Thrombalexin: Use of a Cytotopic Anticoagulant to Reduce Thrombotic Microangiopathy in a Highly Sensitized Model of Kidney Transplantation.

Early activation of coagulation is an important factor in the initiation of innate immunity, as characterized by thrombotic microangiopathy (TMA). In transplantation, systemic anticoagulation is difficult due to bleeding. A novel "cytotopic" agent, thrombalexin (TLN), combines a cell-membrane-bound (myristoyl tail) anti-thrombin (hirudin-like peptide [HLL]), which can be perfused directly to the donor organ or cells. Thromboelastography was used to measure time to clot formation (r-time) in both rhesus and human blood, comparing TLN versus HLL (without cytotopic tail) versus negative control. Both TLN- and HLL-treated rhesus or human whole blood result in significantly prolonged r-time compared to kaolin controls. Only TLN-treated human endothelial cells and neonatal porcine islets prolonged time to clot formation. Detection of membrane-bound TLN was confirmed by immunohistochemistry and fluorescence activated cell sorter. In vivo, perfusion of a nonhuman primate kidney TLN-supplemented preservation solution in a sensitized model of transplantation demonstrated no evidence of TLN systemically. Histologically, TLN was shown to be present up to 4 days after transplantation. There was no platelet deposition, and TMA severity, as well as microvascular injury scores (glomerulitis + peritubular capillaritis), were less in the TLN-treated animals. Despite promising evidence of localized efficacy, no survival benefit was demonstrated.

Development of a rapid, quantitative method for LDL subfractionation with use of the Quantimetrix Lipoprint LDL System.

BACKGROUND: Recent evidence suggests that the presence of small, dense LDL is independently associated with increased risk of developing coronary artery disease. Current methods to subfractionate LDL are time-consuming and/or technically demanding. Therefore, we have sought the development of a less complex LDL subfractionation procedure. METHODS: LDL subfractions were separated using the Quantimetrix Lipoprint(TM) LDL System. High-resolution 3% polyacrylamide gel tubes were scanned densitometrically (610 nm) with a Helena EDC system. A computerized method to identify and quantitatively score the resolved LDL subfractions was developed. Results from the Quantimetrix method were compared using 51 plasma samples with values obtained by nondenaturing gradient gel electrophoresis (NDGGE) and nuclear magnetic resonance (NMR) spectroscopy. RESULTS: LDL subfractionation scores correlated significantly (P <0.05) with triglyceride, HDL-cholesterol, apolipoprotein B100, and LDL-cholesterol/apolipoprotein B100 (r = 0.591, -0.392, 0.454, and -0.411, respectively). For 51 samples, the Quantimetrix method classified 21 with small, 14 with intermediate, and 16 with large LDL. Of the 21 samples classified as small by Quantimetrix, 20 (95%) were classified as small (n = 18) or intermediate (n = 2) by NDGGE. All of the 16 specimens classified as large by Quantimetrix were either large (n = 14) or intermediate (n = 2) by NDGGE. LDL score was inversely correlated (r = -0.674; P <0.0001) with LDL particle size determined by NMR spectroscopy. CONCLUSIONS: A quantitative method for the assessment of LDL particle size phenotype was developed using the Quantimetrix Lipoprint LDL System. The method can be performed in less than 3 h in batch mode and is suitable for routine use in clinical laboratories.

Human thiopurine methyltransferase pharmacogenetics: gene sequence polymorphisms.

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs. TPMT activity is regulated by a common genetic polymorphism that is associated with large individual variations in thiopurine toxicity and efficacy. We previously cloned the functional gene for human TPMT and reported a common variant allele for low enzyme activity, TPMT*3A, that contains point mutations at cDNA nucleotides 460 and 719. In the present study, we set out to determine the number, types, and frequencies of TPMT variant alleles associated with low enzyme activity in clinical laboratory samples in the United States and to compare those results with data obtained from two different ethnic groups. We identified a total of six different variant alleles for low TPMT activity in the 283 clinical laboratory samples studied. The most common variant was *3A; the second most frequent variant allele, *3C, contained only the nucleotide 719 polymorphism; and four other variant alleles were detected. TPMT*3A also appeared to be the most common variant allele in a Norwegian white population sample, but it was not found in a population sample of Korean children. However, *3C was present in samples from the Korean children, as was novel allele, *6. Characterization of variant alleles for low TPMT enzyme activity will help make it possible to assess the potential clinical utility of deoxyribonucleic acid-based diagnostic tests for determining TPMT genotype.

