2 (FljB), or no endogenous flagellin. The data reported here indicate that mice orally immunized with recombinant S. Typhimurium strains do not mount significant systemic or secreted antibody responses to FliCi, FljB or heterologous B-cell epitopes genetically fused to FliCi. These findings are particularly relevant for those interested in the use of flagellins as molecular carriers of heterologous antigens vectored by attenuated S. Typhimurium strains.

Effects of rat anti-VEGF antibody in a rat model of corneal graft rejection by topical and subconjunctival routes.

PURPOSE: To compare the effect of a rat anti-VEGF antibody, administered either by topical or subconjunctival (SC) routes, on a rat model of corneal transplant rejection.METHODS: Twenty-four rats underwent corneal transplantation and were randomized into four treatment groups (n=6 in each group). G1 and G2 received six SC injections (0.02 ml 10 µg/ml) of denatured (G1) or active (G2) anti-VEGF from Day 0 to Day 21 every third day. G3 and G4 were instilled three times a day with denatured (G3) or active (G4) anti-VEGF drops (10 µg/ml) from Day 0 to Day 21. Corneal mean clinical scores (MCSs) of edema (E), transparency (T), and neovessels (nv) were recorded at Days 3, 9, 15, and 21. Quantification of neovessels was performed after lectin staining of vessels on flat mounted corneas.RESULTS: Twenty-one days after surgery, MCSs differed significantly between G1 and G2, but not between G3 and G4, and the rejection rate was significantly reduced in rats receiving active antibodies regardless of the route of administration (G2=50%, G4=66.65% versus G1 and G3=100%; p<0.05). The mean surfaces of neovessels were significantly reduced in groups treated with active anti-VEGF (G2, G4). However, anti-VEGF therapy did not completely suppress corneal neovessels.CONCLUSIONS: Specific rat anti-VEGF antibodies significantly reduced neovascularization and subsequent corneal graft rejection. The SC administration of the anti-VEGF antibody was more effective than topical instillation.

An anti-CD19 antibody coupled to a tetanus toxin peptide induces efficient Fas ligand (FasL)-mediated cytotoxicity of a transformed human B cell line by specific CD4+ T cells.

Treatment of B cell lymphoma patients with MoAbs specific for the common B cell marker (CD20) has shown a good overall response rate, but the number of complete remissions is still very low. The use of MoAbs coupled to radioisotopes can improve the results, but induces undesirable myelodepression. As an alternative, we proposed to combine the specificity of MoAbs with the immunogenicity of T cell epitopes. We have previously shown that an anti-Ig lambda MoAb coupled to an MHC class II-restricted universal T cell epitope peptide P2 derived from tetanus toxin induces efficient lysis of a human B cell lymphoma by a specific CD4+ T cell line. Here we demonstrate that the antigen presentation properties of the MoAb peptide conjugate are maintained using a MoAb directed against a common B cell marker, CD19, which is known to be co-internalized with the B cell immunoglobulin receptor. In addition, we provide evidence that B cell lysis is mediated by the Fas apoptosis pathway, since Fas (CD95), but not tumour necrosis factor receptor (TNFr) or TNF-related receptors, is expressed by the target B cells, and FasL, but not perforin, is expressed by the effector T cells. These results show that B cell lymphomas can be 'foreignized' by MoAb-peptide P2 conjugates directed against the common B cell marker CD19 and eliminated by peptide P2-specific CD4+ T cells, via the ubiquitous Fas receptor. This approach, which bridges the specificity of passive antibody therapy with an active T cell immune response, may be complementary to and more efficient than the present therapy results with unconjugated chimeric anti-CD20 MoAbs.

In vivo targeting of an anti-tumor antibody coupled to antigenic MHC class I complexes induces specific growth inhibition and regression of established syngeneic tumor grafts.

The concept of antibody-mediated targeting of antigenic MHC/peptide complexes on tumor cells in order to sensitize them to T-lymphocyte cytotoxicity represents an attractive new immunotherapy strategy. In vitro experiments have shown that an antibody chemically conjugated or fused to monomeric MHC/peptide can be oligomerized on the surface of tumor cells, rendering them susceptible to efficient lysis by MHC-peptide restricted specific T-cell clones. However, this strategy has not yet been tested entirely in vivo in immunocompetent animals. To this aim, we took advantage of OT-1 mice which have a transgenic T-cell receptor specific for the ovalbumin (ova) immunodominant peptide (257-264) expressed in the context of the MHC class I H-2K(b). We prepared and characterized conjugates between the Fab' fragment from a high-affinity monoclonal antibody to carcinoembryonic antigen (CEA) and the H-2K(b) /ova peptide complex. First, we showed in OT-1 mice that the grafting and growth of a syngeneic colon carcinoma line transfected with CEA could be specifically inhibited by systemic injections of the conjugate. Next, using CEA transgenic C57BL/6 mice adoptively transferred with OT-1 spleen cells and immunized with ovalbumin, we demonstrated that systemic injections of the anti-CEA-H-2K(b) /ova conjugate could induce specific growth inhibition and regression of well-established, palpable subcutaneous grafts from the syngeneic CEA-transfected colon carcinoma line. These results, obtained in a well-characterized syngeneic carcinoma model, demonstrate that the antibody-MHC/peptide strategy can function in vivo. Further preclinical experimental studies, using an anti-viral T-cell response, will be performed before this new form of immunotherapy can be considered for clinical use.

