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The effects of hydroxyethyl starch (HES) on hemostasis were investigated extensively. In order to simulate acute blood loss due to surgery or trauma, one unit (450 ml) of blood was drawn from normal healthy men. This was followed by a 1-liter infusion over 60 minutes of either 6 percent HES, 5 percent albumin, or 0.9 percent sodium chloride (NaCl) as replacement. Coagulation studies were performed before phlebotomy, before infusion and at 0, 4, 20, 27, and 92 hours following infusion. Following infusion of HES and albumin, plasma fibrinogen and antithrombin-III levels fell slightly due to plasma volume expansion and hemodilution. In subjects receiving HES, partial thromboplastin times (PTTs) were significantly (p less than .05) prolonged and factor VIII activities were significantly (p less than .05) decreased when compared to the albumin and NaCl groups. These findings could not be attributed solely to hemodilution. The effects of HES on PTT and factor VIII could not be correlated with plasma HES levels; neither could they be reproduced in vitro by mixing HES with normal plasma. Mean values of the following studies remained normal after infusion of all replacement fluids: prothrombin time, bleeding time, fibrin monomer, fibrin-fibrinogen degradation products, platelet adhesion, circulating platelet aggregates, and platelet count.(ABSTRACT TRUNCATED AT 250 WORDS)

The etiology of abortion, which is still a moot question, is now under intense investigation. The present study discussed the possible role of local fibrinolysis (villous tissue fibrinolysis) in the pathogenesis of abortion by studying tissue fibrinolytic activity in tissue culture as well as blood coagulation, fibrinolysis and the kinin system. Subjects studied include: 1) normal villous tissue (n=46), early pregnancy loss, whose fetal cardiac activity had once been identified (n=22) designated as Group A, and Group B whose fetal cardiac activity had never been identified (n=82). The villous tissues were cultured for 24 hours and then the urokinase (UK) inhibition activity in the medium was determined. 2) Threatened abortion resulted in abortion (poor prognosis, n=42) or the pregnancy continued (good prognosis, n=81). In these patients the following substances in plasma were determined: Prekallikrein, alpha 2-plasmin inhibitor, alpha 1-antitrypsin, alpha 2-macroglobulin, C1-inactivator, antithrombin-III, plasminogen, fibrinogen, fibrin degradation products (FDP). The results are: 1) UK inhibition activity (against UK 2.5 iu), normal villous tissue 26.3 +/- 14.6%, Group A 26.0 +/- 10.3%, Group B 9.6 +/- 10.7%, a significant difference between the normal villous tissue and Group B villous tissue was observed. This suggested that the increased villous tissue fibrinolytic activity due to decreased UK inhibition activity may be one of the causes of abortion. 2) Prekallikrein was significantly reduced in patients with abdominal pain. Also, a significant reduction in plasminogen and elevated C1-inactivator was observed in patients with poor prognosis. The significance of these findings was discussed.

Nephrotic syndrome characterized by hypoalbuminemia and hyperlipidemia is associated with an increased incidence of thromboembolism and increased platelet hyperaggregability. Although plasma coagulation proteins are also abnormal, changes are too inconsistent to attribute thromboembolic complications to the coagulation cascade alone. Antithrombin III (ATIII) has been shown to be deficient in nephrotic syndrome. There is, however, an increase in alpha 2 macroglobulin. It is clear that platelet to platelet interactions require exposure of platelet fibrinogen receptors, the binding of fibrinogen to these receptors, platelet crossbridging, and subsequent platelet aggregation. Fibrinogen is consistently elevated in nephrotic syndrome. Hyperlipidemia and hypoalbuminemia in nephrotic syndrome increases the availability of thromboxane A2 (TxA2) by increasing the availability of TxA2 precursors and the removal of TxA2 inhibitors. Thromboxane A2 is a known inducer of platelet aggregation probably through the exposure of platelet fibrinogen receptors. Recently, fibronectins a group of adhesive proteins, were implicated in platelet to platelet interactions. Since thrombin increases the expression of platelet surface fibronectin, fibronectin may be involved in thrombus formation in nephrotic syndrome. Thromboembolic formation in nephrotic syndrome is a composite mechanism involving the coagulation cascade, platelet-platelet interactions, and platelet-surface interactions.

The nephritogenic effector T cell response producing interstitial nephritis in mice can be largely inhibited by the adoptive transfer of suppressor T cells before or after the induction of disease. These suppressor T cells are harvested from donor mice primed with tubular antigen-derivatized syngeneic lymphocytes, and two subsets of suppressor cells can be characterized within this donor cell population. The first suppressor cell in this network is an L3T4+, I-J+, RE-Id+ cell (Ts-1). Ts-1 cells are antigen-binding suppressor cells that inhibit afferent phase immune responses and, in the presence of tubular antigen, specifically induce Lyt-2+, I-J+ cells (Ts-2) that are antiidiotypic (RE-Id-binding) suppressors. The Ts-2 cell is functionally restricted in its suppressive effect by I-J and Igh-V gene products, and acts on the effector limb of the cell-mediated anti-tubular basement membrane immune response. These studies provide an experimental basis for further efforts to use immunoregulatory modulation in the control of autoimmune renal disease.