Detection of autoantibodies to amylase by ELISA: comparison of detection of macroamylase and free autoantibody.

New ELISAs for detecting macroamylase or free autoantibodies to amylase were tested with 48 samples that had been characterized by gel chromatography and electrophoresis. The macroamylase ELISA, with anti-IgG or anti-IgA for detection, detected macroamylase in 28 of 33 samples known to contain macroamylase (85% sensitivity), whereas the ELISA for free autoantibody to amylase was positive for only 11 samples. Specificities of both ELISAs were 93%. Among 28 true positives detected with the macroamylase ELISA, 22 contained IgA, 3 contained IgG, and 3 contained both immunoglobulins. Detection of IgM added no true positives. ELISA responses (y) were proportional to log [macroamylase concentration by chromatography (x)] from 0 to 1200 U/L: y = 5.15 x + 1.66; r = 0.72; Sy x = 1.65. As new tools for detecting macroenzymes consisting of enzyme-autoantibody complexes, the ELISAs show that some autoantibodies are detected more sensitively as antibody-antigen complexes than as free antibody.

Clinical evaluation of high-performance affinity chromatography for the separation of bone and liver alkaline phosphatase isoenzymes.

The newly described high-performance (HPLC) affinity chromatography method for the separation of human bone and liver alkaline phosphatase (ALP, EC 3.1.3.1) isoenzymes was clinically evaluated. The improved resolution of bone from liver isoenzyme and lower detection limit was achieved by conjugation of wheat-germ lectin (WGL) to a diol-bonded silica gel column, stepwise elution with N-acetylglucosamine (NAG) and post column derivatization using para-nitrophenyl phosphate substrate. To establish a reference interval, we measured bone ALP in 86 healthy women, ages 33 to 95 years. The normal reference interval is described by a piecewise linear regression on age (R2 = 0.20, P less than 0.01). For women less than or equal to 45 years, bone ALP, U/l = 8.495. For normal women between ages of 45 to 55 years, bone ALP, U/l = -12.765 + 0.472* age. If age greater than or equal to 55 years, then bone ALP, U/l 13.219. In all 10 patients with primary biliary cirrhosis, serum bone ALP levels were elevated. In addition, sera from 43 patients with diverse metabolic bone diseases were evaluated. As expected, the sera from all 6 patients with Paget's disease and 2 with osteolytic metastasis had bone ALP activity which was greater than 3 standard deviations (SD) from the mean. In all 10 patients with hypoparathyroidism, bone ALP levels were depressed. Only 1 of the 9 patients with glucocorticoid excess and 2 of the 7 patients with primary hyperparathyroidism had elevated bone ALP when compared to the 95% confidence interval for the normal range.(ABSTRACT TRUNCATED AT 250 WORDS)

Liver- and bone-derived isoenzymes of alkaline phosphatase in serum as determined by high-performance affinity chromatography.

To separate liver and bone alkaline phosphatase (ALP) isoenzymes in human serum, we used high-performance affinity chromatography (HPAC) on a column of wheat-germ lectin conjugated to 7-microns-diameter silica particles and an eluent containing N-acetyl-D-glucosamine (NAG). On-line spectrophotometric detection of ALP involved pumping diethanolamine-buffered p-nitrophenyl phosphate solution post-column. Bone and liver isoenzymes could be separated into two peaks with only 10% overlap when an exponential gradient was used. A linear-step gradient separated 80.9% of liver ALP and 91.6% of bone ALP in two distinct peaks. True bone and liver ALP peak areas for the linear-step gradient were determined by using correction factors, because each peak contained a co-eluted portion of the other ALP isoenzyme. The detection limit improved 10-fold over those of other techniques for ALP isoenzymes, owing to the relatively large sample that could be applied to the column. Correlation with a urea-inactivation procedure was reasonable for patients' serum samples (r = 0.98 and 0.79 for liver ALP and bone ALP, respectively).

