Correlation between major histocompatibility complex class I molecules and CD8+ T lymphocytes in prostate, and quantification of CD8 and interferon-gamma mRNA in prostate tissue specimens.

OBJECTIVE: To investigate the immunology of host-tumour interaction, critical for the development of immunotherapy against cancers, by assessing the major histocompatibility complex (MHC) class I expression in both benign and malignant prostate disease, and the relationship between their expression and degree of tumour-infiltrating lymphocytes. MATERIALS AND METHODS: Direct serial analysis of gene expression in tumours is an extremely sensitive and powerful tool for monitoring immunological changes in the immunotherapy of solid tumours. Most previous monitoring protocols rely mainly on the analysis of patient's peripheral blood but in the present study the direct molecular analysis of small tissue samples was used, and its accuracy compared with that of conventional immunohistochemical analysis. Twenty-four formalin-fixed, paraffin-embedded prostate samples (11 benign and 13 carcinoma) were used for the immunohistochemical analysis of CD8+ T lymphocytes and MHC class I expression. CD8+ T lymphocytes were counted using an ocular grid and MHC class I measured using digital image-analysis software. Twenty-seven frozen prostate tissue samples (12 benign and 15 carcinoma) were used for direct gene measurements of CD8 and interferon-gamma using a quantitative real-time polymerase chain reaction. RESULTS: There were significantly fewer CD8+ T lymphocytes in prostate carcinoma nests than in benign prostate. There was a significant correlation between the number of CD8+ T lymphocytes and MHC class I expression in the prostate. There was a strong correlation between the immunohistochemical estimates of CD8+ T lymphocytes and CD8 gene by polymerase chain reaction, but no significant difference between benign prostate and prostate carcinoma tissue in gene measurements. CONCLUSION: Down-regulation of MHC class I expression by prostate cancer cells is associated with fewer CD8+ T lymphocytes and hence might be important in cancer growth. In addition, the measurement of gene expression in small tissue samples might be useful for monitoring the efficacy of treatment throughout cancer therapy.

Antithrombotic effects of recombinant human soluble thrombomodulin in a rat model of vascular shunt thrombosis.

Thrombomodulin on endothelial cells is a cofactor for thrombin-catalyzed activation of protein C. We have investigated the anticoagulant function of recombinant human soluble thombomodulin (rsTM) in a rat model of arterio-venous (AV)-shunt thrombosis. A bolus injection of rsTM 30 s before the induction of AV-shunt thrombosis inhibited the thrombus formation in a dose-dependent manner. The dose of anticoagulant that inhibited thrombus formation by 50% was 0.4 mg/kg rsTM alpha, 0.15 mg/kg rsTM beta, and 13 U/kg heparin. Recently, we characterized three monoclonal antibodies (moAbs) against human TM whose epitopes are located in the TM epidermal growth factor-like domain (Nawa et al., 1994). moAb 2A2 inhibited thrombin binding to rsTM, and abolished both TM functions as a cofactor in thrombin-catalyzed activation of protein C and as an anticoagulant by modifying thrombin-induced fibrinogen clotting and platelet aggregation. moAb 1F2 preserved the latter activities as an anticoagulant, but inhibited cofactor activity. moAb 10A3 had no inhibitory effect on either activity. Analysis of the in vivo anticoagulant mechanism of rsTM was facilitated by the availability of these moAbs. After incubation at rsTM/moAb molar ratios of 1:1.25, the effect of the mixtures were examined in the AV-shunt thrombosis model. An injection of 0.8 mg/kg rsTM alpha or 0.4 mg/kg rsTM beta resulted in a significant reduction on thrombus formation, as expected. moAb 10A3 had no effect on rsTM activity. However, co-injection of rsTM with moAb 1F2 resulted in a significant decrease of the inhibitory activity on thrombus formation. moAb 2A2 essentially abolished the inhibitory effect of rsTM.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of calcium ionophore A23187 on tissue factor activity and mRNA in endothelial cells.

