Molecular and genetic characterizations of five pathogenic and two non-pathogenic monoclonal antiphospholipid antibodies

Antiphospholipid syndrome (APS) is an autoimmune disease that is characterized by thrombosis, recurrent fetal loss and thrombocytopenia. Antiphospholipid antibodies, detected by enzyme-linked immunoabsorbent assays (aCL) and/or in vitro blood clotting assays (LAC) are strongly associated with APS. Both the molecular structures used by pathogenic antiphospholipid antibodies and the genetic mechanisms leading to their production are unknown. We describe here the variable region genes of seven IgG antiphospholipid antibodies derived from two APS patients. Of these, five are pathogenic as defined in a mouse model of thrombosis and two are not. Analyses of the expressed variable region genes show no preferential V gene usage. However, similar to anti-DNA antibodies, pathogenic antiphospholipid antibodies contain an increased number of arginine residues in the third complimentarity-determining region (CDR3) of their H chains. The increased accumulation of arginine residues in the V(H) CDR3 may act to enhance antigen binding, promote disease and point to the importance of the H chain in the pathogenic potential of certain antiphospholipid antibodies.     

Altered GABAB receptor immunoreactivity in the gerbil hippocampus induced by baclofen and phaclofen, not seizure activity

The present study was performed to determine whether the effects induced by GABA(B) receptor-acting drugs would be related with the alteration in GABA(B) receptor expression in the hippocampus using Mongolian gerbil, a genetic epilepsy model. The distribution patterns of both GABA(B) receptor 1A/B and GABA(B)receptor 2 immunoreactivities were similarly detected in the hippocampi of normal and seizure-prone gerbils. Following baclofen (GABA(B) receptor agonist) or phaclofen (GABA(B) receptor antagonist) treatment, GABA(B) receptor immunoreactivities were decreased or increased by dose-dependent manners, respectively. Vigabatrin (GABA transaminase inhibitor) or 3-mercaptopropionic acid (GAD inhibitor) treatment did not affect GABA(B) receptor expressions. These findings suggest that GABA(B) receptor expression in the gerbil hippocampus may be altered by baclofen or phaclofen treatment.     

SIGN-R1, a novel C-type lectin expressed by marginal zone macrophages in spleen,mediates uptake of the polysaccharide dextran

The marginal zone macrophages of the spleen are implicated in the clearance of polysaccharides, but underlying mechanisms need to be pinpointed. SIGN-R1 is one of five recently identified mouse genes that are homologous to human DC-SIGN and encode a single, external, C-terminal C-type lectin domain. We find that a polyclonal antibody to a specific SIGN-R1 peptide reacts primarily and strongly with a subset of macrophages in the marginal zone of spleen and lymph node medulla. In both sites, SIGN-R1 exists primarily in an aggregated form, resistant to dissociation into monomers upon boiling in SDS under reducing conditions. Upon transfection into three different cell lines, high-mol.-wt forms bearing SIGN-R1 are expressed, as well as reactivity with ER-TR9, a mAb previously described to react selectively with marginal zone macrophages. SIGN-R1-expressing macrophages preferentially sequester dextrans following i.v. injection. Likewise, when phagocytic cells are enriched from spleen and tested in culture, dextran is selectively endocytosed by a subset of very large SIGN-R1(+) cells representing approximately 5% of total released macrophages. Uptake of FITC-dextran by these macrophages in vivo and in vitro is blocked by ER-TR9 and polyclonal anti-SIGN-R1 antibodies. Following transfection with SIGN-R1, cell lines become competent to endocytose dextrans. The dextran localizes primarily to compartments lacking transferrin receptor and the LAMP-1 CD107a panlysosomal antigen. Therefore, SIGN-R1 mediates the uptake of dextran polysaccharides, and it is predominantly expressed in the macrophages of the splenic marginal zone and lymph node medulla.     

