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Immune Sera (immune + sera)
Selected AbstractsDetection of the human GPR50 orphan seven transmembrane protein by polyclonal antibodies mapping different epitopesJOURNAL OF PINEAL RESEARCH, Issue 1 2007Hassina Ould Hamouda Abstract:, GPR50 is an orphan seven transmembrane protein related to the melatonin receptor subfamily comprising MT1 and MT2 receptors. In the absence of any known ligand for GPR50, other tools are critical for the characterization of this protein. Here, we describe the generation, purification and characterization of the first rabbit polyclonal antibodies generated against peptides corresponding to the N-terminus, C-terminus and two additional regions within the intracellular tail of GPR50. Immune sera were purified on peptide-antigen affinity columns. Antibodies specifically recognized a GPR50-YFP fusion protein on the plasma membrane of HEK 293 cells in immunofluorescence experiments. In Western blot experiments, the monomeric and dimeric forms of GPR50 were detected as proteins of 66 and 130 kDa, respectively. In addition, these new antibodies were sufficiently sensitive to detect GPR50 in brain slices of the rat pituitary and human hippocampus. In conclusion, we successfully produced antibodies against the orphan GPR50 protein that will become valuable tools for functional studies of this protein. [source] Role of Immune Serum in the Killing of Helicobacter pylori by MacrophagesHELICOBACTER, Issue 3 2010Stacey Keep Abstract Background:,Helicobacter pylori infection can lead to the development of gastritis, peptic ulcers and gastric cancer, which makes this bacterium an important concern for human health. Despite evoking a strong immune response in the host, H. pylori persists, requiring complex antibiotic therapy for eradication. Here we have studied the impact of a patient's immune serum on H. pylori in relation to macrophage uptake, phagosome maturation, and bacterial killing. Materials and Methods:, Primary human macrophages were infected in vitro with both immune serum-treated and control H. pylori. The ability of primary human macrophages to kill H. pylori was characterized at various time points after infection. H. pylori phagosome maturation was analyzed by confocal immune fluorescence microscopy using markers specific for H. pylori, early endosomes (EEA1), late endosomes (CD63) and lysosomes (LAMP-1). Results:, Immune serum enhanced H. pylori uptake into macrophages when compared to control bacteria. However, a sufficient inoculum remained for recovery of viable H. pylori from macrophages, at 8 hours after infection, for both the serum-treated and control groups. Both serum-treated and control H. pylori phagosomes acquired EEA1 (15 minutes), CD63 and LAMP-1 (30 minutes). These markers were then retained for the rest of an 8 hour time course. Conclusions:, While immune sera appeared to have a slight positive effect on bacterial uptake, both serum-treated and control H. pylori were not eliminated by macrophages. Furthermore, the same disruptions to phagosome maturation were observed for both serum-treated and control H. pylori. We conclude that to eliminate H. pylori, a strategy is required to restore the normal process of phagosome maturation and enable effective macrophage killing of H. pylori, following a host immune response. [source] Subunits of the epithelial sodium channel family are differentially expressed in the retina of mice with ocular hypertensionJOURNAL OF NEUROCHEMISTRY, Issue 1 2005Frank M. Dyka Abstract Glaucoma is a prevalent cause of blindness, resulting in the apoptotic death of retinal ganglion cells and optic nerve degeneration. The disease is often associated with elevated intraocular pressure, however, molecular mechanisms involved in ganglion cell death are poorly understood. To identify proteins contributing to this pathological process, we analysed the retinal gene expression of DBA/2J mice that develop an elevated intraocular pressure by the age of 6 months with subsequent ganglion cell loss. In this study, we identified subunits of the epithelial sodium channel (ENaC) family that are specifically expressed under elevated intraocular pressure. Using reverse transcriptase polymerase chain reaction we observed a significant increase of ,-ENaC in the neuronal retina of DBA/2J mice when compared with control animals, while ,-ENaC and ,-ENaC were not detectable in this tissue. Specific immune sera to ENaC subunits showed up-regulation of ,-ENaC in synaptic and nuclear layers of the retina, and in the retinal pigment epithelium. Consistent with our polymerase chain reaction data, ,-ENaC was not detected by specific antibodies in the retina, while ,-ENaC was only present in the retinal pigment epithelium under ocular hypertension. Finally, the increase of ,-ENaC gene expression in the neuronal retina and the retinal pigment epithelium was not observed in other tissues of DBA/2J mice. Since the intraocular pressure is regulated by the transport of aqueous humour across epithelial structures of the eye that in turn is associated with ion flux, the specific up-regulation of ENaC proteins could serve as a protecting mechanism against elevated intraocular pressure. [source] Distribution and characterization of hemolytic activity by an oral anaerobe from the Streptococcus milleri groupMOLECULAR ORAL MICROBIOLOGY, Issue 2 2004T. Yamaguchi Some oral anaerobes from the Streptococcus milleri strain group were found to secrete human specific hemolytic toxin, which was detected when bacteria were cultured in Todd-Hewitt broth and Brain Heart