The anti-lymphoma activity of APO866, an inhibitor of nicotinamide adenine dinucleotide biosynthesis, is potentialized when used in combination with anti-CD20 antibody.

Abstract APO866 is an inhibitor of nicotinamide adenine dinucleotide (NAD) biosynthesis that exhibits potent anti-lymphoma activity. Rituximab (RTX), an anti-CD20 antibody, kills lymphoma cells by direct apoptosis and antibody- and complement-dependent cell-mediated cytotoxicities, and has clinical efficacy in non-Hodgkin cell lymphomas. In the present study, we evaluated whether RTX could potentiate APO866-induced human B-lymphoma cell death and shed light on death-mediated mechanisms associated with this drug combination. We found that RTX significantly increases APO866-induced death in lymphoma cells from patients and lines. Mechanisms include enhancement of autophagy-mediated cell death, activation of caspase 3 and exacerbation of mitochondrial depolarization, but not increase of reactive oxygen species (ROS) production, when compared with those induced by each drug alone. In vivo, combined administration of APO866 with RTX in a laboratory model of human aggressive lymphoma significantly decreased tumor burden and prolonged survival over single-agent treatment. Our study demonstrates that the combination of RTX and APO866 optimizes B-cell lymphoma apoptosis and therapeutic efficacy over both compounds administered separately.

Radioimmunotherapy combined with maintenance anti-CD20 antibody may trigger long-term protective T cell immunity in follicular lymphoma patients.

Growing evidence suggests that the patient's immune response may play a major role in the long-term efficacy of antibody therapies of follicular lymphoma (FL). Particular long-lasting recurrence free survivals have been observed after first line, single agent rituximab or after radioimmunotherapy (RIT). Rituximab maintenance, furthermore, has a major efficacy in prolonging recurrence free survival after chemotherapy. On the other hand, RIT as a single step treatment showed a remarkable capacity to induce complete and partial remissions when applied in recurrence and as initial treatment of FL or given for consolidation. These clinical results strongly suggest that RIT combined with rituximab maintenance could stabilize the high percentages of patients with CR and PR induced by RIT. While the precise mechanisms of the long-term efficacy of these 2 treatments are not elucidated, different observations suggest that the patient's T cell immune response could be decisive. With this review, we discuss the potential role of the patient's immune system under rituximab and RIT and argue that the T cell immunity might be particularly promoted when combining the 2 antibody treatments in the early therapy of FL.

Anti-HLA antibody repertoire after IVIg infusion in highly sensitized patients waiting for kidney transplantation.

Polyclonal intravenous immunoglobulin (IVIg) treatment reduces crossmatch positivity and increases rates of transplantation in highly sensitised patients (HS). We quantified the panel reactive antibody (PRA) by microlymphocytotoxicity (MLCC), and we analysed anti-HLA class I and class II IgG specific antibody repertoire by Luminex before and after IVIg infusion alone in HS patients awaiting kidney transplantation. Five patients received three monthly infusions of 1 g/kg of IVIg. Serum samples collected pre and post IVIg treatment were submitted for PRA analysis by MLCC. Anti-class I and anti-class II antibody specificities were then tested by Luminex. We focused on the anti-HLA class I and class II antibodies directed against HLA expressed by a previous graft. We also analysed the anti-HLA antibody repertoire in three patients who had not received IVIg infusion. The PRA level determined by MLCC decreased significantly in one of the five patients, dropping from 40% to 17%. The Luminex assay showed fluctuations of the anti-HLA antibody levels over time, but no significant longterm modifications of the anti-HLA antibody repertoire were observed, even in the patient with a strong and prolonged reduction of the PRA determined by MLCC. Our results show that IVIg at 1 g/kg is not sufficient to reduce PRA and does not modify the repertoire of specific anti-HLA antibody determined by Luminex.

Glial reactivity in dogs with visceral leishmaniasis: correlation with T lymphocyte infiltration and with cerebrospinal fluid anti-Leishmania antibody titres

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Immunocytochemical localization of heparin in secretory granules of rat peritoneal mast cells using a monoclonal anti-heparin antibody (ST-1)

We performed immunogold labeling with an ST-1 monoclonal antibody (IgM), specific for intact heparin, to define the subcellular localization of heparin in mast cells. Rat peritoneal mast cells were fixed by a modified Karnovsky method and embedded in Araldite. Ultrathin sections were first treated with sodium periodate and then sequentially incubated with MAb ST-1, rabbit anti-mouse IgM, and protein A-gold. By transmission electron microscopy, gold particles were localized inside cytoplasmic granules of peritoneal mast cells. In contrast, with the same procedure, no labeling was observed in mast cells from rat intestinal mucosa. Control sections of rat peritoneal or intestinal mucosa mast Mast cells cells treated with an irrelevant MAb (IgM) did not show any labeling. Treatment with nitrous Heparin acid abolished the reactivity of MAb ST-1 with peritoneal mast cells. These results Granules show that different mast cells can be identified regarding their heparin content by immunochemical procedures using MAb ST-1.