Comparison between electrophysiologic and morphologic changes in lead induced peripheral neuropathy in rats

Compound nerve action potential (CNAP) of the mixed peripheral nerve is composed of A alpha beta, A delta, and C potentials. All components of CNAPs in the sciatic nerve were recorded by stimulating the tibial nerve of both control and lead-poisoned rats. Marked decrease of nerve conduction velocity and prolonged duration were found in A alpha beta and A delta fibers especially in large myelinated A alpha beta fibers. The amplitude decreased in A alpha beta potential, but the area did not change. In C potential produced by activation of unmyelinated fibers, nerve conduction velocity slightly decreased, but the amplitude and area did not significantly change. Pathologic correlates revealed prominent segmental demyelination with significant decrease of large myelinated fiber densities. Minimal axonal degeneration of unmyelinated fibers was present. We can conclude that electrophysiologic changes in the lead-poisoned rats correlate with pathologic changes in them.     

Thyroglobulin synthesis of oxyphilic cells in various types of neoplastic and autoimmune thyroid diseases.

To determine the content of thyroglobulin in oxyphilic cells of the thyroid, which have been considered as non-thyroglobulin producing cells, the degree of stainability of the various oxyphilic cells for thyroglobulin was compared with that of non-oxyphilic follicular cells in either same or different lesion. A total of 13 oxyphilic lesions, including three follicular adenomas containing oxyphilic cell nodules, four pure oxyphilic cell adenomas, and six Hashimoto's thyroiditis were compared with 16 of non-oxyphilic lesions such as, seven follicular adenomas, four chronic lymphocytic thyroiditis, and five Graves' disease. Many oxyphilic cells stained positively for thyroglobulin regardless of their morphologic variation, but less intensely than the usual follicular cells in follicular adenomas, chronic lymphocytic thyroiditis, and Graves' disease. The stainability of oxyphilic cells for thyroglogulin did not show any significant correlation with morphologic features, whereas in follicular adenomas, the non-oxyphilic follicular cells forming microfollicles stained less strongly for thyroglobulin than the same cells lining large mature follicles in a reproducible way. With above findings, we concluded that oxyphilic cells maintain the functional activity in terms of thyroglobulin synthesis, although the content of the thyroglobulin is less than that of non-oxyphilic colloid forming follicular cells.     

Structure, secretion, and bacterial specificity of an endogenous lectin from cystic fibrosis lung.

Endogenous heparin-binding lectin purified from postmortem lung samples of two cystic fibrosis (CF) patients was compared to lectin derived from normal tissue with respect to structure, carbohydrate specificity, interaction with alginate derived from CF isolates of Pseudomonas aeruginosa, and secretion within the lung. Lectin was purified from extracts of lung tissue by gel filtration on Sepharose CL-2B followed by affinity chromatography on heparin-Sepharose. Lectin purified from either CF lung or control tissue ran as two peptides of approximately 16,000 and 13,000 molecular weight on electrophoresis in sodium dodecyl sulfate. The lectins displayed similar carbohydrate specificity and interacted in much the same way with bacterial alginate. An increase in lectin secretion was seen in CF lungs affecting the bronchial epithelial cells and the mucosal glands. The data suggest that the major changes seen in endogenous heparin-binding lectin in CF are related to the quantity and distribution of lectin secretion.     

Skin thickness of Korean adults

Skin thickness varies considerably between different races and age-groups, between men and women, and between different regions of the body surface. A few authors reported the skin thickness of different regions of the body, but no detailed study have been performed on Asian. We performed 452 biopsies on 28 different regions of the normal skin of Korean men and women. The specimens were stained with hematoxylin-eosin and measured microscopically. The thickness of the skin (epidermis plus dermis) ranged from 521 to 1977 microm; the eyelid, prepuce, and inguinal skin was thinnest (521-626 microm), and the back was thickest (1977 microm). The thickness of the epidermis varied from 31 to 637 microm; skin thickness in the prepuce, eyelid, supraclavicular region, postauricular region, and axilla ranged from 31 to 71 microm; the buttock, dorsum of the hand, and dorsum of the foot were relatively thick (138-189 microm); the palm and sole were thickest (601-637 microm). The thickness dermis varied from 469 to 1942 microm; skin thickness in the eyelid, prepuce, inguinal region, and postauricular region ranged from 469 to 645 microm; the buttock, chest, and anterior neck were relatively thick (1318-1586 microm); the back was thickest (1942 microm). The epidermis accounted for 3.7-16.8% of the entire skin in most regions, except in the palm and sole (40.6-44.6%). Thickness data may be useful in harvesting full- or split-thickness skin grafts.     

