Degradation of human calcitonin in human plasma

The degradation in vitro of human calcitonin (HCT) in plasma from normal subjects and patients with disorders of calcium metabolism was studied. As measured by radioimmunoassay, the hormone was stable at 37°C in phosphate buffer for 24 hr but progressively disappeared when incubated in normal human plasma. The loss was temperature and pH dependent, being maximal at 37°C between pH 6.0 and 7.0. Under these conditions ¹²⁵I-HCT appeared to be degraded to at least one labeled fragment that behaved on polyacrylamide gels as if it were smaller than 1000 daltons. The rate of formation of this fragment correlated well with the rate of loss of immunoreactive HCT. Compared to plasma from normal individuals, plasma from patients with hypercalcemia due to causes other than hyperparathyroidism degraded HCT significantly more rapidly. The mean loss of HCT in 5 hr in plasma from nine such patients was 54% ± 17% (± 2 SEM), compared to 22% ± 5% in plasma from 22 healthy volunteers (p < 0.001). Those patients whose plasma most rapidly degraded HCT had milkalkali syndrome, metastatic carcinoma of the colon, metastatic oat cell carcinoma of the lung, and metastatic breast carcinoma. Rates of HCT degradation in plasma from eight hypercalcemic patients with hyperparathyroidism and seventeen normocalcemic patients with malignancy were within the normal range. From these findings we conclude that human plasma contains one or more enzymes that degrade HCT and that the hormone is degraded more rapidly in plasma from some patients with hypercalcemia.     

Interaction of human chorionic gonadotropin and human luteinizing hormone with human thyroid membranes

In an attempt to identify a possible pathogenetic role for the hCG molecule in the mechanism of the hyperthyroidism which occurs in choriocarcinoma, we have looked for evidence that the hCG molecule has a thyrotropic action on the human thyroid. The thyrotropic activity of various hCG preparations on the human thyroid was assessed by measuring the stimulation of adenylate cyclase activity in human thyroid plasma membranes purified by sucrose density gradient centrifugation. The highly purified hCG CR119 preparation stimulated human thyroid adenylate cyclase activity. Its activity was more than 654 times greater than could be accounted for by human TSH (hTSH) contamination of the preparation, as determined by RIA. The thyrotropic activity intrinsic to 1.0 IU hCG was equivalent to roughly 0.27 microU hTSH. Significant saturable binding of the 125I-labeled highly purified hCG preparation to human thyroid membranes was demonstrated, and the bound component was characterized. Its apparent molecular size, subunit composition, and testis receptor-binding characteristics were those of the hCG molecule. Examination of a crude urinary hCG preparation in adenylate cyclase and TSH radioligand assays using human thyroid membranes showed no evidence of any molecule other than hCG with a thyrotropic action on the human thyroid. Given that hCG binds to and stimulates adenylate cyclase activity in human thyroid tissue, as the above data indicate, then human LH (hLH) would be expected to do the same, since hLH and hCG have such strong structural and functional similarities. As anticipated, a highly purified hLH preparation exhibited TSH binding inhibition and adenylate cyclase stimulation. Its activity was more than 1030 times greater than could be accounted for by hTSH contamination of the preparation. The thyrotropic activity intrinsic to 1.0 IU hLH was equivalent to roughly 44 microU hTSH. Thus, in addition to other shared properties, the hLH molecule and the hCG molecule share the ability to interact with human thyroid tissue. These results strongly indicate that the hCG molecule has a thyrotropic action on the human thyroid and support the hypothesis that hCG is the thyrotropic factor that mediates the hyperthyroidism which occurs in patients with hCG-secreting neoplasms.     

Degradation of human calcitonin in human plasmas.

