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Monoclonal antibodies to the estrogen receptor protein were used to evaluate the specificity of the peroxidase antiperoxidase (PAP) method for localization of estrogen receptors (ER) in human breast carcinoma. Twenty-five cases of breast carcinoma were selected, and they were roughly classified as ER-rich and ER-poor tumors based on ER levels as determined by dextran-coated charcoal (DCC) assay. ER-rich tumors showed strongly positive cytoplasmic and, to a lesser degree, nuclear staining. Both the cytoplasmic and nuclear staining were less intense in ER-poor tumors, and the staining intensity varied from one to another cell population in the same tumor. Completely negative cells were also observed. The results of the PAP and DCC methods correlated fairly well in ER-rich tumors but not in ER-poor tumors. The PAP method provided visual localization of ER combined with excellent tissue morphology. Application of both methods (DCC and PAP) may provide better assessment of the estrogen receptor levels in breast carcinomas.

In the present study, we extended the analysis of the regulation of inflammatory lymphokine production in mice with schistosomiasis mansoni. Splenic lymphocytes of chronically infected mice were briefly pulsed in vitro by soluble egg antigens, washed, and then cultured overnight. The supernatant culture fluid added to cultures of splenic cells of acutely infected or peritoneal lymphocytes of antigen-sensitized mice inhibited the production of migration inhibition factor (MIF). Elaboration of MIF suppressor factor (MIF-SF) required the Lyt-1-,2+,3+ subset of T lymphocytes. MIF-SF acted only on egg antigen-primed cells and required H-2 compatibility with the target cell for its suppressive effect. Further analysis with recombinant strains revealed that the factor interacted with I-AB or I-C subregion-compatible target cells. Experiments using immunoadsorbent columns with bound anti-I subregion alloantisera indicated that MIF-SF contained I-C subregion-encoded determinants. Extrapolation of this in vitro model to in vivo conditions would indicate that the granulomatous response is modulated by I region-derived suppressor factor(s) that regulate lymphokine production by TDH effector cells.

Prostatic steroid-binding protein, whose expression is stimulated by androgens, consists of two subunits, one containing the polypeptides C1 and C3 and the other containing the polypeptides C2 and C3. We have isolated and sequenced cDNA clones specific for C3 mRNA and used them to isolate and characterize genomic clones for two C3 genes. Both genes are 3.2 kilobases with identical exon/intron arrangements, which is similar to the organization of the C1 and C2 genes, suggesting that they may have arisen by duplications of an ancestral gene. Finally, homologous human genes have not been detected.

The kinetics of antibody synthesis was investigated after intraperitoneal, subcutaneous, and footpad infection of various strains of mice with herpes simplex virus. Immunoglobulin M antibodies appeared 5 days after and immunoglobulin G antibodies appeared 10 to 12 days after intraperitoneal infection with herpes simplex virus type 1. The major histocompatibility complex and the background genome of inbred mice were not found to have a systematical influence on antibody synthesis. Female mice, however, consistently produced more antibodies than did male if the infection was done intraperitoneally, but not if it was done subcutaneously or into footpads. Castration considerably increased the amount of antibodies produced by male mice. The difference in antibody formation between females and males could be abolished by injection of silica; moreover, antibody titers were enhanced by this treatment. This has also been found by immunization with a Formalin-inactivated herpes simplex virus vaccine. The effect of silica in enhancing antibody formation could be observed up to 12 days after infection. Infectious virus could be detected up to 2 days after infection, and herpes simplex virus type 1 antibody-stimulating antigens could be detected up to 4 days in ultrasonicates of macrophages. The assumption is made that androgen-sensitive cell populations, including macrophages and their soluble products, are involved in antibody-depressing mechanisms.

The effect of venous occlusion on blood fibrinolytic activity and platelet activation was studied in 10 normal human volunteers. The procedure was used as a model to study the pharmacological effects of 1-(4-isopropylthiophenyl)-2-n-octylaminopropanol (suloctidil, Sulocton) in a double-blind cross-over trial comparing suloctidil 200 mg three times per day versus identical placebo, each given for one week at random, after an initial one-week placebo run-in; blood sampling was done at the end of each period. Statistically significant changes in plasminogen, alpha 2-anti-plasmin, fibrinogen and antithrombin-III were consistently obtained on each occasion and could be ascribed to local haemoconcentration while a much higher increase of plasminogen activator activity occurred. The influence of venous occlusion on plasma beta-thromboglobulin and platelet factor-4 levels was found to be less consistent and under the trial conditions no obvious difference between the regimens studied could be objectified. On the contrary platelet aggregation by collagen was significantly reduced at the end of the suloctidil period.

