Molecular and evolutionary analyses of formyl peptide receptors suggest the absence of VNO-specific FPRs in primates

Formyl peptide receptors (FPRs) were observed to expand in rodents and were recently suggested as candidate vomeronasal chemosensory receptors. Since vomeronasal chemosensory receptors usually underwent positive selection and evolved concordantly with the vomeronasal organ (VNO) morphology, we surveyed FPRs in primates in which VNO morphology is greatly diverse and thus it would provide us a clearer view of VNO-FPRs evolution. By screening available primate genome sequences, we obtained the FPR repertoires in representative primate species. As a result, we did not find FPR family size expansion in primates. Further analyses showed no evolutionary force variance between primates with or without VNO structure, which indicated that there was no functional divergence among primates FPRs. Our results suggest that primates lack the VNO-specific FPRs and the FPR expansion is not a common phenomenon in mammals outside rodent lineage, regardless of VNO complexity.

抗 LDH－C4 IgA 和IgG 抗体在粘膜免疫系统的分布及其抗生育作用的研究

已有的研究表明，小鼠背部携带能分泌抗原特异的IgA 单克隆抗体的杂交细 胞瘤，可以保护小鼠抵抗微生物和病毒等多种病原体经粘膜途径感染机体。我们 利用背部携带能分泌抗精子特异抗原（LDH-C4）的IgA 和IgG 杂交细胞瘤、以 及抗DNP 的IgA 骨髓细胞瘤的小鼠为动物模型，采用定量ELISA 法研究了抗 LDH－C4 IgA 与抗DNP IgA 单克隆抗体在呼吸道、肠道及生殖道内转运和分布， 抗LDH-C4 IgG2b 在肠道内转运与分布，以及抗LDH-C4 IgA 和IgG 单克隆抗体 在体内抗生育作用。 研究结果表明，带瘤小鼠血液中含有较高水平抗原特异的 IgA 和IgG 单克 隆抗体。PA4 和MOPC IgA 单克隆抗体在呼吸道、肠道以及雌性生殖道分泌物 内有较高的分布水平。在肠道，PA4 和MOPC IgA 单克隆抗体的分布水平显著 高于IgG（p<0.01 和p<0.05）。在肠道和生殖道的不同部位，IgA 抗体的分布水 平不同。在肠道，结肠分泌物中的IgA 单克隆抗体显著高于其它肠道部位 （p<0.01）。在生殖道，IgA 单克隆抗体分布水平以子宫角分泌物中最高。雄性 的前列腺也有较高的IgA 抗体水平。在呼吸道、肠道以及雌性生殖道相应部位的 分泌物内，PA4 IgA 单克隆抗体的水平显著高于MOPC IgA 单克隆抗体的分布水 平（p<0.05）。PA4 和MOPC IgA 单克隆抗体在粘膜分泌物内的分布水平差异可 能与其IgA 聚合形式的不同有关。另外，除气管外，在两时间点间分泌物中的 IgA 抗体水平没有显著差异。 检测背部带瘤小鼠交配后的两细胞胚胎期，发现携带PA4 和G2b 杂交细瘤 的雌性小鼠的受精率与对照组并没有显著的差异，这表明抗LDH-C4 IgA 和IgG 单克隆抗体在体内不能明显抑制小鼠的精子和卵子的结合或受精过程。注射细 胞后的27 天，检测鼠着床胚胎时，发现带瘤两性小鼠均携带PA4 时或者只有 雌性携带PA4 杂交瘤时，以及雌雄两性小鼠均携带G2b 杂交瘤细胞时，交配后的怀孕率与能分泌抗DNP 抗体的MOPC 的骨髓瘤细胞瘤的相应组别相比，显 著降低（p<0.01）。但PA4 各组与G2b 各组之间无显著差异(p>0.05）。然而，雌 雄的小鼠均带瘤时，最高怀孕减少率未能达100%。这些结果提示，抗LDHC4 IgA 和IgG 单克隆抗体在小鼠体内不能有效地抑制小鼠的精子与卵子的结 合，但能显著地抑制小鼠受精后胚胎的发育。抗LDH-C4 的IgA 和IgG 单克隆 抗体单独存在时，在体内均具有抗生育作用，但不能完全抑制生育。