The degradation in vitro of human calcitonin (HCT) in plasma from normal subjects and patients with disorders of calcium metabolism was studied. As measured by radioimmunoassay, the hormone was stable at 37°C in phosphate buffer for 24 hr but progressively disappeared when incubated in normal human plasma. The loss was temperature and pH dependent, being maximal at 37°C between pH 6.0 and 7.0. Under these conditions ¹²⁵I-HCT appeared to be degraded to at least one labeled fragment that behaved on polyacrylamide gels as if it were smaller than 1000 daltons. The rate of formation of this fragment correlated well with the rate of loss of immunoreactive HCT. Compared to plasma from normal individuals, plasma from patients with hypercalcemia due to causes other than hyperparathyroidism degraded HCT significantly more rapidly. The mean loss of HCT in 5 hr in plasma from nine such patients was 54% ± 17% (± 2 SEM), compared to 22% ± 5% in plasma from 22 healthy volunteers (p < 0.001). Those patients whose plasma most rapidly degraded HCT had milkalkali syndrome, metastatic carcinoma of the colon, metastatic oat cell carcinoma of the lung, and metastatic breast carcinoma. Rates of HCT degradation in plasma from eight hypercalcemic patients with hyperparathyroidism and seventeen normocalcemic patients with malignancy were within the normal range. From these findings we conclude that human plasma contains one or more enzymes that degrade HCT and that the hormone is degraded more rapidly in plasma from some patients with hypercalcemia.     

Effect of human recombinant Endostatin protein on human angiogenesis

Tumor growth and metastasis are dependent on the development of new blood vessels. Inhibitors of new vessel growth have been widely investigated as anti-tumor agents. Endostatin, a 20 kDa C-terminal fragment of collagen XVIII inhibits endothelial cell proliferation, induces endothelial cell apoptosis, and can both inhibit and reverse tumor growth in mice. However, human recombinant endostatin has had limited testing against human tissue targets. To investigate the effect of human endostatin on a human vessel target over a broad range of concentrations (10^–12–10^–4 M), human placental vein disks were grown for a period of 2 weeks in a 0.3% fibrin clot overlayed with growth medium. Disks from five individual placentas were tested. For each placenta utilized, a control (medium and 20% fetal bovine serum [FBS]) group and a group treated with heparin (300 g/ml) and hydrocortisone 21-phosphate (350 g/ml) (heparin-steroid) at a dose known to inhibit angiogenesis were included. Endostatin was tested at concentrations of 10^–12–10^–4 M in medium containing 20% FBS. The rate of initiation and the angiogenic growth index (on a visually graded semi-quantitative scale of 0–16) were determined for all experimental conditions. Endostatin inhibited angiogenesis in our model only in high concentrations. At 10^–5 M, endostatin did not alter the percent of wells that initiated an angiogenic response, but significantly inhibited subsequent vessel growth. At 10^–4 M, endostatin was able to inhibit both initiation and subsequent new vessel growth. Human endostatin can inhibit the initiation of a human angiogenic response and inhibit the subsequent proliferation of human neovessels when used at high doses in a continuous exposure model.     

Absence of a directly causative role for human herpesvirus 7 in human lymphoma and a review of human herpesvirus 6 in human malignancy

 In search of a (new) viral etiological agent, we screened 64 lymph node samples from Hodgkin's disease (HD) and 43 samples (32 lymph node and 11 skin biopsies) from non-Hodgkin's lymphoma (NHL) for human herpesvirus 7 (HHV-7). Twenty-nine control samples were tested as well, including 17 with benign lymphadenopathy. None of the samples tested positive by Southern blot hybridization using HHV-7-specific probes. We conclude that there is no major HHV-7 load in human lymphoma and that HHV-7 is not likely to be directly involved in its etiology. This is in contrast to a small minority of human lymphoproliferative diseases in which HHV-6 can be found at high copy number, but in which an etiological role is still uncertain. Received: September 8, 1998 / Accepted: November 2, 1998     

Comparison of recombinant human thrombin and plasma-derived human alpha-thrombin

Bleeding can be a serious complication of surgery, and topical thrombin is widely used as an adjunct to hemostasis in diverse surgical settings. The potent hemostatic properties of thrombin derive from its ability to activate platelets directly to aggregate and adhere to damaged vessels and to catalyze the formation simultaneously of a fibrin matrix. Application of exogenous thrombin bypasses the physiological process of generating a thrombin burst by directly initiating the terminal reactions of blood clot formation. Currently, thrombin used to control surgical bleeding is primarily from bovine plasma, with a small percentage from human plasma. Human thrombin isolated from pooled plasma carries the risk of transmitting plasma-borne pathogens or prion diseases. The bovine preparations have been associated with protein and preparative contaminants that pose potential risks of developing cross-reacting antibodies. There is a need for a pure therapeutic preparation of human thrombin. Recombinant human thrombin (rhThrombin) has been efficiently produced from a prethrombin-1 precursor obtained from Chinese hamster ovary cell culture. This rhThrombin is substantially free of process-derived contaminants and has been characterized extensively in terms of composition, primary, secondary, and tertiary structure, enzymatic activity; and in vivo pharmacology. In vivo studies of topically applied rhThrombin have shown it is effective in achieving hemostasis in a rabbit liver excisional wound model. Clinical studies are ongoing to evaluate the safety and efficacy of rhThrombin as an adjunct to hemostasis in patients undergoing surgery.     

