Protective genes expressed in endothelial cells of second hamster heart transplants to rats carrying an accommodated first graft

Abstract: Accommodation refers to survival of a xenograft despite the presence of anti-donor organ antibodies and complement. We have recently shown that accommodation of a hamster heart transplanted to a rat receiving short-term cobra venom factor (CVF) and continuing cyclosporine A (CyA) therapy is associated with i) the expression in the endothelial cells (EC) and smooth muscle cells of the graft of a number of "protective" genes, ii) a prominent intragraft Th2 cytokine profile, and iii) the relatively heavy deposition of IgG2c antibodies on the EC of the graft. In contrast, rejecting grafts do not express the protective genes, have a Th1 cytokine profile, and apparently have lesser amounts of IgG2c. These findings are consistent with host factors (Th2 cytokines and IgG2c) contributing to xenograft accommodation. To test whether these host factors may predispose to the development of accommodation, we placed a second hamster heart into each of 12 rats carrying a surviving first heart; recipients were, at the time, receiving only CyA. Whereas first grafts transplanted to rats receiving only CyA survive for 3 to 4 days, 11 out of 12 second transplants survived more than 20 days, and the other survived for 7 days. Nine of the twelve were not rejected: of these, four were removed between day 35 and 132 for study, and the remainder are still beating at 35 to 52 days. The surviving second hearts we studied had accommodated in that the picture on immunopathology was the same as for surviving first hearts. We suggest that the Th2 cytokines and perhaps the IgG2c response are factors in allowing prolonged survival of the second grafts and, further, that these factors contribute to the expression in the EC and smooth muscle cells of the surviving second hearts of the protective genes.     

Selective cytotoxicity associated with in vitro exposure of fresh rat renal fragments and continuous cell lines to atractyloside

The consumption of plants containing the diterpenoid atractyloside (ATR) causes selective proximal tubule injury, renal failure and death in humans. We have compared the effects of ATR in freshly isolated renal proximal tubules and glomeruli from rat and also in cell lines: NRK, derived from the proximal tubules, and MDBK and MDCK more closely representing the distal nephron. The effects of ATR (10– 500?μM) on proximal tubules and glomeruli were assessed by changes in lipid peroxidation, de novo protein synthesis and the leakage of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), glutamate dehydrogenase (GDH) and N-acetyl-β-D-glucosaminidase (NAG). The susceptibility of NRK, MDBK and MDCK cell lines to ATR was assessed by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, measuring mitochondrial reduction. Enzyme leakage was the most sensitive of the markers of cell injury in fresh fragments and ranked LDH>GDH>ALP>NAG in proximal tubules. As little as 20?μM ATR caused significant enzyme leakage from proximal tubules, but there were no increases in enzyme leakage from glomeruli at concentrations ?500?μM ATR. De novo protein synthesis was only inhibited 50% at ATR concentration >5?mM in the proximal tubules, but there were no effects in glomeruli. Malondialdehyde production was significantly elevated at 1?mM ATR for proximal tubules, and 500?μM for glomeruli. NRK cells were sensitive to ATR (IC₅₀, 120?μM), but MDBK or MDCK cells were unaffected by?1?mM of this diterpenoid. Both freshly isolated fragments and continuous cell lines representing the proximal tubules are more sensitive to ATR than either glomeruli or cells representing the distal nephron. These data also show that protein synthesis is a less specific and sensitive measure of ATR cytotoxicity than enzyme leakage in fragments. MTT reduction to formazan was the most sensitive in the NRK cell line. The low levels of lipid peroxidation products in proximal tubular fragments or sensitive renal cell lines at toxic levels of ATR suggest that oxidative injury is not a key mechanism.     

Protection against cyclophosphamide-induced alopecia by sulfhydryl-containing agents in the newborn rat animal model

Background Alopecia is a common side-effect of cancer chemotherapy. Although this complication has been known for many decades, little progress has been made in its prevention or treatment. Previously, we made the following observations: ( a ) treatment of 8-day-old rats with 1-0-n-arabinofuranosylcytosine (ara-C), doxorubicin, and cyclophosphamide (CYC) produced either total body alopecia (ara-C and CYC) or alopecia confined to the head and proximal part of the neck (doxorubicin); ( b ) Imuvert, a biological response modifier, and interleukin-1 protected against alopecia-induced by ara-C; and (c) neither Imuvert or interleukin-1 protected against CYC-induced alopecia. Objective Experiments were designed to test for agents to protect against CYC-induced alopecia. Methods Agents were tested in the 8-day-old rats as a model for chemotherapy-induced alopecia. Results Mesna and S-2-(3-aminopropylamino)-ethylphospnorothioic acid (WR-2721) did not offer any protection against chemotherapy-induced alopecia. N-Acetylcysterine offered very good protection against alopecia induced by CYC but not that produced by ara-C in the newborn rate animal model. Conclusion N-Acetylcysteine may prove to be important in the prevention of CYC-induced alopecia, but this needs to be tested in the clinical setting.     

