Antibodies to acetylcholine receptor in parous women with myasthenia: evidence for immunization by fetal antigen

The weakness in myasthenia gravis (MG) is mediated by autoantibodies against adult muscle acetylcholine receptors (AChR) at the neuromuscular junction; most of these antibodies also bind to fetal AChR, which is present in the thymus. In rare cases, babies of mothers with MG, or even of asymptomatic mothers, develop a severe developmental condition, arthrogryposis multiplex congenita, caused by antibodies that inhibit the ion channel function of the fetal AChR while not affecting the adult AChR. Here we show that these fetal AChR inhibitory antibodies are significantly more common in females sampled after pregnancy than in those who present before pregnancy, suggesting that they may be induced by the fetus. Moreover, we were able to clone high-affinity combinatorial Fab antibodies from thymic cells of two mothers with MG who had babies with arthrogryposis multiplex congenita. These Fabs were highly specific for fetal AChR and did not bind the main immunogenic region that is common to fetal and adult AChR. The Fabs show strong biases to VH3 heavy chains and to a single Vk1 light chain in one mother. Nevertheless, they each show extensive intraclonal diversification from a highly mutated consensus sequence, consistent with antigen-driven selection in successive steps. Collectively, our results suggest that, in some cases of MG, initial immunization against fetal AChR is followed by diversification and expansion of B cells in the thymus; maternal autoimmunity will result if the immune response spreads to the main immunogenic region and other epitopes common to fetal and adult AChR

A New Peptide Ligand for Targeting Human Carbonic Anhydrase IX, Identified through the Phage Display Technology

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy

Optimization of a Novel Peptide Ligand Targeting Human Carbonic Anhydrase IX

BACKGROUND: Carbonic anhydrase IX (CA IX) is a hypoxia-regulated transmembrane protein over-expressed in various types of human cancer. Recently, a new peptide with affinity for human carbonic anhydrase IX (CaIX-P1) was identified using the phage display technology. Aim of the present study is to characterize the binding site in the sequence of CaIX-P1, in order to optimize the binding and metabolic properties and use it for targeting purposes. METHODOLOGY/PRINCIPAL FINDINGS: Various fragments of CaIX-P1 were synthesized on solid support using Fmoc chemistry. Alanine scanning was performed for identification of the amino acids crucial for target binding. Derivatives with increased binding affinity were radiolabeled and in vitro studies were carried out on the CA IX positive human renal cell carcinoma cell line SKRC 52 and the CA IX negative human pancreatic carcinoma cell line BxPC3. Metabolic stability was investigated in cell culture medium and human serum. Organ distribution and planar scintigraphy studies were performed in Balb/c nu/nu mice carrying subcutaneously transplanted SKRC 52 tumors. The results of our studies clearly identified amino acids that are important for target binding. Among various fragments and derivatives the ligand CaIX-P1-4-10 (NHVPLSPy) was found to possess increased binding potential in SKRC 52 cells, whereas no binding capacity for BxPC3 cells was observed. Binding of radiolabeled CaIX-P1-4-10 on CA IX positive cells could be inhibited by both the unlabeled and the native CaIX-P1 peptide but not by control peptides. Stability experiments indicated the degradation site in the sequence of CaIX-P1-4-10. Biodistribution studies showed a higher in vivo accumulation in the tumor than in most healthy tissues. CONCLUSIONS: Our data reveal modifications in the sequence of the CA IX affine ligand CaIX-P1 that might be favorable for improvement of target affinity and metabolic stability, which are necessary prior to the use of the ligand in clinical approaches

Isolation and characterization of a laminin-binding protein from rat and chick muscle.

A major laminin-binding protein (LBP), distinct from previously described LBPs, has been isolated from chick and rat skeletal muscle (Mr 56,000 and 66,000, respectively). The purified LBPs from the two species were shown to be related antigenically and to have similar NH2-terminal amino acid sequences and total amino acid compositions. Protein blots using laminin and laminin fragments provided evidence that this LBP interacts with the major heparin-binding domain, E3, of laminin. Studies on the association of this LBP with muscle membrane fractions and reconstituted lipid vesicles indicate that this protein can interact with lipid bilayers and has properties of a peripheral, not an integral membrane protein. These properties are consistent with its amino acid sequence, determined from cDNAs (Clegg et al., 1988). Examination by light and electron microscopy of the LBP antigen distribution in skeletal muscle indicated that the protein is localized primarily extracellularly, near the extracellular matrix and myotube plasmalemma. While a form of this LBP has been identified in heart muscle, it is present at low or undetectable levels in other tissues examined by immunocytochemistry indicating that it is probably a muscle-specific protein. As this protein is localized extracellularly and can bind to both membranes and laminin, it may mediate myotube interactions with the extracellular matrix