抗LDH-C4 IgA和IgG抗体在带瘤小鼠生殖道和其它粘膜分泌物内分布特性的研究

中文摘要 　 已有的研究表明，小鼠背部携带能分泌抗原特异的IgA单克隆抗体的杂交细胞瘤，可以保护小鼠抵抗微生物和病毒等多种病原体经粘膜途径感染机体。我们利用背部携带能分泌抗精子特异抗原（LDH-C4）的IgA和IgG杂交细胞瘤、以及抗DNP的IgA骨髓细胞瘤的小鼠为动物模型，采用定量ELISA法研究了抗LDH-C4 IgA与抗DNP IgA单克隆抗体在呼吸道、肠道及生殖道内转运和分布，抗LDH-C4 IgG2b在肠道内转运和分布，以及抗LDH-C4 IgA和IgG单克隆抗体与体内抗生育作用的关系。研究结果表明，带瘤小鼠血液中含有较高水平抗原特异的IgA和IgG单克隆抗体。PA4和MOPC IgA单克隆抗体在呼吸道、肠道以及雌性生殖道分泌物内有较高的分布水平。在肠道，PA4和MOPC IgA 单克隆抗体的分布水平显著高于IgG（p < 0.01和p < 0.05）。在肠道和生殖道的不同部位，IgA抗体的分布水平不同。在肠道，结肠分泌物中的IgA单克隆抗体显著高于其它肠道部位（p < 0.01）。在生殖道，IgA单克隆抗体分布水平以子宫角分泌物中最高。雄性的前列腺也有较高的IgA抗体水平。在呼吸道、肠道以及雌性生殖道相应部位的分泌物内，PA4 IgA单克隆抗体的水平显著高于MOPC IgA单克隆抗体的分布水平（<0.05）。PA4和MOPC IgA单克隆抗体在粘膜分泌物内的分布水平差异可能与其IgA聚合形式的不同有关。另外，除气管外，在两时间点间分泌物中的IgA抗体水平没有显著差异。检测背部带瘤小鼠交配后的两细胞胚胎期，发现携带PA4或G2b杂交细胞瘤的雌雄小鼠的受精率与对照组并没有显著性差异，这表明抗LDH-C4 IgA和IgG单克隆抗体在体内不能显著抑制小鼠的精子和卵子的结合或受精过程。注射细胞后的27天，检测着床胚胎时，发现带瘤两性小鼠均携带PA4时或者只有雌性携带PA4杂交瘤时，以及雌雄性小鼠均携带G2b杂交瘤时，交配后的怀孕率与带能分泌抗DNP抗体的MOPC骨髓瘤细胞瘤的相应组别相比，显著降低（p < 0.01）。但PA4各组与G2b各组之间无显著差异（p < 0.05）。然而，雌雄小鼠均带瘤时，最高怀孕减少率也未达到100%。这些结果提示，抗LDH-C4 IgA和IgG单克隆抗体在小鼠体内不能有效地抑制小鼠的精子和卵子的结合，但能显著地抑制小鼠受精后胚胎的发育。抗LDH-C4 的IgA或者IgG单克隆抗体单独存在时，在小鼠体内均具有抗生育作用，但不能完全抑制生育。

Amplified immunoassay of human IgG using real-time biomolecular interaction analysis (BIA) technology

An automated biomolecular interaction analysis instrument (BI-Acore) based on surface plasmon resonance (SPR) has been used to determine human immunoglobulin G (IgG) in real time. Polyclonal anti-human IgG antibody was covalently immobilized to a carboxymethyldextran modified gold film surface. The samples of human IgG prepared in HBS buffer were poured over the immobilized surface. The signal amplification antibody was applied to amplify the response signal. After each measurement, the surface was regenerated with 0.1 mol/L H3PO4. The assay was rapid, requiring only 30 min for antibody immobilization and 20 min for each subsequent process of immune binding, antibody amplification and regeneration. The antibody immobilized surface had good response to human IgG in the range of 0.12-60 nmol/L with a detection limit of 60 pmol/L. The same antibody immobilized surface could be used for more than 110 cycles of binding, amplification and regeneration. The results demonstrate that the sensitivity, specificity and reproducibility of amplified immunoassay using real-time BIA technology are satisfactory.

口服牦牛血清IgG对小鼠免疫功能的影响

用环磷酰胺（Cy）制造小鼠免疫功能抑制模型，以口服扶正女贞素片的小鼠为阳性对照组，试验组小鼠连续口服耗牛血清IgG，10d后测定小鼠免疫水平的变化。结果，口服牦牛血清IgG的小鼠，抗Cy引起的免疫器官萎缩效果极显著（P＜0．01），抗Cy引起的小鼠血清IgG含量降低（P＜0．05），能提高小鼠血清IgG水平（P＜0．05）；牦牛血清IgG对小鼠急性毒性试验属无毒级。证明口服牦牛血清IgG能显著提高小鼠的免疫功能。

Selective defect of anti-pneumococcal IgG in a patient with persistent polyclonal B cell lymphocytosis.

BACKGROUND: Persistent polyclonal B cell lymphocytosis (PPBL) is a rare condition characterized by increased IgM and large excess of B cells with an IgD(+) CD27(+) phenotype. In normal individuals, these cells play a central role in the defense against pneumococcal infection. So far, few studies have characterized humoral immune responses in PPBL patients. We therefore measured IgG directed against S. pneumoniae antigens in a 51 yr-old woman with PPBL before and after vaccination with a pneumococcal 23-valent polysaccharide vaccine. METHODS: Antibodies against pneumococcal antigens were measured first with an overall immunoassay using microplates coated with the 23-valent pneumococcal vaccine. A serotype-specific test was also performed according to the WHO consensus protocol. RESULTS: Despite a large number of IgD(+) CD27(+) cells, our patient had low baseline titers of IgG directed against pneumococcal antigens and did not significantly respond to a 23-valent polysaccharide vaccine against S. pneumoniae. On the contrary, she had good titers of IgG directed against tetanus toxoid. CONCLUSION: IgM(+) IgD(+) CD27(+) cells which accumulate in this patient with typical PPBL patient failed to perform IgG isotype switch after a polysaccharide vaccine. The potential mechanisms and relationships with the main features of PPBL are discussed. Further studies on a larger number of similar patients are needed.