Studies on the mechanism of 3-deazaguanine cytotoxicity in L1210-sensitive and -resistant cell lines

Summary 3-Deazaguanine (3-DG), a purine analogue, has unusual antitumor activity against experimental mammary tumor models and a number of other solid tumors. Others have shown that mutant CHO cells deficient in hypoxanthine guanine phosphoribosyl transferase (HGPRTase) or adenine phosphoribosyl transferase (APRTase) are resistant to 3-DG. We developed a L1210 cell line resistant to 3-DG, L1210/3-DG, by subculturing the parent L1210/0 cells in the presence of increasing concentrations of 3-DG. The IC₅₀ was 3.5 M and 620 M for L1210/0 and L1210/3-DG, respectively. Cytotoxicity studies proved the resistance to be stable. Examination of the baseline-specific activity of HGPRTase and APRTase showed that the former was 118-fold lower in L1210/3-DG than in L1210/0, and the latter demonstrated no difference. A 4-h treatment of the cell lines at IC₅₀ doses showed 48% and 23% reductions in IMP dehydrogenase in L1210/0 and L1210/3-DG, respectively. The rate of de novo purine biosynthesis was studied by using [¹⁴C]formic acid. Formate flux increased 2-fold in L1210/3DG in concert with the observed deficiency of HGPRTase in the cell line. 3-DG uptake was studied with [¹⁴C]-labelled compound. The total radioactivity was 9-fold higher in L1210/0 than in L1210/3-DG at 2 h. Subsequent chromatographic separation of radioactivity showed the 3-DG and 3-deazaguanosine pools of the drug to be equal in both lines. However, 3-DG nucleotide pools at 1 min and 2 h were 2.5-fold and 16-fold lower, respectively, in L1210/3-DG than in L1210/0. 3-DG incorporation studies with radiolabelled drug demonstrated that 3-deazaguanine is incorporated in the acid-insoluble fraction of the cell. These studies conclude that HGPRTase, and not APRTase, is required for the activation of drug. Inhibition of IMP dehydrogenase is partially responsible for antitumor activity of the drug. The incorporation of drug into nucleic acids may be a major mechanism for its antitumor activity. Further studies using a cloned cDNA probe for hypoxanthine guanine phosphoribosyltransferase (HGPRT) demonstrated no change in the DNA arrangements of the L1210/3-DG cell line, and Northern blot analysis showed approximately equal expression of mRNA in both cell lines.Abbreviations used APRTase adenine phosphoribosyltransferase - HGPRTase Hypoxanthine guanine phosphoribosyltransferase - IMPD Inosine mono-phosphate dehydrogenase - PRPP 5-Phosphorylribose-1-pyrophosphate - AOPCP , -Methyleneadenosine 5-diphosphate - NAD Nicotineamide dinucleotide - EDTA Ethylenediamine tetra acetic acid Presented at annual meeting of American Association of Cancer Research in May, 1986Supported in part by Warner-Lambert Company, Ann Arbor, Michigan     

"Empty wall" and "vertical strut" signs of ACL insufficiency

We report our observation on the "empty wall" and "vertical strut" signs of anterior cruciate ligament (ACL) insufficiency. ACL tears most commonly occur in the midsubstance; arthroscopic evaluation of patients with these tears often reveals minimal evidence of previous ACL tissue along the intercondylar wall, thus giving the appearance of an "empty wall." In proximal ACL tears, the long remnant of ACL tissue may adhere to adjacent PCL tissue. Arthroscopically, one may see this vertically oriented strut of tissue, which to the casual arthroscopist may mimic a normal-appearing ACL except for orientation and tension. In addition, the "empty wall" sign will be noted because the lateral intercondylar wall becomes easily visible following ACL injury. In two separate prospective studies of 84 such patients, the combined incidence of the empty wall sign was 82%, and the incidence of the vertical strut sign was 50%. These findings should be sought for meticulously at the time of arthroscopic evaluation. The vertical strut should not be misinterpreted as an aberrantly oriented ACL or partial ACL tear.