Alkaline phosphatase isoenzymes of liver and bone origin are incompletely resolved by wheat-germ-lectin affinity chromatography.

We used wheat-germ-lectin affinity chromatography as a tool to investigate the structure of alkaline phosphatase (ALP, EC 3.1.3.1) and to obtain fractions enriched in either bone or liver ALP activity. Liver and bone isoenzymes in serum samples were incompletely resolved except that the activity in the nonretained fraction (fraction 1) always represented pure liver isoenzyme and constituted a larger percentage of total activity in pooled sera with increased liver ALP activity than in pooled sera with increased bone activity. In contrast, a more avidly retained ALP activity, presumably with high glycosylation, was found in human serum with high activity of bone ALP. Using a solid-phase immunoassay, we examined the fractions obtained from the wheat-germ-lectin-Sepharose 4B column to determine whether the isoenzyme preference of the monoclonal antibody was markedly influenced by the degree of glycosylation. Whether samples contained high proportions of liver or of bone isoenzyme activity, the nonretained fraction contained a higher percentage of liver ALP, whereas the more strongly bound fraction contained a higher percentage of bone ALP. Except for eluted fractions that either contained no detectable N-acetylglucosamine or the highest percentage of it, the avidity of the liver-isoenzyme-specific monoclonal antibody for ALP seemed to be independent of the degree of glycosylation, suggesting that the epitope for monoclonal antibody may be expressed in some structure other than the carbohydrate moieties.

Effect of two anticoagulants on leukocyte yield and function, and on lysosomal enzyme activity.

We compared acid citrate-dextrose (ACD-B) and heparin to determine which anticoagulant better preserves leukocytes for lysosomal enzyme assays if processing was done immediately or delayed for 24 h or more. Twenty normal subjects had blood drawn into tubes containing either ACD-B or heparin. The leukocytes were isolated by sedimentation in dextran (50 g/L) less than 2, 24, 48, and 72 h later. The most apparent difference was that cell counts indicated a 30% reduction in the number of leukocytes for ACD-B and a 95% reduction for heparin-treated cells at 48 h. The neutrophil function assay indicated that leukocyte processing must be done in less than 24 h regardless of the anticoagulant used, and that heparin is to be preferred. A comparison of heparin and ACD-B for maintenance of the activity of arylsulfatase A (EC 3.2.6.1) and hexosaminidase (EC 3.2.1.50) indicates that there is no effect of anticoagulant. However, at 48 h after venipuncture, there is an 80% reduction in the number of heparin-treated samples that are suitable for use in the assay. Those laboratories doing lysosomal enzyme tests on mailed specimens, which are most often greater than 24 h old, should use ACD-B as anticoagulant.

Inhibition of DNA synthesis in rat hepatocytes by platelet-derived type beta transforming growth factor.

Platelet-derived type beta transforming growth factor (TGF beta) is a potent inhibitor of DNA synthesis in primary monolayer cultures of adult rat hepatocytes. TGF beta induced a 50% inhibition of epidermal growth factor (EGF)-mediated DNA synthesis at approximately 5 X 10(12) M. This inhibition did not appear to be due to a delay in the peak of DNA synthesis or a toxic action, nor could it be overcome by increasing concentrations of the mitogens EGF, insulin, or glucagon. Inhibition was observed either when TGF beta and EGF were continuously present together in the culture medium or when TGF beta was added to the hepatocyte cultures after removal of the EGF stimulant. This observation together with a lack of an inhibitory effect of TGF beta on the binding of 125I-labeled EGF to hepatocytes in culture, suggests that the inhibitory action of TGF beta was not caused by a direct competition with EGF at the cell surface. TGF beta could not inhibit DNA synthesis once it had begun; however, the inhibitory action of TGF beta could be partially overcome by increasing amounts of conditioned medium produced by normal hepatocytes. Specific saturable receptors for TGF beta were found on the normal rat hepatocytes, but specific binding could not be detected on hepatocytes from regenerating liver. TGF beta is thus a potent inhibitor of EGF-induced DNA synthesis in adult rat hepatocytes. Its significance for growth control in vivo has yet to be assessed.

