Molecular identification of T cell-specific antigens on human T lymphocytes and thymocytes

We have identified membrane glycoproteins which carry T cell-specific antigens on human T lymphocytes and thymocytes. Purified cells were surface-labeled with NaB³H₄ after treatment with neuraminidase and galactose oxidase. Immunoprecipitations were performed with rabbit anti-human T cell-specific antibodies using co precipitation with protein A-containing staphylococci strain Cowan I. The labeled membrane glycoproteins and the precipitates were subjected to polyacrylamide slab gel electrophoresis and visualized by fluorography. The antibodies specifically precipitated 4 proteins called GP200, GP180, GP165 and GP160 (mol. wts. = 200000, 180000, 165000 and 160000) from surface-labeled T lymphocytes and low-density (medullary) thymocytes. The GP200 and GP180 were not labeled on high-density (cortical) thymocytes. A protein with a mol. wt. of 45000 was precipitated from thymocytes. Another glycoprotein on T lymphocytes and thymocytes with a mol. wt. similar to that of mouse and rat Thy-1 or Θ antigen (mol. wt. 25000) reacted with the antibodies.     

A novel inserter instrument (Crossbow) for installation of self-reinforced bioabsorbable arrows into meniscus tissue

A novel inserter, Crossbow, was developed for arthroscopic installation of self-reinforced bioabsorbable meniscus arrows into meniscus tissue. The Crossbow comprises a reservoir cartridge for up to four arrows, a triggering mechanism, and an interchangeable (curved or straight) trocar for guiding and installation of an arrow. A randomized biomechanical study was carried out by installing 13-mm-long meniscus arrows into fresh porcine menisci with the Crossbow and with the standard manual inserter trocar system. The mean pull-out force for Crossbow-installed arrows was 56.8 +/- 14.2 N, and that for arrows installed with the manual system was 43.7 +/- 13.8 N; the difference was statistically significant. While Crossbow is easy and rapid to use and gives good grip of arrows into pig meniscus, it may be an attractive alternative instrument to treat bucket-handle lesions of the menisci arthroscopically.     

Pharmacokinetics of Clodronate after Single Intravenous,Intramuscular and Subcutaneous Injections in Rats

Abstract: The pharmacokinetics of clodronate was studied in rats after single intravenous, intramuscular and subcutaneous doses of a mixture of unlabelled and ¹⁴C-labelled disodium clodronate (25 mg/5 μCi/kg). The peak clodronate concentration in plasma was reached within 5 min., and the drug was eliminated with a half-life of about 1.5 hr regardless of administration route. Bioavailabilities after intramuscular and subcutaneous administration were 105% and 89%, respectively. During the 72 hr collection period, the mean share of clodronate recovered from the urine was about 53% of the dose regardless of administration route. Most of the drug was excreted during the first 24 hr. The amount of clodronate in bone (femur) was 186 μg/g tissue at 2 hr after intravenous administration, 188 μg/g after intramuscular administration and 157 μg/g after subcutaneous administration. It is concluded that absorption of clodronate after intramuscular and subcutaneous administration is rapid and good, and that the concentrations of the drug in bone after 2 hr are about the same as after intravenous administration.     

The Influence of Dichloromethylene Bisphosphonate on the Healing of a Long Bone Fracture,Composition of Bone Mineral and Histology of Bone in the Rat

Abstract: The effects of dichloromethylene bisphosphonate (clodronate) on the composition of bone mineral, morphology and histology of a long bone with an artificial femoral fracture were studied in a 22 week experiment. Two hundred twenty-four female rats were allocated to dose groups of 0, 3, 10, and 30 mg/kg clodronate daily subcutaneously. Bone calcium, phosphorus and magnesium concentrations remained stable and fluoride concentration rose with time. There were no statistical differences between different groups. Clodronate did not alter the histology of the callus nor delayed the healing of the fracture. It caused mild to moderate prominence of the metaphyseal area in the fractured bone in a dose- and time-dependent manner. Serum osteocalcin levels were lowered in the treated animals dose-dependently. Other serological as well as haematological values were within normal range. Clodronate seems in this experimental arrangement to be a safe agent to administer in different pathological conditions of bone even when they are complicated by fractures of long bones.     

Distribution and epitope analysis of the cell membrane glycoprotein (HPCA-1) associated with human hemopoietic progenitor cells

Monoclonal antibodies My10, BI.3C5, 12.8, and ICH3 identify a monomeric cell surface glycoprotein (HPCA-1) of 100-120 kD, which is selectively expressed on human hemopoietic progenitor cells. Other tissues are nonreactive with the exception of capillary endothelia and basement membrane in some sites. In addition, the antigen can be detected on cell lines that exhibit characteristics associated with early T cell precursors. HPCA-1 is therefore associated with myeloid, B, and T lineage precursors. Sequential immunoprecipitation and Western blotting studies demonstrate that BI.3C5, ICH3, My10, and an antibody directed against endothelial cells, 188.27, all react with the same glycoprotein species, although the epitopes involved may be distinct. The epitope recognized by BI.3C5 is sialic acid dependent, whereas that recognized by ICH3 is not. The My10 epitope has partial sensitivity to neuraminidase. Competitive/additive binding experiments suggest that these epitopes, although probably distinct, may be closely associated.     