Receptors for secretin, helodermin, VIP (vasoactive intestinal peptide), PHI and GRF (growth hormone releasing factot) in the rat pancreas

info:eu-repo/semantics/published

Equal IgG antibody response to pneumococcal vaccination in all stages of human immunodeficiency virus disease.

To evaluate the immunogenicity and safety of a 23-valent pneumococcal vaccine in human immunodeficiency virus (HIV)-seropositive patients, 80 men and 18 women received 1 dose of the vaccine (Pneumo 23; Pasteur Mérieux MSD, Brussels). The total IgG antibody response against all 23 Streptococcus pneumoniae capsular antigens was measured. Antibody levels were expressed in arbitrary units per microliter, referring to a standard curve. Geometric mean titers of the total IgG capsular antibodies on the day of vaccination and 30-45 days later were compared. The ratios of titers after and before vaccination in patients with > 500, 200-500, and < 200 CD4 lymphocytes/microL were 10, 10, and 12.6, respectively. Nonresponse (ratio < 4) occurred in 17% of patients and was unrelated to CD4 cell count. The vaccine was well tolerated; no serious side effects occurred. In 83% of the patients with HIV infection, the total antipneumococcal IgG level was higher after vaccination.

An entirely cell-based system to generate single-chain antibodies against cell surface receptors.

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.

Normal IgG response to dermatophagoïde P1 antigen, bovin betalactoglobulin and soya protein

info:eu-repo/semantics/published

Detection of amino-terminal extracellular domain of somatostatin receptor 2 by specific monoclonal antibodies and quantification of receptor density in medulloblastoma.

Somatostatin receptor 2 (SSTR2) is expressed by most medulloblastomas (MEDs). We isolated monoclonal antibodies (MAbs) to the 12-mer (33)QTEPYYDLTSNA(44), which resides in the extracellular domain of the SSTR2 amino terminus, screened the peptide-bound MAbs by fluorescence microassay on D341 and D283 MED cells, and demonstrated homogeneous cell-surface binding, indicating that all cells expressed cell surface-detectable epitopes. Five radiolabeled MAbs were tested for immunoreactive fraction (IRF), affinity (KA) (Scatchard analysis vs. D341 MED cells), and internalization by MED cells. One IgG(3) MAb exhibited a 50-100% IRF, but low KA. Four IgG(2a) MAbs had 46-94% IRFs and modest KAs versus intact cells (0.21-1.2 x 10(8) M(-1)). Following binding of radiolabeled MAbs to D341 MED at 4 degrees C, no significant internalization was observed, which is consistent with results obtained in the absence of ligand. However, all MAbs exhibited long-term association with the cells; binding at 37 degrees C after 2 h was 65-66%, and after 24 h, 52-64%. In tests with MAbs C10 and H5, the number of cell surface receptors per cell, estimated by Scatchard and quantitative FACS analyses, was 3.9 x 10(4) for the "glial" phenotype DAOY MED cell line and 0.6-8.8 x 10(5) for four neuronal phenotype MED cell lines. Our results indicate a potential immunotherapeutic application for these MAbs.

Functional evolution of mammalian odorant receptors.

The mammalian odorant receptor (OR) repertoire is an attractive model to study evolution, because ORs have been subjected to rapid evolution between species, presumably caused by changes of the olfactory system to adapt to the environment. However, functional assessment of ORs in related species remains largely untested. Here we investigated the functional properties of primate and rodent ORs to determine how well evolutionary distance predicts functional characteristics. Using human and mouse ORs with previously identified ligands, we cloned 18 OR orthologs from chimpanzee and rhesus macaque and 17 mouse-rat orthologous pairs that are broadly representative of the OR repertoire. We functionally characterized the in vitro responses of ORs to a wide panel of odors and found similar ligand selectivity but dramatic differences in response magnitude. 87% of human-primate orthologs and 94% of mouse-rat orthologs showed differences in receptor potency (EC50) and/or efficacy (dynamic range) to an individual ligand. Notably dN/dS ratio, an indication of selective pressure during evolution, does not predict functional similarities between orthologs. Additionally, we found that orthologs responded to a common ligand 82% of the time, while human OR paralogs of the same subfamily responded to the common ligand only 33% of the time. Our results suggest that, while OR orthologs tend to show conserved ligand selectivity, their potency and/or efficacy dynamically change during evolution, even in closely related species. These functional changes in orthologs provide a platform for examining how the evolution of ORs can meet species-specific demands.