Specific binding to cultured cells of 125I-labeled type beta transforming growth factor from human platelets.

Purified type beta transforming growth factor from human platelets (TGF beta) radioiodinated with 125I-labeled Bolton and Hunter reagent was found to bind to a variety of cultured cells of both epithelial and mesenchymal origin, including normal human fibroblasts and keratinocytes. TGF beta binding sites have also been found on three mouse embryo-derived fibroblast-like cell lines with lower levels of TGF beta binding on the chemically transformed derivatives of these cell lines. A variety of human tumor cell lines was shown to have an inverse correlation between their level of TGF beta binding and their ability to form colonies in soft agar. The mouse embryo-derived AKR-2B (clone 84A) cells reached maximal binding of 125I-labeled TGF beta after 2 hr at 22 degrees C. Scatchard analysis of the equilibrium binding of TGF beta to AKR-2B (clone 84A) cells gives a Kd of 33 pM with approximately equal to 10,500 binding sites per cell. This Kd for TGF beta binding to AKR-2B (clone 84A) cells agreed well with the ED50 of 40 pM for stimulation of colony formation of these cells by TGF beta. The TGF beta binding sites on the AKR-2B cells were shown to be specific for TGF beta with no significant competition with epidermal growth factor, fibroblast growth factor, or insulin and only a small level of competition with high concentrations of platelet-derived growth factor. Partially purified preparations with TGF beta-like activity from mouse embryos and medium conditioned by mouse embryo-derived cells competed effectively for binding to the TGF beta receptor.

Comparison of intra- and extracellular transforming growth factors from nontransformed and chemically transformed mouse embryo cells.

Multiple transforming growth factors (TGFs) have been isolated from the serum-free conditioned media and acid-ethanol cell extracts of nontransformed AKR-2B and chemically transformed AKR-MCA mouse cells. The TGF activity was analyzed using Bio-Gel P-60 chromatography in 1 M acetic acid and tested for colony-stimulating activity in soft agar using AKR-2B, AKR-2B (clone 84A), NRK, and AKR-MCA cells as indicators. Both intracellular and extracellular TGF activity from AKR-MCA and AKR-2B cells show a major peak of AKR-2B and epidermal growth factor-potentiated NRK colony-stimulating activity that coelute in the 13,000 +/- 2,000 molecular weight region. In the 24,000 +/- 7,000 molecular weight range, AKR-MCA cells produce intracellular and extracellular NRK colony-stimulating activity that is not potentiated by epidermal growth factor, while the intracellular and extracellular NRK colony-stimulating activity produced by AKR-2B cells requires added epidermal growth factor for colony formation. Also important in determining the growth and morphological characteristics of a cell line, besides the production of a TGF, is the ability of a cell to respond to TGF activity. We have shown that the transformed AKR-MCA cells form more colonies than AKR-2B cells in response to certain TGF activities. This suggests that the increased responsiveness of AKR-MCA cells to TGFs may be important in determining its phenotype.

Cell cycle variation in 125I-labeled epidermal growth factor binding in chemically transformed cells.