A clustering outbreak of hand, foot, and mouth disease (HFMD) occurred from July, 1981 to January, 1982 in Matsue City and Gotsu City, Shimane Prefecture. Thirty-seven patients with clinical HFMD were virologically and serologically examined, and Coxsackie virus A10 (CA10) was isolated in 18 patients from vesicles (7/16), throat-swabs (9/31) and feces (6/7). During the period, no CA16 or enterovirus 71 were isolated from HFMD patients or from other diseases such as pharyngitis, febrile diseases, and aseptic meningitis. Serological diagnosis was performed employing an African green monkey kidney cell (AG-1)-adapted CA10 which demonstrated cytopathogenic effects on the cells. Paired sera from seven patients including three cases in which isolation failed showed a significant increase of neutralizing antibody titer against CA10. Finally, an etiological diagnosis was made in 21 out of 37 patients with clinical HFMD. This is the first report of a clustering outbreak of HFMD caused by CA10 in Japan.

New analogues of cAMP, namely 2'-phosphate of cAMP and its esters, were obtained by direct phosphorylation of N6-benzoyladenosine 3',5'-cyclic phosphate with 2-cyanoethyl phosphate in the presence of DCC followed by the removal of protecting groups.

The inhibition of thrombin by antithrombin-III involves formation of a 1:1 covalent complex between protease and inhibitor and concomitant cleavage of the antithrombin-III peptide chain after Arg-385. The resultant fragment remains connected to the complex via a disulfide bond. This complex spontaneously breaks down into a fragment of approximately 55,000 daltons and smaller peptides. Breakdown is prevented by the presence of hydroxylamine or diisopropylflurophosphate, or by denaturation with urea. It occurs even if the purified complex is treated with diisopropylflurophosphate prior to purification, and can be greatly accelerated by the presence of small amounts of active thrombin. The initial sites of proteolytic attack on the complex are after Arg-13 of the thrombin A chain and Arg-68 of the thrombin B chain. These data indicate that active thrombin can be released from the antithrombin-thrombin complex, and that thrombin becomes more susceptible to proteolytic attack when complexed with antithrombin.

Antithrombin III concentrates offer a new therapeutic possibility for immediately increasing the inhibitory potential of plasma. Although heparin activates ATIII, thereby accelerating its consumption, it cannot increase the overall inhibitory capacity towards coagulation. The anticoagulant effect of heparin is in relation with the presence of sufficient amounts of ATIII. The intensive care of patients with liver failure, amniotic fluid embolism, heavy bleedings and septicemia was greatly aided by the substitution of ATIII. Disseminated intravascular coagulation was prevented or interrupted and there are observations suggesting that also shock lung and shock kidney are avoided. The substitution of ATIII allows an extremely extensive therapeutic plasmapheresis without the risk of thromboses.

Since the introduction of synthetic chromogenic and fluorogenic peptide substrates from serine proteases, the testing of coagulation has undergone a dramatic conceptual and methodological change. The concept of coagulation profiling has emerged and automated methodologies are being introduced. Synthetic substrate methods for the evaluation of antithrombin-III; progressive antithrombin; plasminogen; antiplasmin; prekallikrein; antikallikrein ; alpha 1-antitrypsin; prothrombin; heparin; platelet factor IV; urokinase; tissue activator of plasminogen; factor assays; amidolytic equivalents of prothrombin time; and partial thromboplastin time have been developed. Studies on antithrombin-III indicate that immunological methods evaluate the total immunoreactive-antithrombin-III (antigenic) level and do not discriminate between functionally active forms and the AT-III serine protease complex. The clinical significance of AT-III measured by immunological methods is highly questionable. The coagulant assays for the measurement of AT-III require purified alpha-thrombin preparations. The noncoagulant forms of thrombin (beta- and gamma-) result in falsely low antithrombin-III quantitation. The molecular heterogeneity in a given thrombin preparation if standardized in terms of its amidolytic activity does not produce any errors in the quantitation of AT-III levels with synthetic peptide methods. None of the immunological methods provide clinically relevant information except in normal plasma where the immunological and functional activities are identical. Analysis of pathologic plasma samples using laser nephelometry, radial immunodiffusion and radioimmunoassay methods revealed that the functional activity of various serine protease inhibitors is greatly reduced but the reduction in the immunological quantities is minimal. Since coagulation proteins are functional, a ratio between their functional activity and absolute protein levels may be a useful parameter. Employing human and bovine thrombin; bovine and human Xa with their respective substrates, the absolute quantitation of heparin is satisfactorily carried out, however, these assays only measure heparin concentrations and do not reflect the overall anticoagulant effect of heparin. Using the synthetic substrates, the value of measuring absolute concentrations of heparin in a patient on heparin therapy is questionable. With the introduction of fluorogenic substrates, the presence of activated coagulation factors may be demonstrated in patients with thrombotic disorders.(ABSTRACT TRUNCATED AT 400 WORDS)