Tissue factor (TF) is an integral membrane glycoprotein that serves as a cofactor for blood coagulation factor VIIa. The induction of TF on the surface of endothelial cells is initiated by various kinds of stimuli including lipopolysaccharide (LPS), interleukin-1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF alpha). The mechanisms leading to induction of TF are largely unknown and the present study explores the influence of calcium influx on TF induction in LPS-stimulated human umbilical vein endothelial cells. TF cofactor activity was measured on cell surfaces and in lysates by a two-stage chromogenic assay after the cells were incubated under a variety of conditions. TF activity of cell surfaces increased 3.3-fold above control values after LPS stimulation (100 ng/ml, 4 h). Addition of 20 microM A23187, a calcium ionophore, to the LPS-stimulated cells just before the TF assay, resulted in an additional 8.8-fold enhancement. TF activity of lysed cells increased 10.5-fold above control values after LPS stimulation (100 ng/ml). Incubation with lower concentrations of A23187 and 100 ng/ml-1 micrograms/ml LPS for 4 h resulted in activity twice that of LPS stimulation alone. The TF mRNA signal of LPS plus 1 microM A23187-treated cells was also increased in addition to LPS treated cells.

Monoclonal antibodies against human thrombomodulin whose epitope is located in epidermal growth factor-like domains.

Thrombomodulin (TM) on endothelial cells is a glycoprotein that functions as a cofactor for thrombin-catalyzed activation of protein C. The structural requirement for thrombin binding and cofactor activity were investigated using monoclonal antibodies (moAbs) against TM and site-directed mutagenesis of recombinant human soluble TM (rsTM). Results showed that moAb 2A2 inhibited thrombin binding to rsTM and also abolished its functions as a cofactor in thrombin-catalyzed activation of protein C and as an anticoagulant by modifying thrombin-induced fibrinogen clotting and platelet aggregation, moAb 1F2 did not affect its activity as an anticoagulant, but inhibited its cofactor activity, and moAb 10A3 did not inhibit either activity. Epitope analysis was carried out by site directed mutagenesis of rsTM expressed in CHO cells. Some proteins with mutations within the second disulfide loop of the fourth EGF-like domain showed reduced affinity for moAb 1F2, but retained cofactor activity. These results suggest that the epitope of moAb 1F2 includes the second disulfide loop of the fourth EGF-like domain, which is close to a region required for cofactor activity. Mutant proteins of the third disulfide loop of the fifth EGF-like domain showed loss of interaction with moAb 2A2. Thus the epitope of moAb 2A2 may include the third disulfide loop of the fifth EGF-like domain. Furthermore, replacement of Asn-439 by Gln decreased the cofactor activity and anticoagulant activity, and resulted in low affinity for either moAb 1F2 or 2A2, suggesting that Asn-439, which is located in the second disulfide loop of the sixth EGF-like domain, is critical for determining the functional conformation of the EGF-like domains 4-6.

Up-regulation of tissue factor mRNA in human umbilical vein endothelial cells by calmodulin inhibitor W7.

Tissue factor (TF) is an integral membrane glycoprotein that serves as a cofactor for the blood coagulation factor VIIa. The induction of TF synthesis and activity on the surface of endothelial cell membrane is initiated by lipopolysaccharide (LPS), phorbol 12-myristate 13-O-acetate (PMA), and inflammatory factors such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha). Treatment of cells with 1 micrograms/ml LPS induced an 8.7-fold increase in total TF activity compared with nontreated cells. Co-incubation with 1 micrograms/ml LPS and 30 microM W7, a potent calmodulin inhibitor, resulted an additional 2.0 fold increase in total TF activity. A similar tendency was observed after treatment with either TNF alpha plus W7, or IL-1 beta plus W7. The effect of W7 appeared to be synergistic since incubation with 30 microM W7 alone increased TF activity levels to only 1.5-fold that of control cells. Northern blot analysis showed that W7 and LPS-treated endothelial cells expressed about three times higher levels of TF mRNA compared to LPS-treated cells. Treatment with W7 and LPS resulted in a slow but large calcium influx into endothelial cells. This result suggest that the contribution of W7 may be dependent mainly on calcium influx by unknown mechanisms rather than direct inhibition of calmodulin, because calcium ionophore treatment also showed a synergistic effect on TF mRNA and activity expression.