Infusion broth. The toxin elicited by the Streptococcus intermedius strain was partially fractionated by ammonium sulfate precipitation. Preincubation with glutathione or cysteine showed significant inhibiting effects; however, no effects were seen with dithiothreitol or ,-mercaptoethanol, and cholesterol was a weak inhibitor. Five kinds of protease inhibitor had no effect on the hemolytic activity, and rabbit preimmune and immune sera against the bacterial cells showed weak inhibition at a similar level. Digestion with trypsin, chymotrypsin, proteinase-K, subtilisin and pronase-P brought about a rise in activity, followed by a decrease during long-term incubation. Other enzymes tested showed no effects. Further, the presence of the intermedilysin gene in the portion with hemolytic activity was not identified by polymerase chain reaction. [source] Functional characteristics of antibodies induced by Arg-gingipain (HRgpA) and Lys-gingipain (Kgp) from Porphyromonas gingivalisMOLECULAR ORAL MICROBIOLOGY, Issue 4 2001T. Nakagawa Arginine-specific gingipain (HRgpA) and lysine-specific gingipain (Kgp), enzymes produced by Porphyromonas gingivalis, may be candidates for an anti,P. gingivalis vaccine. The purpose of our study was to determine whether HRgpA and Kgp have opsonic target sites and whether these sites are available and accessible on intact P. gingivalis cells. Rabbits were used to generate polyclonal antibodies to both proteins. Animals were immunized and immunoglobulin G (IgG) fractions were isolated from preimmune and immune sera. Functional characteristics of the antibodies were assessed by determining antibody titers by enzyme-linked immunosorbent assay (ELISA), generating Western immunoblots, and measuring antibody enhancement of P. gingivalis opsonization, phagocytosis and killing by polymorphonuclear leukocytes (PMN) of intact cells of strains of P. gingivalis representative of the four serotypes. Strains studied included 33277 (serotype A), A7A1,28 (serotype B), W50 (serotype C) and 381 (serotype D). Both HRgpA and Kgp induced high titers of IgG antibody. Anti-HRgpA and anti-Kgp bound to both HRgpA and Kgp demonstrating a large proportion of shared antigenic epitopes. The two antibodies bound equally well to all four P. gingivalis serotypes with titers ranging from 77 to 205 ELISA units when compared to preimmune IgG set at 1 ELISA unit. The immunoblot patterns of binding of the two antibodies to HRgpA and Kgp and to sonicates of the four P. gingivalis serotypes were virtually identical. Both antibodies detected components in HRgpA at 27, 35 and 45 kDa and in Kgp at 27, 32, 35, 40 and 55 kDa. The antibodies also detected components at or near these same positions in addition to multiple high molecular mass components in the cell sonicates of P. gingivalis. Both proteins induced antibodies that significantly enhanced opsonization as assessed by chemiluminescence, with values ranging from 130 mV to 375 mV for anti-HRgpA IgG and from 240 mV to 475 mV for anti-Kgp IgG. Both antibodies significantly enhanced PMN-mediated bacterial killing of the four P. gingivalis serotypes, although the percentage of killing varied among the serotypes (24,81% for anti-HRgpA and 37,89% for anti-Kgp). Thus, both HRgpA and Kgp express opsonic target sites and induce high titers of antibodies that opsonize and enhance killing of all four serotypes of P. gingivalis. These two proteins appear to be potential candidate antigens for an anti,P. gingivalis vaccine. [source] Antigenic cross-reactivity among Porphyromonas gingivalis serotypesMOLECULAR ORAL MICROBIOLOGY, Issue 3 2000Q. Fan The goal of our research program is to develop a Porphyromonas gingivalis vaccine. Vaccine development requires identification of antigenic components shared by the many clonal types of P. gingivalis. The purpose of the present study was to evaluate the extent and nature of antigenic cross-reactivity among serotypes of P. gingivalis and to identify shared antigenic components. Strains selected to represent serotypes A,D were 33277, A7A1-28 W50 and 381, respectively. Using intact cells, antibodies were raised in rabbits. Titers were assessed by enzyme-linked immunosorbent assay (ELISA) using intact cells as antigen, Western blots were prepared and biologic activity was measured as opsonization (chemiluminescence expressed as mV) and enhancement of phagocytosis and killing by polymorphonuclear leukocytes. Extensive cross-reactivity that varied greatly among serotypes was observed by ELISA. The Western blots showed an even greater extent of cross-reactivity, with shared protein components at approximately 140, 130, 37, 32 and 28 kDa and a shared variable molecular mass smear considered to be lipopolysaccharide and other carbohydrate. Additional protein components at 110, 85, 35 and 20 kDa appeared to be shared by some but not all serotypes. In the functional assays, strains 33277 and 381 were equally well opsonized by anti-33277 and anti-381 (500,650 mV) but opsonized to a much lesser extent by anti-A7A1-28 and anti-W50 (roughly 125 mV and 350 mV respectively). A7A1-28 and W50 were opsonized by all four immune sera almost equally but to a much lower extent (roughly 400 mV and 250 mV respectively). Enhancement of phagocytosis and killing in the presence of active complement mirrored opsonization with the exception that 381 was reasonably well opsonized by anti-A7A1-28 (400 mV) and anti-W50 (350 mV), but poorly killed. The protein components at 140, 130, 37 and 28 kDa shared by all of the four serotypes appear to have potential as vaccine candidate antigens. [source] | |||||||||||||