Hemochromatosis is a syndrome which, when fully expressed, is manifested by melanoderma , diabetes mellitus, and liver cirrhosis, with iron overload involving parenchymal and reticuloendothelial cells in many organ systems. This clinical presentation may arise as a consequence of either hereditary or acquired abnormalities of iron overload, although the mechanisms are quite different. In hereditary hemochromatosis (also known as primary, or idiopathic, hemochromatosis), increased intestinal iron absorption leads to excessive accumulations of iron, throughout the body, particularly in parenchymal cells. In secondary forms of iron overload including transfusional hemosiderosis, alcoholic cirrhosis, thalassemia, sideroblastic anemia, and porphyria cutanea tarda, iron accumulates in the reticuloendothelial system initially, but with increasing amounts of total body iron, excessive iron deposits eventually accumulate in parenchymal cells throughout the body producing a picture indistinguishable from hereditary hemochromatosis. In this article, the course, prognosis, and therapy of iron overload will be reviewed in detail. Clinical and experimental data concerning the pathogenesis of the different forms of iron overload will be examined critically. In particular, information relating to possible abnormalities of reticuloendothelial function, intestinal mucosal iron transport, and alterations in serum and tissue isoferritin patterns in hereditary hemochromatosis will be analyzed, and possible directions for future research will be suggested. The mode of inheritance and linkage with the major histocompatibility (HLA) complex will be discussed. Theories on the pathogenesis of tissue damage by excess iron will be evaluated. Methods for measuring the extent of iron overload in clinical practice will be described, including measurements of serum iron, serum ferritin, iron absorption, cobalt excretion, desferrioxamine excretion, liver biopsy and tissue iron determinations, and HLA typing. Finally, unresolved problems in the understanding of the disease process, diagnosis, and therapy will be delineated.

Ferritin is the intracellular iron storage protein. Tissue ferritin stores are markedly increased in hemochromatosis, a disease of iron overload that has been linked to chromosome 6. In order to provide further information concerning the genetics of ferritin synthesis and to determine if the structural gene for ferritin was on chromosome 6, studies were performed to identify the human chromosome that contains the ferritin gene. Ferritin immunoassays were performed on extracts of Chinese hamster ovary somatic cells that were hybridized with human lymphocytes and fibroblasts and contained various human chromosomes in different combinations. None of the 13 cell lines that lacked immunoreactive human ferritin contained chromosome 19, and all 9 of the cell lines that produced human ferritin contained chromosome 19. No other human chromosome shared this association with human ferritin. In studies of subclones of ferritin-positive cell lines, immunoreactive ferritin consistently segregated only with chromosome 19. Immunoprecipitation studies performed on cells that had been incubated with 59Fe-containing transferrin indicated that chromosome 19-containing cells incorporated iron into intact and functional molecules of human ferritin. The necessary and exclusive association of chromosome 19 with human ferritin indicates that a defect in the structural gene for human ferritin cannot account for the abnormalities of hemochromatosis. Moreover, this hamster-human hybrid cell system should prove useful in further studies of regulation of ferritin concentration and composition.