Characterization of new monoclonal antibodies to human insulin-like growth factor-II and their application in western immunoblot analysis.

Four immunoglobulin G1 class monoclonal antibodies (mAbs; 1D5, 1D9, 2B11, and 2H11) were produced against recombinant human insulin-like growth factor-II (rhIGF-II). Enzyme-linked immunosorbent assay established that these four mAbs specifically recognized rhIGF-II and hIGF-II, but not rhIGF-I. mAbs 1D5, 1D9, and 2H11 did not cross-react with mouse rIGF-II, although there are only six amino acid differences between mouse IGF-II and human IGF-II. The epitope for each mAb was partially defined by enzyme-linked immunosorbent assay using mouse-human chimera IGF-II mutants and other IGF-II mutants that were prepared by site-directed mutagenic procedures. These results indicated that the epitopes of mAbs 1D5, 1D9, and 2H11 are in the C-domain of hIGF-II around Ala32 to Ser36, and that of 2B11 is in the carboxyl-terminal end of the B- and A-domains of hIGF-II. A specific and sensitive RIA was developed using mAb 2H11. In this RIA, IGF-II variant ([RLPG/S29]IGF-II) and rhIGF-II competed equally with [125I]IGF-II for binding to mAb 2H11. Similar results were produced when mAbs 1D5, 1D9, and 2B11 substituted for 2H11. The potential usefulness of mAb 2H11 in an immunoblot procedure to characterize the heterogeneity of IGF-II in the sera and tumor tissues of patients with nonislet cell tumor hypoglycemia was evaluated. A procedure that combined acid-ethanol extraction of serum or tumor tissues and immunoaffinity concentration of the extracted IGF-II with mAb 2H11-immobilized resin was found to be an effective way to prepare the samples. In Western immunoblots, a quantity of rhIGF-II as low as 3 ng could be identified, whereas 200 ng rhIGF-I or rat IGF-II were not recognized. The levels of IGF-II in the sera of 12 patients with nonislet cell tumor hypoglycemia varied from normal to about twice normal. The mol wt (M(r)) of this IGF-II was between 10-17K. There was little of the processed 7.5K M(r) IGF-II in the sera of these patients. Finally, the source of the high M(r) forms of IGF-II was the tumor, because the ratios of high M(r) forms of IGF-II to 7.5K IGF-II changed dramatically from 99:1 and 91:9 to 4:96 and 32:68 in two patients after successful excision of their tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

[A study on hemorheology of pregnant women with obesity].

The hemorheological properties of women with obesity were studied during pregnancy. Blood viscosity at a shear rate of 0.5/sec was measured with a Contraves Low Shear 100 viscometer and that at a shear rate of 115/sec was measured with a Well-Brookfield viscometer. The levels of blood viscosity (both shear rates) of non-pregnant and pregnant women with obesity were significantly higher compared than those of control women. Plasma viscosity was similar in both groups. The filterability of erythrocytes was determined with a St. George's Filtrometer. The initial relative filtration rate, which represented the deformability of each red cell, was similar in both groups. The clogging rate which represented the properties of red clogging capillaries was increased in non-pregnant women with obesity as compared in non-pregnant control women. However, it was similar in pregnant women in both groups. Hematocrits were increased significantly in women with obesity both in non-pregnant and pregnant states. There was no significant difference between fibrinogen levels in the two groups. The results of the present study suggested that the high frequency of pre-eclampsia among pregnant women with obesity was due to increased Ht and blood viscosity which were factors predisposing to pre-eclampsia.

High-level expression of recombinant human soluble thrombomodulin in serum-free medium by CHO-K1 cells.