Previous studies have shown that the nontransformed AKR-2B cells when arrested in the G1 phase of the cell cycle due to low-molecule-weight nutrient (amino acid) deficiency exhibit a 5- to 10-fold lower level of epidermal growth factor (EGF) receptor activity than do the same cells in the rapidly growing state or arrested in G1 due to growth factor deficiency. The chemically transformed AKR-MCA and C3H/MCA-58 cell lines spontaneously arrest growth in G1 due to nutrient deficiency when grown to saturation density in medium with 10% fetal bovine serum. An examination of 125I-labeled EGF binding in rapidly growing and G1-arrested AKR-MCA and C3H/MCA-58 cells showed that the G1-arrested chemically transformed cells also have a 10- to 20-fold reduction in the amount of 125I-labeled EGF binding relative to the same cells in the rapidly growing state. Stimulation of DNA synthesis in the arrested cells by the addition of serum-free medium caused a 6- to 10-fold increase in 125I-labeled EGF binding. This recovery of receptor activity was inhibited by actinomycin D and cycloheximide, suggesting that new messenger RNA synthesis as well as increased protein synthesis is necessary for the recovery of EGF binding. A comparison of EGF binding in C3H/MCA-58 cells and the nontransformed parent line (C3H/10T 1/2) in the rapidly growing state showed the same approximate level of receptor activity. However, the rapidly growing AKR-MCA cells had approximately one-tenth the amount of EGF binding as did the rapidly growing nontransformed parent line (AKR-2B). Scatchard analysis of binding data showed a 10-fold greater number of receptors in the AKR-2B cells relative to the AKR-MCA cells with a lesser difference in apparent receptor affinity. The chemically transformed BP-3T3, like the other two chemically transformed lines, was also demonstrated to arrest growth spontaneously due to nutrient deficiency with an associated 100-fold decrease in EGF binding. Rapidly growing BP-3T3 cells had only slightly less 125I-labeled EGF binding than did the nontransformed parent line (BALB-3T3) in the rapidly growing state. The data indicate that one mechanism for reduction of EGF binding in chemically transformed cells is the propensity of these cells to arrest growth in G1 at saturation density due to low-molecular-weight nutrient deficiency, a state associated with decreased EGF binding.

Photodynamic effect of hematoporphyrin derivative as a function of optical spectrum and incident energy density.

Tumor cell killing effect of hematoporphyrin derivative (HPD) and light was studied in culture to determine the dependence of this effect on treatment variables. Particular attention has been given to the spectral characteristics of the light and the absorption properties of hematoporphyrin. A human tumor cell line was treated using HPD and three broad bands of light ranging from the short- to the long-wavelength end of the visible spectrum. Cell killing was assessed by trypan blue exclusion. A transformed mouse embryo cell line was treated in a similar manner, and its reproductive efficiency was determined following treatment. Results of both studies are consistent with the hypothesis that the photocytotoxic action of HPD plus light is directly proportional to the number of light quanta absorbed by the HPD in each cell. For thin layers of cells, such as in situ carcinoma, it appears that short-wavelength radiation falling in the porphyrin Soret band around 400 nm may have from 12 to 30 times the killing power as does red light.

Transforming growth factor production by chemically transformed cells.

Evidence is presented indicating that the chemically transformed AKR-MCA and C3H/MCA-58 murine cell lines produce "transforming growth factor(s)" capable of inducing a transformed morphology and the ability to grow in soft agar in nontransformed, anchorage-dependent indicator cells. Serum-free medium conditioned by exposure to the chemically transformed cells was chromatographed on a Bio-Gel P-60 column after dialysis and lyophilization. Using the nontransformed mouse AKR-2B cells as the indicator cells, a peak of soft agar growth-stimulating activity was detected in the molecular weight range of 10,000 to 12,000. The soft agar growth-stimulating activity in pooled fractions from the AKR-MCA cells was shown to be trypsin and dithiothreitol sensitive and relatively heat stable; the activity was not destroyed by heating to 56 degrees for 30 min or to 100 degrees for 3 min. The pooled material also caused stimulation of growth in the soft agar of rat NRK cells and stimulation of DNA synthesis in the AKR-2B cells. The quantity required to give significant competition for binding to the epidermal growth factor receptor was about one order of magnitude greater than that required for stimulation of soft agar growth. Further separation of these polypeptide(s) by carboxymethylcellulose chromatography revealed three apparent peaks of soft agar growth-stimulating activity. Epidermal growth factor receptor-competing activity cochromatographed with the early minor soft agar growth-stimulating peak, whereas the two major peaks of soft agar growth-stimulating activity had no associated detectable competition for epidermal growth factor binding to its receptor. The data indicate that at least a major portion of the transforming growth factors produced by the chemically transformed cells is different from those described previously in murine sarcoma virus-transformed mouse cells and human tumor cells.