Jaws of human

Some of main morphological differences between living humans and apes are seen in the size and overall proportions of mandibles;for example, in mandibular dental arch, angle of mandible, height of coronoid process and condyle, and development of chin. Here I review the comparative anatomy of mandible in the living humans and great apes and introduce briefly the morphological features in fossil hominid mandibles.     

Effects of zymosan-activated human granulocytes on isolated human airways

In asthma a temporal association exists between the late allergic reaction (LAR), the influx of granulocytes into the airway wall, and an increase in bronchial responsiveness. We therefore tested the hypothesis that activated human granulocytes constrict isolated human airways and increase their sensitivity to cholinergic stimuli. Bronchial rings were dissected from 23 lung tissue specimens collected at thoracotomy and studied isotonically in organ baths. Airways were incubated with 1, 2, 5, 10, or 20 x 10(6) granulocytes from normal or atopic donors. Activation of the cells with serum-treated zymosan (STZ, 0.2 mg/ml), which itself did not alter baseline airway caliber, resulted in a bronchoconstriction proportional to the number of zymosan-activated granulocytes (ZAG) present (rs = 0.79, p less than 0.001). This contraction was reduced by about 70% with the leukotriene C4/D4 receptor antagonist FPL 55712 (11.5 microM; p less than 0.001) or with the lipoxygenase inhibitor nordihydroguaiaretic acid (10 microM; p less than 0.001). The scavengers of activated oxygen molecules superoxide dismutase (300 U/ml) and bovine catalase (5,000 U/ml), the cyclooxygenase inhibitor indomethacin (10 microM), or the histamine (H1) receptor antagonist mepyramine (2.8 microM) had no effect. Granulocyte suspensions from atopic donors contained more eosinophils (p less than 0.001), and the magnitude of the contraction to 10 x 10(6) ZAG was related to the proportion of eosinophils (rs = 0.66, p less than 0.01). The sensitivity of the airways to methacholine was unchanged in the presence of 1, 2, or 5 x 10(6) ZAG and decreased with 10 or 20 x 10(6) ZAG (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Establishment of clonal human placental cells synthesizing human choriogonadotropin.

Seven clonal human placental cell lines were established by transformation of human first-trimester placental cells with simian virus 40. These transformed cells synthesized native human choriogonadotropin (chorionic gonadotropin) (hCG) as well as the free alpha and beta subunits of hCG. The amount of native hCG synthesized by these cells was, however, lower than the amount of free beta subunit. (Both hCG and the beta subunit are detected by the radioimmunoassay for beta subunit, but only hCG is detected by the radioreceptor assay.) The alpha and beta subunits produced by these transformed placental cells were heterogeneous in size; the sizes of the predominant alpha and beta species, however, corresponded to those of urinary alpha and beta subunits, respectively. The seven cell lines transformed by simian virus 40 had chromosome numbers from the near diploid to the near tetraploid range. Fluorescent staining demonstrated the Y chromosome in all the transformants. Furthermore, B-type glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase; EC 1.1.1.49) was present in all seven lines. These characteristics ruled out possible HeLa contamination of the transformed lines. Regulation of the synthesis of alpha and beta subunits plus hCG in these transformed human placental cells differed from the regulation in choriocarcinoma cells.     

