Epiglycanin-immunoreactive glycoproteins in mouse fetal tissues and fetal cells in culture

Fetal tissues from time-pregnant female A/J mice of 16- and 19-day pregnancies and from neonates 1 day after birth, as well as from fetal cells in culture, absorbed significant amounts of anti-epiglycanin antibody. Detergent-solubilized glycoproteins, with epiglycanin activity, from fetal tissues and cells were separated by polyacrylamide gel electrophoresis, and the protein bands electroblotted onto a nitrocellulose gel. After the antigens were labeled with rabbit anti-epiglycanin antiserum and [125I]epiglycanin, autoradiography revealed two major bands containing the antigenic determinant at Mr 90,000 and 82,000. Bands of similar molecular weights, but with no demonstrated immunologic cross-reactivity, were observed by fluorography, if intact cells prior to solubilization were labeled by galactose oxidase followed by sodium borotritiide. Immunoreactive epiglycanin activity could be destroyed by Pronase, endo-N-acetyl-alpha-D-galactosaminidase (Diplococcus pneumoniae), or periodate oxidation. Activity was enhanced with neuraminidase. The spleen, liver, or erythrocytes from adult A/J mice did not possess the antigen, but incubation of adult spleen or liver with neuraminidase (Vibrio cholerae) exposed the epitope.     

T cell depletion with anti-CD5 immunotoxin in histocompatible bone marrow transplantation. The correlation between residual CD5 negative T cells and subsequent graft-versus-host disease.

Twenty-nine patients with advanced leukemias (median age 34 years) received histocompatible sibling marrow that had been depleted of T cells by ex vivo incubation with anti-CD5 monoclonal antibody-ricin immunotoxin (T101-R) for the purpose of graft-versus-host disease prophylaxis. Donor cell engraftment was documented in 28/29 patients by DNA restriction fragment length polymorphisms. In this pilot study the dose of T101-R incubated with donor marrow was increased in a stepwise manner from 300 ng (10 patients) to 600 ng (5 patients) to 1000 ng immunotoxin (IT)/10(7) bone marrow mononuclear cells (14 patients) in an attempt to achieve more effective GvHD prophylaxis. A statistically significant reduction in acute GvHD was achieved for patients receiving marrow pretreated with 1000 ng of immunotoxin (34%) compared to recipients of BM treated with 300 ng immunotoxin (100%, P = 0.0004). T-depleted marrow samples were evaluated for residual T cell activity using several in vitro assays including proliferation to the purified mitogen PHA (HA-17) and in mixed lymphocyte culture (MLC), T cell cytotoxicity, a limiting dilution assay for detecting precursors of proliferating T cells (LDApPTL), and phenotypic analysis of viable T cells expanded in 16-day culture with interleukin 2. The extent of T cell depletion determined by LDA assay varied widely at each immunotoxin concentration used. Thus, there was no correlation between the dose of T cells infused and subsequent GvHD. Phenotyping of lymphocytes recovered from immunotoxin-treated marrow demonstrated that residual T cells were CD5 negative in all cases tested. The only in vitro parameter that predicted subsequent acute or chronic GvHD was the demonstration of viable CD5 negative lymphocytes with T cell phenotype (CD2, CD3, and/or CD7 positive) after 16-day culture with IL-2 of the T-depleted bone marrow. We observed that such CD5 negative cells expressing other T cell markers have cytotoxic function and speculate that these cells may be capable of mediating GvHD in allogeneic transplantation.     

Lectin-like cell adhesion molecule 1 mediates leukocyte rolling in mesenteric venules in vivo

During the inflammatory response, granulocytes and other leukocytes adhere to and emigrate from small venules. Before firm attachment, leukocytes are observed rolling slowly along the endothelium in venules of most tissues accessible to intravital microscopy. The molecular mechanism underlying this early type of leukocyte-endothelial interaction is unknown. Leukocyte rolling was investigated in venules (diameter, 40 microns) of the exposed rat mesentery. Micro-infusion of a recombinant soluble chimera (LEC-IgG) of the murine homing receptor lectin-like cell adhesion molecule 1 (LEC-CAM 1; gp90MEL) into individual venules reduced the number of rolling leukocytes by 89% +/- 2% (mean +/- SEM, n = 20 venules), while a similar CD4 chimera (CD4-IgG) had no effect (inhibition 14% +/- 7%, n = 25). Rolling was also greatly reduced by a polyclonal serum against LEC-CAM 1 (inhibition 84% +/- 3%, n = 35); preimmune serum was ineffective (11% +/- 13% inhibition, n = 28). These findings indicate that LEC-CAM 1 mediates the adhesive interaction underlying leukocyte rolling and thus may play an important role in inflammation and in pathologic conditions involving leukocytes.     

Case Report: Giant choledochal cyst

We report an adult female with a rare giant choledochal cyst. The patient presented following a normal pregnancy with the classical triad of an abdominal mass associated with jaundice and right upper quadrant abdominal pain. The cyst was excised using an intramural technique and biliary reconstruction achieved with a Roux-en-Y hepaticojejunostomy. Our patient has remained well with no evidence of malignancy over a 12 year review period. The aetiology and current management of this condition are discussed.     

Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa

The objective was to test the hypothesis that the optimal cryoprotective agent for cryopreservation of human spermatozoa would be a solute for which cells have the highest plasma membrane permeability, resulting in the least amount of volume excursion during its addition and removal. To test this hypothesis, theoretical simulations were performed using membrane permeability coefficients to predict optimal procedures for the addition and removal of a cryoprotectant. Simulations were performed using data from four different cryoprotectants: (i) glycerol, (ii) dimethyl sulphoxide, (iii) propylene glycol and (iv) ethylene glycol. Thermodynamic formulations were applied to determine approaches for the addition and removal of 1 M and 2 M final concentrations of cryoprotectant, allowing the spermatozoa to maintain a cell volume within their osmotic tolerance limits. Based on these data, ethylene glycol was predicted to be optimal for minimizing volume excursions among the solutes evaluated. These predictions were then experimentally tested using glycerol as the control cryoprotectant and ethylene glycol as the experimental cryoprotectant. The results indicate that there was a higher (P < 0.05) recovery of motile spermatozoa after cryopreservation when using 1 M ethylene glycol than with 1 M glycerol, supporting the hypothesis that use of the cryoprotectant for which the cell has the highest permeability will result in higher cell survival.