Fc-independent cross-linking of a novel platelet membrane protein by a monoclonal antibody causes platelet activation

A monoclonal antiplatelet antibody (MA-13G8E1) is described that dose- dependently induces platelet aggregation and serotonin release in an Fc- independent fashion. Whereas platelets were equally aggregated by F(ab')2 fragments of this monoclonal antibody (MoAb), its Fab fragments, on the other hand, were inactive, indicating that divalent interaction is an essential requirement to induce platelet activation by MA-13G8E1. In addition, we could show that platelet epitope cross- linking by MA-13G8E1 occurred on the same platelet. MA-13G8E1 stimulated platelet phospholipase C (PLC) and induced activation of protein kinase C (PKC), both of which were almost unaffected by aspirin pretreatment. Furthermore, PLC activation appeared to be a direct antibody-mediated effect, since intracellular Ca2+ rises were not inhibited by EGTA, cytochalasin B, or aggregation-blocking MA-16N7C2 (antiglycoprotein [anti-GP]IIb/IIa). The MA-13G8E1 antigen is constitutively expressed on resting platelets of different species (7,100 +/- 800 molecules per human platelet), but not on other cell types tested. Both immunoprecipitation and affinity isolation by MA- 13G8E1 showed two low-molecular weight proteins (45 and 36 kD), having slightly acidic isoelectric pH levels (4.5 to 5.5) and forming multimolecular complexes. In conclusion, we found an MoAb that is able to induce platelet activation in an Fc-independent fashion. The mechanism involves cross-linking of a hitherto undescribed platelet membrane protein, leading to PLC and PKC stimulation.     

Malignant uveal melanoma and simulating lesions: MR imaging evaluation

Twenty-one patients with intraocular disease were studied by magnetic resonance (MR) imaging and computed tomography (CT). In 13 cases, malignant uveal melanoma was considered the likely diagnosis. Both imaging methods were accurate in determining the location and size of uveal melanomas. MR imaging was superior for the assessment of possible associated retinal detachment, for assessment of vitreous change, and for differentiating uveal melanoma from choroidal hemangioma and choroidal detachment. A case of retinal gliosis could not be differentiated from uveal melanoma by either technique. Uveal melanomas appeared as hyperintense lesions on T1-weighted images and as hypointense lesions on T2-weighted images. High signal intensity of the vitreous was observed in patients with vitritis and in those who were thought to have protein leaking into the vitreous as a result of impairment of the retinal-blood barrier.     

Evaluation of a rapid stoolantigen test for the diagnosis of Helicobacter pylori infection in Chinese patients

OBJECTIVE: To evaluate the accuracy of a rapid assay that wasdeveloped to detect Helicobacter pylori antigen in the stool,using the principle of immunochromatography, in the Chinese population. METHODS: Eligible patients without prior treatment of H.pylori were recruited. An in‐house rapid urease test (RUT) andhistology were used as the gold standard. The results of the rapidstool antigen test were compared with the gold standard. RESULTS: Valid rapid stool antigen test results for interpretationwere obtained from 94 consecutive patients (mean age: 52.5, range:22?82 years). Sensitivity, specificity, positive predictivevalue, negative predictive value and accuracy were, respectively, 77.5%,87.0%, 81.6%, 83.9% and 83.0%.The test was easy to perform and results were available within 15 min. CONCLUSION: The rapid stool antigen test using immunochromatography accuratelydiagnoses H. pylori infection in Chinese patients.     

