Elvax 40P implants: Sustained,local release of bioactive molecules influencing mammary ductal development

Elvax 40P (EVX), an ethylene vinyl-acetate copolymer, has been well characterized as an implant material that causes no inflammatory response and is capable of the sustained, local release of a wide variety of undenatured macromolecules in vivo. To investigate the usefulness of this material in developmental studies we examined the effect of EVX implants containing either deoxycorticosterone acetate (DCA) or testicular hyaluronidase on alveolar differentiation and ductal growth in the mouse mammary gland. DCA implants produced localized alveolar differentiation on ducts, while implants containing Thase caused basal lamina disruption at the duct's growing tip, resulting in epithelial dysplasias. We conclude that EVX implants allow assessment of the primary (nonsystemic) effects of biologically active molecules on developing tissue and should therefore have a variety of interesting experimental uses.     

DF3 antigen, a human epithelial cell mucin, inhibits adhesion of eosinophils to antibody-coated targets

Although mucins provide lubrication and physical protection for epithelial cell surfaces, other functional roles for these large glycoproteins are unknown. One human mucin, designated DF3 Ag, is detectable on apical surfaces of normal epithelial secretory cells and in normal milk, urine, and plasma. The present studies have examined the effects of DF3 Ag purified from both normal and malignant sources on the antibody-dependent cytotoxicity of Schistosoma mansoni by eosinophils (ADCC-E) and on the adherence of eosinophils to inert antibody-coated targets. DF3 Ag purified from tissue culture supernatant of a human breast carcinoma cell line or from human milk inhibited ADCC-E in a concentration-dependent manner, with half-maximal inhibitory activity at 3 to 10 micrograms/ml. Inhibition of ADCC-E was specific for the DF3 mucin, because no inhibition was observed with two other unrelated, circulating glycoproteins: carcinoembryonic Ag and alpha 1-acid glycoprotein. Inhibition was not a result of direct cytotoxicity of the DF3 Ag for eosinophils, as demonstrated by the lack of detectable effect of the mucin on cellular trypan blue exclusion or PMA-induced H2O2 release. The inhibitory effect was time dependent, requiring the presence of DF3 Ag in the ADCC-E culture for at least 4 h, beginning within the first 2 h of eosinophil-schistosomula interaction. Furthermore, inhibition was not a result of interaction between DF3 Ag and the activating lymphokine. These data suggest that inhibition of ADCC-E by DF3 Ag is a result of interference of adhesion of eosinophils to Ig-coated targets. In this regard, purified DF3 tumor Ag prevents eosinophil adherence to human Ig-conjugated Sepharose 4B beads. Preincubation of the inert Ig-coated targets with DF3 Ag did not inhibit subsequent eosinophil adherence, suggesting that DF3 Ag interacts with a moiety present on the eosinophil. Inhibition of adhesion occurred at 37 degrees C, but was also observed at 4 degrees C. These results suggest that DF3 Ag acts as an immunomodulating agent. Because activated eosinophils can damage surrounding normal tissues as well as infectious organisms, DF3 Ag may serve to protect secretory epithelium from the cytotoxic effects of activated inflammatory cells.     

THE ROLE OF NERVE GROWTH FACTOR IN THE RAMIFICATION OF SYMPATHETIC NERVE FIBRES INTO THE RAT IRIS IN ORGAN CULTURE

Small amounts of nerve growth factor (NGF) were present in the superior cervical ganglion and the iris of the rat. The observations that NGF content in each of the tissues was depleted during organ culture and that more NGF appeared in the media than was originally present in the tissues indicated that synthesis or activation of NGF had occurred in organ culture. Antibody to NGF or the depletion of endogenous NGF retarded growth of new sympathetic axons into irides in organ culture. Exogenously added NGF appeared to enhance the initiation of axonal sprouting and the rate of the ramification of nerve fibres.     