Estrogen receptor (ER) in human breast cancer tissues was demonstrated in paraffin sections as well as in frozen sections by immunoperoxidase methods using monoclonal antibody (H222) against ER. The avidin-biotin-peroxidase complex method was used for the paraffin sections fixed in cold buffered formalin, and the peroxidase-antiperoxidase method was used for the fixed frozen sections. The results were compared with the ER content in the respective tumor tissue determined by dextran-coated charcoal assay. The specific staining for ER was located exclusively in the nuclei of cancer cells in both paraffin and frozen sections. Differences in the intensity and distribution of nuclear staining within a section were often observed, suggesting heterogeneity of the ER content of individual breast cancer cells. In 24 breast cancer tissues studied simultaneously by both paraffin and frozen section methods, 21 (88%) showed similar evaluation of the presence of ER. The results of immunocytochemical staining agreed with those of the dextran-coated charcoal assay in 89 (82%) of the 109 paraffin-sectioned tumor tissues and in 24 (86%) of the 28 frozen-sectioned tissues, indicating that ER can be demonstrated immunocytochemically by use of paraffin as well as frozen sections.

Human antithrombin III (ATIII) is a plasma inhibitor of several serine proteases of the blood coagulation system. Previous investigations have reported that the presence of heparin has a multifold accelerating effect on the inhibition of factor XIIa and XIIf, the active species derived from factor XII. Recent studies from our laboratories have confirmed that ATIII inactivates factor XIIa and factor XIIf, but only contributes 2% to 3% to the inhibition of activated factor XII species in plasma. The major inhibitor is C1 inhibitor. Therefore, we have reexamined the heparin effect on the rate of inhibition of factor XIIa and factor XIIf in purified systems. We also have studied the effect of heparin on the inactivation of both factor XII-derived active species by various plasmas. Using purified factor XIIa and ATIII, we found that heparin (0.7 to 34.0 U/mL) increased the rate of inhibition of Factor XIIa. However, at heparin concentrations usually achieved during anticoagulant therapy (0.7 U/mL), the inhibition was accelerated only fourfold. This implies only a 6% contribution to the inhibitory effect of plasma. This suggestion was confirmed by the observation that heparin (1.5 U/mL) added to factor XII-deficient plasma and reconstituted with factor XIIa did not produce a detectable enhancement of the rate of inhibition of factor XIIa. Furthermore, using purified factor XIIf and antithrombin III, heparin (3.6 to 57.2 U/mL) increased the inactivation rate constant of factor XIIf by 1.6 to 14.0 times. This small effect was confirmed by the observation that heparin at a concentration greater than that sufficient for anticoagulation (1.4 U/mL) did not modify the inactivation rate of factor XIIf by prekallikrein-deficient plasma, and thus C1 inhibitor remains the major inhibitor even in the presence of heparin. From this study and our previous investigations on the effect of heparin on the inhibition of kallikrein and factor XIa, we conclude that heparin does not significantly affect the protease activity of purified contact activation factors or the activities expressed by these proteases in plasma.

In the present communication, we describe the unique observation that Lyt-1+2-, L3T4+ T cells but not Lyt-2+ T cells isolated from the spleens of normal, unimmunized H-2d mice proliferate strongly and directly to an irradiated, syngeneic, Lyt-1+2-, L3T4+, Ia- autoreactive T cell line/clone. In contrast, Lyt-1+2- T cells failed to proliferate when stimulated by long-term, antigen-specific, H-2d-restricted T cell lines. Supernatants from the cultures of autoreactive T cells or recombinant interleukin 2 (IL-2) alone, failed to induce proliferation of the Lyt-1+2- T cells, suggesting that cell-cell interaction is essential for growth. In addition, the proliferative response of Lyt-1+2- T cells was independent of Ia+ antigen-presenting cells and was not blocked by either anti-Iad or anti-H-2d antibodies, but was inhibited by anti-L3T4 antibodies. All these observations suggested that the responding Lyt-1+2- T cells were recognizing the anti-self-Ia receptor expressed on autoreactive T cells and that such T cells might therefore express the internal image of self-Ia determinants. We suggest that the T-T interactions observed may represent interactions between two helper T cell subpopulations at the idiotope level and that autoreactive T cells may function as an important feature of the regulatory network and/or lead to the expansion of the T cell repertoire.