Expression of blood group A antigens in human bone marrow cells

We have studied the appearance of blood group A-activity during hematopoiesis in human bone marrow cells by the use of the blood group A-specific lectin from Vicia cracca. Cells that bound the lectin were identified using antiserum against the lectin followed by rosetting with protein A-containing Staphylococcus aureus cells. Only cells of the erythroid lineage from blood group A individuals formed staphylococcal rosettes. A-activity occurred in basophilic normoblasts and later stages of erythropoiesis, whereas pronormoblasts were negative. The appearance of blood group A-activity coincided roughly with the onset of hemoglobin synthesis and slightly later than the expression of the major sialoglycoprotein of erythrocytes, glycophorin A. Glycophorin A did not, however, contain blood group A-activity when analyzed by immunoprecipitation and gel electrophoresis.     

Pulmonary inactivation of 5-hydroxytryptamine is decreased during cigarette smoke ventilation of rat isolated lungs.

1 The effect of cigarette smoke ventilation on the inactivation of [14C]-5-hydroxytryptamine (5-HT) was studied in isolated perfused lungs of the rat. 2 [14C]-5-HT 9.6 nmol was infused into the pulmonary circulation of rat lungs in 3 min. The nonrecirculating perfusion effluent was collected during the 5-HT infusion in three consecutive 1 min fractions. The amount of metabolites of 5-HT was determined from the perfusion effluent and from the perfused lungs. 3 The amount of metabolites of 5-HT in the perfused lungs was also decreased by cigarette smoke ventilation, although the total amount of radioactivity in the lung tissue was not significantly changed. 5 The decreased pulmonary inactivation of 5-HT may cause increased circulating levels of 5-HT, which would explain some cardiovascular changes during smoking.     

The correlation of breakpoint cluster region rearrangement and p210 phl/abl expression with morphological analysis of Ph-negative chronic myeloid leukemia and other myeloproliferative diseases

The chromosome 22 derivative, the Philadelphia (Ph) chromosome, results from a reciprocal translocation t(9;22) (q34;q11) and is associated with chronic myeloid leukemia (CML). The translocation can be identified at the DNA level in Ph-positive CML by using a probe to the breakpoint cluster region (bcr). In addition, as a result of this translocation an abl-related 210-kd protein with protein tyrosine kinase (PTK) activity is produced. We analyzed 28 cases of Ph-negative CML for rearrangement of the chromosome 22 sequences and found that eight of the 28 show rearrangement of the bcr. When 12 of the Ph-negative cases were independently reviewed, five were indistinguishable from Ph-positive CML on the basis of morphology, peripheral blood film and clinical details. These five also showed bcr rearrangement. The other seven were reclassified as six atypical CML (aCML) and one chronic myelomonocytic leukemia (CMML). None of these seven showed bcr rearrangement. In addition 11 cases of bcr- CML were assayed for abl-related PTK, and no detectable activity was present, whereas p210 phl/abl PTK was observed both in Ph-positive (three cases examined) and Ph-negative, bcr + (four cases examined) CML. Therefore, bcr + CML, whether or not the Ph chromosome is cytogenetically apparent, involves a similar molecular alteration and produces the 210-kd protein with enhanced PTK activity. Furthermore, these cases can be distinguished from Ph-negative bcr- CML by careful evaluation of clinical and hematologic data.     

Pharmacokinetics of clodronate after single intravenous, intramuscular and subcutaneous injections in rats.

The pharmacokinetics of clodronate was studied in rats after single intravenous, intramuscular and subcutaneous doses of a mixture of unlabelled and 14C-labelled disodium clodronate (25 mg/5 muCi/kg). The peak clodronate concentration in plasma was reached within 5 min., and the drug was eliminated with a half-life of about 1.5 hr regardless of administration route. Bioavailabilities after intramuscular and subcutaneous administration were 105% and 89%, respectively. During the 72 hr collection period, the mean share of clodronate recovered from the urine was about 53% of the dose regardless of administration route. Most of the drug was excreted during the first 24 hr. The amount of clodronate in bone (femur) was 186 micrograms/g tissue at 2 hr after intravenous administration, 188 micrograms/g after intramuscular administration and 157 micrograms/g after subcutaneous administration. It is concluded that absorption of clodronate after intramuscular and subcutaneous administration is rapid and good, and that the concentrations of the drug in bone after 2 hr are about the same as after intravenous administration.     

Structural and partial amino acid sequence analysis of the human hemopoietic progenitor cell antigen CD34

Monoclonal antibodies of the CD34 class all recognize a monomeric cell surface antigen of approximately Mr 110,000 which is selectively expressed on human hemopoietic progenitor cells. This structure can be readily surface-labeled with [125I]actoperoxidase and by periodate-[3H]borohydride, but it labels only weakly with [35S]methionine, [35Sl]cysteine, 3H-amino acids, or 3H-mannose, even after prolonged labeling periods. However, the antigen is more efficiently labeled by [3H]glucosamine. Lectin binding studies, sensitivity to certain glycosidases, and gel filtration analysis of glycans released by alkaline hydrolysis indicate that this glycoprotein contains several complex-type N-linked glycans as well as several highly sialylated O-linked glycans. Western blotting experiments show that various CD34 antibodies fail to efficiently detect desialylated and/or de-N-glycosylated forms of the antigen. Experiments involving the use of tunicamycin, together with metabolic labeling studies, strongly suggest that this structure "turns over" very slowly in vivo. The CD34 antigen is not detectably labeled by 32P-phosphate in vivo, nor are immune complexes containing it associated with phosphokinase activity in vitro. Sequential immunoprecipitation and Western blotting studies indicate that this antigen is not a member of the leukosialin/sialophorin family despite the fact that these molecules share several structural similarities. Partial amino acid analysis of highly purified CD34 antigen revealed no significant sequence similarity with any previously described structures.