Estrogen (ER) - and progestagen (PgR) - receptors were estimated by a DCC method using Scatchard plot analysis In tissue samples of 68 patients with ovarian carcinomas. The mean ER and PgR levels were at 52 and 74 fmol/mg cytosol protein, respectively. ER+ PgR+ records were noted in 32.4%, ER+ PgR- in 26.4%, ER- PgR+ in 7.4%, and 33.8% presented with ER- PgR-. In the surviving group 44.1% had Er+ PgR+, whereas the patients who died had an incidence of only 20.5% ER+ PgR+. In addition, the incidences of ER+ PgR+ in stage I plus II and stage III plus IV patients were 42.6 and 29.5%, respectively. Furthermore, higher incidences of ER+ PgR+ were recorded in patients older than 60 years of age (63%) than in younger subjects (36%). The present data combine to suggest that receptor assays should become a useful tool in the management of patients bearing ovarian carcinomas. In addition, ER and PgR determinations provide a prognostic index and may improve the possibility to predict which well-differentiated stage I ovarian carcinomas are likely to recur.

We have used ammonium sulphate precipitation followed by affinity chromatography to partially purify the estrogen receptor from Xenopus laevis liver which may control the genes for vitellogenin, the precursor of the egg yolk proteins. The rate at which receptor binds estradiol explains the kinetics of the induction of vitellogenin synthesis by estradiol, and the dissociation constant (0.5 X 10(-9) M) explains the concentration dependence of the response, which has a threshold of 10(-9) M estradiol, when 67% of the receptor is bound to estradiol. The estradiol concentration in male liver, which does not make vitellogenin, is 0.18 X 10(-9) M, sufficient to saturate 26% of the receptor, while in female liver, which makes vitellogenin continuously, the estradiol concentration is 3.5 X 10(-9) M, giving 88% saturation of receptor, suggesting that the proportion of occupied receptor decides whether or not the vitellogenin genes are active. In the physiological concentration range, estradiol modulates the level of receptor, which varies between 100 binding sites per nucleus in males and 440 in females, but artificially high concentrations of estradiol raise the level to approximately 1000 sites per nucleus. This suggests that the small increase in vitellogenin mRNA induced by physiological concentrations of estradiol is due to pre-existing receptor and that the much larger increases induced by very high concentrations depends on newly-synthesized receptor.

Twenty-three probands with idiopathic hemochromatosis were assigned the status of homozygotes: 132 of their relatives were classified as homozygotes, heterozygotes or normal individuals using the HLA haplotypes of the probands as markers of the hemochromatosis allele. Only half of the probands sought help because of symptoms or signs of iron overload. Clinical manifestations of iron loading were present, however, 95% of the probands and 67% of the discovered homozygotes. The commonest symptom was joint pain and stiffness. None of the heterozygotes had any clinical symptoms of excess body iron. High transferrin saturation and serum ferritin levels were prevalent in homozygotes: only 1 of 38 homozygotes had values for both of these measurements that were within normal limits. The level of transferrin saturation was increased in 6% of heterozygotes but only 1% had serum ferritin concns greater than 350 ng ml-1. The mean radioiron absorption levels of 27 homozygotes and 28 heterozygotes were similar to those in 44 controls. Radioiron absorption in relation to the respective serum ferritin concn was above the 95% confidence interval of controls in 65% of the homozygotes and 7% of the heterozygotes. The inverse relation between radioiron absorption and the respective serum ferritin concn observed in controls was absent in homozygotes but remained strong in the heterozygotes. Absence of the inverse relation indicates a deregulation of the iron absorptive mechanism in homozygotes which results in the size of body iron stores having no effect on the level of iron absorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Covalently bound conjugates of human serum albumin and heparin were prepared as compounds which could improve the blood-compatibility of polymer surfaces either by preadsorption or by covalent coupling of the conjugates onto blood contacting surfaces. The conjugates (10-16 weight % of heparin) were obtained by a condensation reaction between albumin and heparin using 1-ethyl-3-(dimethylaminopropyl)-carbodiimide. Unreacted albumin and heparin were removed by diethyl-aminoethyl (DEAE)-cellulose and Cibacron Blue Sepharose chromatography respectively. The activity of the heparin component incorporated in the albumin-heparin conjugates (Ac) was compared with that of the heparin used for the synthesis of the conjugates (Anat) by thrombin time, inhibition of Factor Xa and the activated partial thromboplastin time (APTT) assays. The Ac/Anat ratio for the above assays was as follows: Thrombin time 1.25, Factor Xa inhibition 0.5. and APTT 0.5. Gel filtration chromatography showed broad-molecular weight distributions. The conjugates were fractionated using immobilized antithrombin III (ATIII). High ATIII and low ATIII affinity conjugate fractions showed the same behavior as ATIII fractionated heparin with respect to thrombin times and Factor Xa inhibition.