Cultivation of gene-engineered Chinese hamster ovary (CHO-K1) cells that produce recombinant human soluble thrombomodulin (rsTM) was investigated to optimize conditions for high-level expression of the protein in a serum-free medium. For economic protein production, oxygenation of cultures with pure O2 permitted sufficient cell growth for high rsTM production with only 1 g/l of microcarriers and a low foetal bovine serum concentration. A longer growth phase (over 5 days) with serum was important to establish sufficient growth of this cell line on the microcarriers for subsequent serum-free culture, and to support a long-term production phase (about 2 months). In the production phase, a high glucose concentration (6.15 g/l) in the serum-free medium was very effective for prolonging the harvest cycle interval. Under these conditions, up to 100 mg/l rsTM was expressed in the conditioned medium. The rates of glucose consumption (G) and lactate production (L) were measured periodically and their ratio (L/G ratio) correlated with rsTM productivity. When the average L/G ratio was lower, reflecting a lower lactate production rate due to appropriate oxygenation of the culture, the specific rsTM production rate increased. Thus it may be possible to estimate protein productivity from L/G ratios calculated from the glucose and lactate measurements.

The cDNA cloning and mRNA expression of rat protein C.

We cloned a cDNA coding for rat protein C, which provides hybridization probes for the detection of protein C mRNA in several tissues. The cloned cDNA was 1543 bp long and contained a single open reading frame of 1383 nucleotides. The proposed rat protein C precursor contained 461 amino acid residues: a 41 amino acid preproleader sequence, and light (155 amino acids) and heavy (263 amino acids) chains joined by a Lys-Arg dipeptide. Northern blot analysis showed that the rat protein C mRNA was expressed not only in the liver, but also in the kidney.

The glycosaminoglycan of recombinant human soluble thrombomodulin affects antithrombotic activity in a rat model of tissue factor-induced disseminated intravascular coagulation.

Previous studies on recombinant human soluble thrombomodulin (rsTM) from Chinese hamster ovary cells revealed that rsTM was expressed as two proteins that differed functionally in vitro due to the presence (rsTM beta) or absence (rsTM alpha) of chondroitin-4-sulfate. The current study evaluates the in vivo behavior of rsTM in rats and in a rat model of tissue factor-induced disseminated intravascular coagulation (DIC). rsTM beta was more potent than rsTM alpha for prolongation of the activated partial thromboplastin time (APTT) and their in vivo half-lives determined by ELISA were 20 min for rsTM beta and 5.0 h for rsTM alpha. Injection of a tissue factor suspension (5 mg/kg) resulted in DIC as judged by decreased platelet counts and fibrinogen concentrations, prolonged APTT, and increased fibrin and fibrinogen degradation products (FDP) levels. A bolus injection of either rsTM (0.2 mg/kg) 1 min before induction of DIC essentially neutralized effects on platelets, fibrinogen, and FDP levels, and had only a moderate effect on APTT prolongation. The dose of anticoagulant to inhibit the drop in platelet counts by 50% (ED50) was 0.2 mg/kg rsTM alpha, 0.07 mg/kg rsTM beta, and 7 U/kg heparin. The effect of increasing concentrations of rsTM and heparin on bleeding times were compared in experiments involving incision of the rat tail. Doubling of the bleeding times occurred at 5 mg/kg rsTM alpha, 3 mg/kg rsTM beta or 90 U/kg heparin. These values represent a 25-fold increase over the ED50 for rsTM alpha, 43-fold for rsTM beta and 13-fold for heparin.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and refolding of recombinant human IGF II from silkworms infected with recombinant Bombyx mori nuclear polyhedrosis virus.