Identification of the gene encoding the human mitochondrial RNA polymerase (h-mtRPOL) by cyberscreening of the Expressed Sequence Tags database

A gene cloning strategy based on the screening of the Expressed Sequence Tags database (dbEST) using sequences of mitochondrial housekeeping proteins of yeast was employed to identify the cDNA encoding the precursor of the human mitochondrial RNA polymerase (h- mtRPOL). The 3831 bp h-mtRPOL cDNA is located on chromosome 19p13.3 and encodes a protein of 1230 amino acid residues. The protein sequence shows significant homologies with sequences corresponding to mitochondrial RNA polymerases from lower eukaryotes, and to RNA polymerases from several bacteriophages. The mitochondrial RNA polymerase carries out the central activity of mitochondrial gene expression and, by providing the RNA primers for replication- initiation, is also implicated in the maintenance and propagation of the mitochondrial genome. Genes involved in the control of mtDNA replication and gene expression are attractive candidates for human disorders due to abnormalities of nucleo-mitochondrial intergenomic signalling. The availability of the h-mtRPOL cDNA will allow us to test its role in mitochondrial pathology. In addition, we propose the 'cyberscreening' of dbEST, based on yeast/human cross-species comparison, as a powerful, simple, rapid and inexpensive method, that may accelerate several-fold the molecular dissection of the human mitochondrial proteome.      

Interleukin-7 influences osteoclast function in vivo but is not a critical factor in ovariectomy-induced bone loss.

IL-7 is produced by stromal cells in bone marrow and is a major regulator of B and T lymphopoiesis. It is also a direct inhibitor of osteoclastogenesis in vitro. In this study we show that IL-7-deficient mice have increased OC and decreased trabecular bone volume compared with WT mice but mimic WT mice in the amount of trabecular but not cortical bone lost after ovariectomy. INTRODUCTION: Interleukin (IL)-7 is a potent regulator of lymphocyte development, which has significant effects on bone. Bone marrow cell cultures from IL-7 deficient (IL-7KO) mice produced significantly more TRACP(+) osteoclasts (OCs) than did cells from wildtype (WT) mice. A previous study found that treatment of mice with a neutralizing antibody to IL-7 blocked ovariectomy (OVX)-induced bone loss. We examined if differences exist between the bones of WT and IL-7KO mice and if OVX altered bone mass in IL-7KO mice. MATERIALS AND METHODS: Studies were in 2-month-old sham-operated (SHAM) and OVX female mice that were killed 4 weeks after surgery. IL-7KO mice and WT controls were in a C57BL/6 background. Both vertebrae (L(1)) and femora were evaluated by DXA, muCT, and histomorphometry. IL-7KO mice were confirmed as IL-7 deficient by their almost total lack of mature B cells in their bone marrow. RESULTS: There was significantly less trabecular bone volume in the vertebrae of IL-7KO mice than in WT mice. In addition, IL-7KO mice had significantly decreased (p < 0.05) trabecular number (13%) and increased trabecular spacing (15%). OVX decreased vertebral trabecular bone volume (TBV) by 21% (p < 0.05) in WT mice and by 22% (p < 0.05) in IL-7KO mice compared with SHAM. IL-7KO SHAM mice also had significantly less (30%) TBV (TA/TTA) in their femurs, as measured histomorphometrically, than did WT SHAM mice. Femurs from IL-7KO SHAM mice had significantly increased percent OC surface (23%) compared with WT SHAM. As in the vertebrae, OVX significantly decreased femoral TBV in both WT and IL-7KO mice by similar amounts (47% and 48%, respectively, p < 0.05 for both) compared with SHAM. However, OVX decreased cortical bone mass in WT but not in IL-7KO bones. We also examined bone marrow cells from WT and IL-7KO mice. Bone marrow cells from IL-7KO animals showed a significant increase in the number of TRACP(+) osteoclast-like cells (OCLs), which formed in cultures that were stimulated with macrophage-colony stimulating factor (M-CSF) and RANKL (both at 30 ng/ml). However, there was no significant difference in the number of OCLs that formed in B lymphocyte-depleted (B220(-)) bone marrow cell cultures from WT and IL-7KO mice. CONCLUSIONS: IL-7 deficiency in mice caused increased OC number in bone and decreased bone mass. OVX-induced bone loss in IL-7-deficient mice was selective and occurred in trabecular but not cortical bone.