A search for uncharacterized genes of the S region of the murine H-2 major histocompatibility complex was undertaken; a series of cosmid clones previously aligned by overlap hybridizations were used as radiolabeled probes. Sequences hybridizing with liver poly(A)+ RNA were found within a cosmid covering a region 3' to the C4-Slp gene (the gene encoding the hemolytically inactive isoform of the fourth component of serum complement). Radiolabeled, short cDNA complementary to liver poly(A)+ RNA was used to establish the transcriptional polarity of the newly detected gene and to define fragments containing its 3' end. DNA sequence analyses and comparisons with porcine peptides established that the gene encodes the enzyme steroid 21-hydroxylase (EC 1.14.99.10), a cytochrome P-450 often referred to as P-450(C21), whose major site of expression is the adrenal gland. Two copies of the P-450(C21) gene, very similar yet distinguishable by restriction endonuclease analysis, were found individually associated with C4 and C4-Slp, genes that encode isoforms of mouse fourth component of complement. One of the P-450(C21) genes is coamplified with C4-Slp in H-2w7, a haplotype carrying a rare elongation of the S region. Comparisons with other members of the P-450 gene family show that the P-450(C21) genes encode peptides of extraordinary evolutionary conservation. The detection of a liver transcript of P-450(C21) raises the issue of the specific metabolic role of this enzyme in this organ and may have implications for the interpretation of human congenital adrenal hyperplasia.

The distribution of ferritin and lysozyme in 19 normal and abnormal duodenal biopsies was studied by an immunoperoxidase technique. The abnormal biopsies included cases of chronic duodenitis with gastric metaplasia, gastric heterotopia, villous atrophy, and a case of hemochromatosis. Ferritin is demonstrated in duodenal absorptive cells, with the staining being most intense in the hemochromatosis case. It was absent in duodenal cells showing gastric metaplasia and in the surface epithelial cells of most biopsies with villous atrophy and gastric heterotopia. Lysozyme-positive mononuclear inflammatory cells were markedly increased in all abnormal biopsies. Not all lysozyme-positive cells were ferritin positive. The latter were especially abundant in areas with gastric metaplasia. It is suggested that this abundance may be related to passive diffusion of intestinal contents, particularly iron, through the metaplastic areas, and consequently there may be a relationship between the presence of duodenal gastric metaplasia and uncontrolled iron absorption.

Epidemiological and experimental investigations have led to the hypothesis that the growth of malignant melanoma is induced by hormones. The demonstration of free-hormone binding sites in melanoma tissue may help to determine the validity of this hypothesis, as receptors are necessary for the transformation of hormonal action. The DCC assay with subsequent saturation analysis was used for the demonstration of specific cytoplasmatic binding sites of steroid hormones. The presence of free-cytosolic estrogen-, progesterone- and glucocorticosteroid receptors was investigated in 50 melanoma samples from 46 patients. In 20 of the 50 specimens, free estrogen and progesterone receptors were found. Free-glucocorticosteroid receptors we found in 10 cases. The use of four fold-labeled estradiol (2,4,6,7-[3]-17 beta-estradiol) in the DCC assay produced a false-positive demonstration of free-estrogen receptors. Tyrosinase hydroxylates estradiol at the C2 level of the steroid. Using fourfold labeled estradiol, tritium is separated at the C2 position, thus forming radioactive water in the cytosol which mimics high, free-estrogen binding sites. The use of twofold-labeled estradiol or L-dopa in the experiments with fourfold labeled estradiol produced no false-positive determinations of estrogen receptors. The demonstration of receptors in malignant melanomas was unaffected by the sex of the patient. Also, the type of malignant melanoma, the invasion level, and the prognostic index did not show a correlation with the presence of the various hormone receptors. In metastases, free steroid hormone receptors were detected less often than in the primary tumor.

Data collected in a study of ATIII deficiency also suggested familial variability of blood clotting times amongst genotypically normal individuals. A study of this variability under a variety of models demonstrates a clear age effect and within sibship similarity. There is also a clear parental effect, which could be either environmental or genetic. Even when parental effects are taken into account there remains some excess similarity between sibs, but the increase in log-likelihood provided by this component is no longer highly significant.

Mammary tumour cytosols prepared for steroid receptor assay showed pH values above or below 7.3-7.5 in 50% of the samples. An increase of 0.6 pH units was observed after the addition of dextran coated charcoal. To test the sensitivity of the assay systems to pH changes, breast cancer cytosols were adjusted to pH between 6.0-9.0 before incubation with ligand at 0 degrees C. ER was then separated by isoelectric focusing in a pH gradient between 9.5-3.5 in a routine assay including a dextran coated charcoal step before focusing. PgR was assayed by electrofocusing as well as by a conventional DCC method and Scatchard plot. Unadjusted control cytosols were run in parallel. At pH above or below 7.0-8.0 recovery of receptor was reduced, the loss being most prominent at low pH. At low pH levels Scatchard plots appeared to be of curvo-linear shape. The effect of low intracellular tumour pH on receptor-ligand affinity in vitro may be of great importance as well in vivo, and should be taken into consideration in the therapeutic situation.