Mining Genomic Variants And Causal Pathways Linking Hdl And Triglycerides To Coronary Disease

Blood lipids are important biomarkers of risk of coronary heart disease (CHD), the leading cause of death in the world. Myriad data support a causal role of low-density lipoprotein cholesterol (LDL-C) in increasing risk of CHD. Long-standing epidemiology suggests that high-density lipoprotein cholesterol (HDL-C) may protect from disease while high triglycerides (TGs) increase CHD risk. However, the causality of HDL-C and TG to CHD remains controversial. New genetic methodologies have allowed a better look into causal pathways underlying relationships between these traits and disease. Using a combination of approaches for interrogating rare genetic variation in humans, we investigated how HDL and TG may relate to CHD. First, through sequencing and exome-wide genotyping of subjects with extremely high HDL-C, we identified the first homozygote for a loss-of-function (LOF) variant in SCARB1, which encodes scavenger receptor class BI (SR-BI), a hepatic receptor for HDL-C. Despite markedly elevated HDL-C, carriers of this variant had an increased risk of CHD. These findings suggest that HDL functionality in driving cholesterol removal through SR-BI (the reverse cholesterol transport hypothesis) is protective from CHD in humans. Next, we functionally examined one of the first novel loci from genome-wide association studies (GWAS) for HDL-C, GALNT2. Through discovery of humans with genetic GALNT2 LOF and additional studies in rodents and nonhuman primates, we showed that GALNT2 LOF lowers HDL-C across mammals. We also identify one physiological mechanism linking GALNT2 to HDL-C through its enzymatic function. Thirdly, we studied the mechanism of protection of the APOC3 A43T variant recently reported to lower TGs and CHD risk from exome sequencing. Studies in human carriers and animal models suggest that A43T accelerates renal clearance of circulating ApoC-III, thus hindering its function in delaying TG-rich lipoprotein turnover. These data establish ApoC-III clearance mechanisms as potential therapeutic targets for TG lowering. Finally, we adapted a targeted sequencing approach to increase discovery of causal rare coding and noncoding variants at candidate loci influencing HDL-C and TGs. Collectively, this work provides a sampling of approaches for leveraging the spectrum of genomic methods to identify clinically relevant variants impacting HDL, TG and CHD risk

Gamma-ray imaging detector for small animal research

A novel radiation imaging technology for in vivo molecular imaging in small mammals is described. The goal of this project is to develop a new type of imaging detector system suitable for real-time in vivo probe imaging studies in small animals. This technology takes advantage of the gamma-ray and x-ray emission properties of the radioisotope iodine 125 (125I) which is employed as the label for molecular probes. The radioisotope 125I is a gamma-ray emitting radioisotope that can be commercially obtained already attached to biomedically interesting molecules to be used as tracers for biomedical and molecular biology research.;The isotope 125I decays via electron capture consequently emitting a 35 keV gamma-ray followed by the near coincident emission of several 27--32 keV Kalpha and Kbeta shell x-rays. Because of these phenomena, a coincidence condition can be set to detect 125I thus enabling the reduction of any background radiation that could contaminate the image. The detector system is based on an array of CsI(Na) crystal scintillators coupled to a 125 mm diameter position sensitive photomultiplier tube. An additional standard 125 mm diameter photomultiplier tube coupled to a NaI(Tl) scintillator acts as the coincident detector. to achieve high resolution images the detector system utilizes a custom-built copper laminate high resolution collimator. The 125I detector system can achieve a spatial resolution of less than 2 mm FWHM for an object at a distance of 1.5 cm from the collimator. The measured total detector sensitivity while using the copper collimator was 68 cpm/muCi.;Results of in vivo mouse imaging studies of the biodistribution of iodine, melatonin, and a neurotransmitter analog (RTI-55) are presented. Many studies in molecular biology deal with following the expression and regulation of a gene at different stages of an organism\u27s development or under different physiological conditions. This detector system makes it possible for laboratories without access to standard nuclear medicine radiopharmaceuticals to perform in vivo imaging research on small a mammals using a whole range of 125I labeled markers that are obtainable from commercial sources

A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen

<p>Abstract</p> <p>Background</p> <p>We have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients.</p> <p>Methods</p> <p>The current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification.</p> <p>Results</p> <p>By application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein.</p> <p>Conclusion</p> <p>We have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.</p