We have reported that insulin-like growth factor II (IGF II) was produced as a fusion protein in Bombyx mori (silkworm) larval bodies infected with recombinant B. mori nuclear polyhedrosis virus [J. Gen. Virol., 68, 2599-2606 (1987)]. In this study, the purification of IGF II from the infected silkworms is reported. The fusion protein was extracted with 6.0 M guanidine-HCl from the infected larval bodies homogenized in water. The use of organic solvents to remove the impurities, such as lipid derived from the larval bodies, was a very effective method of purification. IGF II was released from the partially purified fusion protein by treatment with CNBr, purified by HPLC, and refolded by air-oxidization. Refolded IGF II had an identical primary structure including disulfide bonds and showed identical thymidine uptake stimulation activity with human IGF II. Furthermore, protein disulfide-isomerase was shown to be able to refold scrambled IGF II rapidly.

The design, expression, and characterization of human insulin-like growth factor II (IGF-II) mutants specific for either the IGF-II/cation-independent mannose 6-phosphate receptor or IGF-I receptor.

Five mutants of recombinant insulin-like growth factor-II (rIGF-II) that bound with high affinity to either the IGF-II/cation-independent mannose 6-phosphate (IGF-II/CIM6-P) or the IGF-I receptor were prepared by site-directed mutagenic procedures, expressed as fusion proteins in the larva of Bombyx mori or Escherichia coli, purified to homogeneity, renatured, and characterized in terms of their receptor binding affinities and specificities as well as their biological activities. Class I mutants in which Phe26, Tyr27, and Val43 were substituted with Ser, Leu, and Leu, respectively, bound to enriched preparations of rat placental IGF-II/CIM6-P receptors with apparent equilibrium dissociation constants (Kd(app)) that were only slightly greater, i.e. 0.10, 0.05, and 0.06 nM, than that of rIGF-II (0.04 nM) or hIGF-II (0.03 nM). In contrast, replacing Phe26 with Ser resulted in 5- and 20-fold decreases in the affinities of this mutant for highly purified human placental IGF-I and insulin receptors, respectively. The affinities of the two other Class I mutants, [Leu27]- and [Leu43]rIGF-IIs, for these two receptors were reduced 80- to 220-fold. The affinities of Class II mutants, i.e. [Thr48,Ser49,Ile50]- and [Arg54,Arg55] rIGF-IIs, for IGF-I receptors were as potent as rIGF-II; however, they bound very poorly or not at all to the IGF-II/CIM6-P receptor. In the binding study of those mutant rIGF-IIs, IGF-II was observed to have an unexpectedly high affinity for pure human placental insulin receptor preparations. For example, the affinities of hIGF-II, rIGF-II, and two Class II rIGF-II mutants for the insulin receptor were only 3-, 9-, and 5-fold less, respectively, than that of porcine insulin. In two biological assay systems, i.e. the stimulation of DNA synthesis in Balb/c 3T3 cells and glycogen synthesis in HepG2 cells, the Kd(app) of the rIGF-II mutants for the IGF-I receptor but not the IGF-II/CIM6-P receptor correlated with their abilities to produce biological responses.

A fluoroquinolone (DR-3355) protects human lymphocyte cell lines from HIV-1-induced cytotoxicity.

HIV-1 infection of human CD4+ lymphocyte cell lines results in cell death. Treatment, but not pretreatment, of infected cells, with a fluoroquinolone antibiotic, DR-3355, protects a significant subfraction of cells from HIV-1-mediated cytolysis. All surviving cells have lost expression of the CD4 antigen, but do (MT-4) or do not (CEM) express viral antigens and produce infective virus. The rescued CEM and MT-4 cells are phenotypically stable and do not require continuous exposure to the drug for survival.

Fluoroquinolones protect the human lymphocyte CEM cell line from HIV-1-mediated cytotoxicity.

Infection of the human lymphocyte CEM cell line with the HIV-1 (human immunodeficiency virus-1, LAV-1 strain) results in cell death. A fluoroquinolone antibiotic, ofloxacin, protected the infected cells from HIV-1-mediated cytolysis. Other fluoroquinolones, e.g. ciprofloxacin, norfloxacin, and enoxacin, also protected the infected cells from HIV-1-mediated cytolysis. The d-isomer of ofloxacin (DR-3354) was about 50-fold less effective than the l-isomer (DR-3355). Almost none of the rescued cells had detectable HIV-antigens and they could be maintained for long periods in vitro without drugs.