MDM2 expression in EBV-infected nasopharyngeal carcinoma cells

To understand whether the p53-regulated mdm2 gene expression was altered by the Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC), the NPC-TW01 cell line was infected by EBV through IgA receptor-mediated endocytosis. The mdm2 gene was expressed only in a small fraction of the NPC cell population and could be enhanced in the EBV-infected (EBV+) cells. In the animals bearing EBV+ and EBV- NPC xenografts, the MDM2+ cells only appeared in clusters in both EBV+ and EBV- tumors with stronger expression in EBV+ cells. Cotransfection of pmdm2-Luc plus pSV40-p53 plus pCMV-LMP1 in the NPC-TW06 line that had p53 heterozygous point mutation showed stronger mdm2 promoter activity than cells cotransfected with pmdm2-Luc plus pSV40-p53, but no mdm2 promoter activity was seen in cells cotransfected with pmdm2-Luc plus pCMV-LMP1. Only the EBV-LMP1 but not the EBV-LMP2A gene could enhance p53 to upregulated mdm2 expression. Tumor cells in NPC biopsy specimens revealed similar mdm2 expression as in the animal model. It is concluded that although EBV can indirectly enhance mdm2 gene expression in tumor cells that express this gene, it cannot turn on or directly regulate mdm2 expression in cells that do not express this gene. In other words, EBV plays a role as an enhancer in NPC tumorigenesis.     

Hypersensitivity to Forcipomyia taiwana (biting midge): clinical analysis and identification of major For t 1, For t 2 and For t 3 allergens

BACKGROUND: Forcipomyia taiwana is a tiny, blood-sucking midge that cause intense pruritis and swelling in sensitive individuals. It is distributed island-wide in rural Taiwan and Southern China. Objective: This study aimed to study the allergic immune responses and identify F. taiwana allergens. METHODS: Crude whole body F. taiwana extracts were prepared with phosphate-buffered saline. The specific IgE antibody was determined by enzyme-linked immunoassay and immunoblotting. Protein was analyzed by electrospray ionization tandem mass spectrometry. RESULTS: Among the 372 subjects that were exposed to F. taiwana bites, 179 (48%) reported an immediate skin reaction with/without delay reaction and 41(11.1%) reported a solely delay reaction. The skin of 21 subjects was tested with F. taiwana extract. Of these 21 subjects, 12 (57.1%) produced immediate skin reactions and contained high levels of specific IgE antibody against F. taiwana. Immunoblotting revealed that 11 allergenic components are able to bind specific IgE. Allergens of 22, 24, 35, 36, and 64 kDa bound 50, 50, 75, 66.7, and 75% of IgE-containing sera tested, respectively. Tryptic fragments of the 24, 35, 36, and 64 kDa allergens were analyzed by ESI-MS/MS. Selected tryptic peptides of 24, 35, and 36, and 64 kDa allergens exhibited significant sequence identity with triosephosphate isomerase of Anopheles merus,Tenebrio molitor,Ochlerotatus togoi, and Chrysops vittatus, fructose 1,6-bisphosphate aldolase of Antheraea yamamai and Homalodisca coagulata, and a slow muscle myosin S1 heavy chain of Homarusamericanus and a protein with unknown function from A. gambiae, respectively. The 35 and 36 kDa proteins may represent different isoforms of the fructose 1,6-bisphosphate aldolase. CONCLUSION: We conclude that immediate reaction to F. taiwana bites is IgE mediated and the 24 (For t 1), 35 (For t 2), and 64 kDa (For t 3) proteins are candidates for major F. taiwana allergens. Further studies are needed to confirm these allergens.     

Determination of biogenic amines in fish implicated in food poisoning by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatography (MECC) method for the simultaneous determination of seven biogenic amines in fish was developed. The peaks of all components were successfully separated within 11.5 min. MECC was performed with 0.06 M sodium deoxycholate in 0.02 M borate buffer (pH 9.2)-methanol (95:5, v/v) solvent. The average recoveries for all components ranged from 84.4 to 100.3%. The application of this method to detect amines in fried marlin fillet implicated in a food poisoning incident indicated that a high level (56.24 mg/100 g) of histamine was present in the sample. Another 10 fish samples collected from markets were also analyzed and did not contain detectable levels of histamine (<2.5 mg/100 g).