Purification of human pancreatic kallikrein and organ-specificities of human glandular kallikreins

Human pancreatic kallikrein (H. Panc. K.) was purified from human pancreas by serial liquid chromatographies. The final preparation had a specific activity of 9.2 AU/A280 (AU: amidase unit for H-Pro-Phe-Arg-MCA) and its N-terminal sequence coincided with the reported sequence determined from cloned cDNA analysis. In HPLC (gel filtration), one symmetrical peak corresponding to a molecular weight of 48,000 was obtained. In SDS-PAGE without 2-mercaptoethanol, one band corresponding to a molecular weight of 52,000 was obtained. Protease inhibitor specificities of H. Panc. K. were the same as those of human urinary kallikrein (HUK) and hog pancreatic kallikrein (hog Panc. K.), while anti-HUK rabbit antibody inhibited the activities of H. Panc. K. and HUK, but not that of hog Panc. K. From the analysis of affinity for concanavalin A and erythroagglutinating phytohemagglutinin, the carbohydrate parts of H. Panc. K. are relatively rich in bi-(or multi-) antennary complex type sugar chains with bisecting GlcNAc compared with those of human salivary kallikrein and HUK. These findings will be a help to clarify the physiological and pathophysiological roles of H. Panc. K. in the pancreas and pancreatic diseases, especially in acute pancreatitis.     

Generation of human monoclonal antibodies to human immunodeficiency virus.

Based on the finding that cells producing antibodies to human immunodeficiency virus (HIV) circulate in the peripheral blood of HIV-infected individuals, attempts were made to immortalize such B cells with Epstein-Barr virus. Mononuclear cells from 58 HIV-seropositive subjects at various stages of HIV infection were transformed, and anti-HIV cell lines were derived from 4 subjects, all of whom were in early stages of infection. Seven of these cell lines have been stable with respect to antibody production for up to 15 months. Three lines are producing IgG antibody to the 41-kDa HIV transmembrane glycoprotein gp41 and 4 produce IgG antibodies to the 24-kDa HIV core protein p24, its precursors and a breakdown product. The antibodies are reactive by ELISA, by radioimmunoprecipitation, and by Western blot, demonstrating the feasibility of producing multiple stable cell lines synthesizing human monoclonal antibodies to HIV by immortalization of peripheral blood cells with Epstein-Barr virus.     

Priming of normal human neutrophils by recombinant human growth hormone

Growth hormone (GH) plays an important role in the development, maintenance and function of the immune system. Previous data has demonstrated that GH is also a newly defined macrophage-activating factor. Activation of polymorphonuclear neutrophils (PMN) by GH has not yet been examined. This paper presents studies demonstrating the effects of GH on the migratory behaviour and respiratory burst of PMN. In a modified Boyden chamber chemotaxis assay, GH did not stimulate PMN locomotion when added directly to the cells but potently inhibited formylpeptide-stimulated chemotaxis with effective concentrations in the picomolar range. The migration inhibition observed is known from studies on PMN priming-factors to be due to enhanced adhesiveness of PMN to artificial surfaces such as nitrocellulose, suggesting that GH stimulates PMN adhesiveness. Priming of PMN by GH was confirmed by direct demonstration of a stimulatory effect on reduction of nitroblue tetrazolium. These findings suggest that GH may be involved in the regulation of PMN functions.     

Binding of human thrombin to human factor VIII:RAg

The binding capacity of radiolabelled human alpha-thrombin (alpha-thrombin), inactivated with phenyl-methyl sulphonyl fluoride (PMSF), to factor VIII related antigen (VIII:RAg) multimers isolated by immune precipitation and resolved by sodium dodecyl sulphate (SDS) agarose gel electrophoresis has been examined. 125I-PMSF alpha-thrombin bound predominantly to four low molecular weight multimers (LMW) of VIII:RAg prepared from either normal or haemophilia A plasma. Two of these VIII:RAg multimers present in serum had lost the capacity to bind thrombin. No bands were observed when either plasma or serum from three patients with severe von Willebrand's disease (vWd) was processed in an identical manner. This binding was not dependent on the presence of an intact catalytic site on the thrombin molecule. A similar pattern of binding to VIII:RAg multimers was observed with 125I-labelled human anti-FVIII antibodies as 125I-alpha-thrombin and appears to involve the same multimers.     

Neoplastic human T-cells capable of responding to multiple human alloantigens

Monoclonally-derived neoplastic T-cells from a patient with cutaneous T- cell lymphoma respond to multiple human HLA-D antigens in mixed lymphocyte culture. The implications of this phenomenon relevant to normal T-cell function and to malignancy are discussed.