Effects of Escherichia Coli Subtilase Cytotoxin and Shiga Toxin 2 on Primary Cultures of Human Renal Tubular Epithelial Cells

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause post-diarrhea Hemolytic Uremic Syndrome (HUS), which is the most common cause of acute renal failure in children in many parts of the world. Several non-O157 STEC strains also produce Subtilase cytotoxin (SubAB) that may contribute to HUS pathogenesis. The aim of the present work was to examine the cytotoxic effects of SubAB on primary cultures of human cortical renal tubular epithelial cells (HRTEC) and compare its effects with those produced by Shiga toxin type 2 (Stx2), in order to evaluate their contribution to renal injury in HUS. For this purpose, cell viability, proliferation rate, and apoptosis were assayed on HRTEC incubated with SubAB and/or Stx2 toxins. SubAB significantly reduced cell viability and cell proliferation rate, as well as stimulating cell apoptosis in HRTEC cultures in a time dependent manner. However, HRTEC cultures were significantly more sensitive to the cytotoxic effects of Stx2 than those produced by SubAB. No synergism was observed when HRTEC were co-incubated with both SubAB and Stx2. When HRTEC were incubated with the inactive SubA_A272B toxin, results were similar to those in untreated control cells. Similar stimulation of apoptosis was observed in Vero cells incubated with SubAB or/and Stx2, compared to HRTEC. In conclusion, primary cultures of HRTEC are significantly sensitive to the cytotoxic effects of SubAB, although, in a lesser extent compared to Stx2.     

Human eosinophil cytotoxicity-enhancing factor. II. Multiple forms synthesized by U937 cells and their relationship to thioredoxin/adult T cell leukemia-derived factor

Recently, our laboratory reported the purification and partial amino acid sequence of a 10-kDa eosinophil cytotoxicity-enhancing factor (ECEF) polypeptide from the U937 cell source. This cytokine enhanced human eosinophil antibody-dependent cytotoxic function by greater than 200% and was half-maximally effective at a concentration of approximately 1 ng/ml. In this study, we describe the conditions required for ECEF synthesis and the use of rabbit antibody raised to 10-kDa ECEF to investigate the existence of related polypeptide species. Unstimulated U937 cells released an immunoreactive 14-kDA species. Cells stimulated with 7.5 micrograms/ml of LPS also released a 13-kDa species. Cells stimulated with 400 ng/ml of PMA also synthesized a 10-kDa species (equivalent in size to the form we had purified). This 10-kDa species remained primarily cell associated, but detectable amounts were released into the supernatant by 48 h of culture. In washed cell pellets, the location of the 10-kDa species was found to be in the plasma membrane, externally oriented, as determined by FACS analysis, iodination with the membrane impermeable reagent 125I-sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate, and by its removal with brief trypsin treatment. Partial amino acid sequence data suggested that the 14-, 13-, and 10-kDa species all share the same N-terminal. The 14- and 10-kDa ECEF species were recovered by electroelution from SDS-PAGE gels and tested for activity in the assay of eosinophil cytotoxic function. Because of the amino acid sequence similarities between the ECEF species and thioredoxin (TRX), rTRX (synthesized in Escherichia coli and purified) was also tested for activity. The 14-kDa ECEF and rTRX induced a slight, but consistent and statistically significant enhancement of eosinophil cytotoxic function. By comparison, lower doses of the 10-kDa ECEF induced a major increase in cytotoxic function. Thus the forms of ECEF differ in size, conditions required for synthesis, trafficking by the U937 cell after synthesis, and biologic activity. It is likely that these considerations bear on the involvement of ECEF in the pathophysiology of eosinophilia in vivo.     

DE-52 and DE-53 cellulose microcarriers

Summary DEAE cellulose anion exchangers having small ion exchange capacity (0.5 to 2.0 meq/g dry material) were tested for cell attachment kinetics and capacity to support growth anchorage-dependent cells. It was found that cells from established cell lines (BHK and MDCK) can grow to confluency on DEAE cellulose particles having exchange capacity of 1 and 2 meq/g dry materials, DE-52 and DE-53, respectively. On the other hand, chick embryo fibroblasts (primary cells) can grow only on DE-53 particles. This research was partially supported by a grant from the National Council for Research and Development, Israel, and the GSF, München, Germany.