Presence and function of chondroitin-4-sulfate on recombinant human soluble thrombomodulin.

We constructed a human soluble thrombomodulin (sTM) expression vector using the RSV promoter. Recombinant sTM (rsTM) was expressed in CHO cells and was recovered from culture medium by ion exchange chromatography. Two active fractions, designated as rsTM alpha (low salt elution) and rsTM beta (high salt elution), were detected and further purified by immunoaffinity chromatography. Purified rsTM beta contained bound chondroitin-4-sulfate as judged by HPLC detection of the chondroitinase ABC and AC I digestion product, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose. The apparent Kd values for thrombin of alpha and beta were 7.4 and 1.4 nM respectively. RsTM beta was more effective at inhibition of thrombin clotting activity and had antithrombin III-dependent anticoagulant activity which was not possessed by rsTM alpha. Both anticoagulant activities were lost after chondroitinase treatment of rsTM beta.

[Hemorheological studies on platelet counts and size in normal pregnancy and pregnancies with preeclampsia and intrauterine growth retardation].

It has been reported that preeclampsia and pregnancy resulting in intrauterine growth retardation (IUGR) are associated with high hematocrits. The relations between hematocrits (Ht) and platelet volumes in normal and abnormal pregnancies were investigated to clarify a hemorheological effect on formation of microthrombus. 1) In normal pregnancy, Ht was decreased from 12-19 weeks gestation and reached its lowest level at 28-31 weeks gestation. The mean platelet volume (MPV) was decreased from 20 to 31 weeks gestation but markedly increased from 38 to 41 weeks gestation. The platelet count (Pl) remained unchanged during pregnancy. 2) In severe type of preeclampsia, at 28-37 weeks gestation Ht and MPV were markedly increased and Pl was markedly decreased at 38-41 weeks gestation as compared with normal pregnancies. 3) Mothers who delivered IUGR had a much higher level of Ht at 28-35 weeks gestation and MPV level from 38 weeks gestation than in normal mothers. As the volume of young platelets is large, increased MPV is suggestive of the occurrence of platelet consumption. From these results, it was suggested that microcirculatory disturbances such as higher blood viscosity due to hemoconcentration and microthrombus formation were related to the onset of preeclampsia or IUGR.

Hyperproduction of polyhedrin-IGF II fusion protein in silkworm larvae infected with recombinant Bombyx mori nuclear polyhedrosis virus.

A gene coding for insulin-like growth factor II (IGF II) was constructed from 16 oligodeoxynucleotides synthesized chemically and cloned into EcoRI-SalI sites of pBR322. In this gene at ATG codon for methionine was introduced for cleavage by CNBr at the beginning of mature IGF II. For expressing foreign genes, a new host-vector system, with Bombyx mori silkworm larvae as the host and B. mori nuclear polyhedrosis virus (BmNPV) as the vector, has been developed. BmNPV genomic DNA codes polyhedrin which is a major protein of inclusion bodies and is mass-produced in infected silkworm larvae. We employed this polyhedrin production system to obtain a large yield of a foreign gene product. The coding region of the carboxy-terminal half of polyhedrin was removed and the remainder was ligated with the IGF II gene in phase to create a fusion protein gene consisting of the coding region of the amino-terminal half of polyhedrin and the IGF II gene. This fusion protein gene was combined in a plasmid with the promoter and 5' and 3' flanking regions of the polyhedrin gene. The resulting plasmid and the wild-type BmNPV genomic DNA were cotransfected into BM-N cells, and a recombinant virus was isolated by the limiting dilution method. The silkworm larvae infected with the recombinant virus produced 3.6 mg of the fusion protein per larva and the infected BM-N cells produced 0.3 mg per ml of culture. IGF II was released from the fusion protein produced by BM-N cells infected with the recombinant virus by CNBr treatment, purified by extraction with guanidine-HCl, column chromatography and HPLC and the correct amino-terminal amino acid sequence confirmed.