Evaluation of the circulating fraction of the HER-2/neu oncogene in patients with cervical cancer.

OBJECTIVE: To evaluate circulating HER-2/neu in cervical cancer patients prior to and following treatment. METHODS: Controls, and patients with either cervical dysplasia or cancer taken from an active gynecologic oncology service were evaluated for the expression of HER-2/neu in serum by ELISA before and following surgery, radiation, chemotherapy or combinations thereof. The resulant data was then evaluated for significance by either ANOVA or non-parametric testing. RESULTS: Mean differences were noted for patients with cervical cancer compared to controls. Patients with a good response to the chemotherapy indicated an increase in the serum oncogene, while those not responding either had no marked change or decreased the level of serum HER-2/neu. CONCLUSIONS: As serum HER-2/neu is a membrane bound portion of the intact molecule, these results suggest that due to the induction of cell death and breakdown, the liberation of this fraction (increased levels in the serum), is a viable indicator of response to treatment in some patients. A more detailed examination of this possibility along with expanded correlation with tissue expression is required.

Improved blood substitute: evaluation of its effects on human endothelial cells.

The authors have previously documented that appropriate chemical and pharmacologic modification of the hemoglobin molecule are required to attenuate certain pathophysiologic reactions of the reticuloendothelium. The current study further investigates the molecular responses of human coronary artery endothelial cells to a high concentration (0.4 mmol) of 1) unmodified bovine hemoglobin; and 2) an improved blood substitute that comprises hemoglobin cross-linked intramolecularly with o-adenosine triphosphate and intermolecularly with o-adenosine, and conjugated with reduced glutathione. In this study, the scavenging effect of hemoglobins toward nitric oxide (NO) was evaluated by the measurement of nitrite (NO2-) and nitrate (NO3-) formation. The pro-oxidant effect of hemoglobin on endothelial cells was examined by the measurement of intracellular reduced glutathione, and by monitoring the formation of lipid hydroperoxides and 8-iso prostaglandin F2alpha, a novel potent vasoconstrictor, which is produced by a noncyclooxygenase mechanism involving free radical catalyzed peroxidation of arachidonic acid. The inflammatory reactions of endothelial cells were evaluated by the expression of the adhesion molecule, intracellular adhesion molecule-1, and the activation of nuclear transcription factor, nuclear factor kappaB. In additional, endothelial cell responses were investigated by analysis of intracellular ionized calcium concentrations. Results indicate that unmodified hemoglobin in a concentration of 0.4 mmol/L can aggravate endothelial cell oxidative and inflammatory responses. This hemoglobin produced a significant (p < 0.01) depletion of reduced glutathione, acceleration of lipid peroxidation, and a greater influx of Ca2+. The formation of 8-iso prostaglandin F2alpha increased compared with the control cells (p < 0.01). Unmodified hemoglobin was found to be a potent scavenger of NO, great activator of nuclear factor kappaB, and a stimulator of intracellular adhesion molecule-1 expression. Contrarily, the improved blood substitute did not appear to induce oxidative stress nor to increase the intracellular Ca2+. The concentration of 8-iso prostaglandin F2alpha was similar to that in the control cells, whereas the formation of NO2-/NO3- was much lower (p < 0.05) than in the unmodified hemoglobin group. The effect of an improved blood substitute can be linked with the anti-inflammatory and cytoprotective properties of adenosine, which is used as a cross-linker and surface modifier, and the type of the chemical modification procedure that lowers hemoglobin pro-oxidant potential.

Modified hemoglobin solution, with desired pharmacological properties, does not activate nuclear transcription factor NF-kappa B in human vascular endothelial cells.

The aim of the present study was to evaluate the role of hemoglobin (Hb) and the contribution of chemically modified Hb solutions on the activation of nuclear transcription factor. NF-kappa B, and propagation of oxidative stress within human vascular endothelial cells. The activation of an oxidative stress-sensitive NF-kappa B can be linked with the propagation of an inflammatory state via rapid induction of genes for several pro-inflammatory mediators. Human coronary artery endothelial cells (HCAEC) were cultured on glass coverslips or cell culture plates to confluence. Then, the cells were incubated for up to 18 hours with endothelial basal medium (EBM) supplemented with 5% FBS and test agents in a concentration of 0.1 and 0.2 mmol: 1) unmodified bovine Hb (UHb): 2) modified Hb solution polymerized with glutaraldehyde (GLUT-Hb), and 3) a novel modified Hb solution (Hb-PP-GSH) prepared according to our patented procedure (U.S. Patent No. 5,439,882). The positive control for the NF-kappa B activation study included a treatment of the cells with: I) endotoxin: IL-1; TNF; and H2O2. Results indicate that Hb's pro-oxidant potential was influenced by the type of chemical modification procedure. The GLUT-Hb autoxidation rate, peroxidase-like activity and reactivity with H2O2/ferryl species formation were higher as compared to UHb, by 15%, 35% and 30%, respectively. However, pro-oxidant potential of Hb-PP-GSH was significantly lower than that of UHb (by 22%, 12% and 28%, respectively). The extent of oxidative stress of the HCAECs was found to be the Hb modification-type and concentration dependent. Although the highest endothelial lipid peroxidation and the largest depletion of intracellular GSH was associated with 0.2 mmol of GLUT-Hb, the Hb-PP-GSH did not produce significant changes when compared to the control cells. The UHb generated a moderate oxidative stress to the endothelium. The immunofluorescent and EMSA results indicate a correlation between the type of Hb chemical modification and the induction of NF-kappa B nuclear translocation. We found that GLUT-Hb rapidly activated NF-kappa B and induced nuclear translocation. Treatment of the cells with an increasing amount of UHb leads to the partial nuclear induction of NF-kappa B. However, Hb-PP-GSH did not activate NF-kappa B directly. In this study, the positive control cells treated with endotoxin, IL-1 or TNF demonstrated full nuclear translocations, whereas H2O2 caused only partial induction. In conclusion, nuclear translocation of NF-kappa B by Hb solutions might be dependent on Hb's pro-oxidant potential and extent of Hb-mediated endothelial oxidative stress. Besides the low oxidative potential of Hb-PP-GSH, the observed lack of NF-kappa B activation by this Hb solution can be also related to the anti-inflammatory properties of adenosine which is used in our novel modification procedure. In this study, only the Hb-PP-GSH, cross-linked intramolecularly with o-adenosine triphosphate and intermolecularly with o-adenosine, and combined with reduced glutathiore, was shown to be non-toxic to the endothelium and promises to be an effective free-Hb based blood substitute.

Expression of adhesion molecules and von Willebrand factor in human coronary artery endothelial cells incubated with differently modified hemoglobin solutions.

Previous studies have established a linkage between free Hb molecules and the production of inflammatory mediators by the reticuloendothelial cells. An important aspect of the endothelial response to the inflammatory stimuli is the expression of adhesion molecules on the luminal surface. Therefore, the present study was designed to investigate the effects of various free-Hb based oxygen carrying solutions on the intracellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1) and also von Willebrand factor (vWF) expression by human endothelium. Human coronary artery endothelial cells (HCAEC) were cultured on glass coverslips until they reached confluence, then incubated for 18 hours with endothelial basal medium (EBM) supplemented with 5% FBS and a 0.1 mmol or 0.2 mmol of the bovine Hb solutions: 1) pure unmodified bovine Hb (UHb); 2) modified bovine Hb solution (Hb-PP-GSH) prepared according to our newly developed procedure (U.S. Patent No. 5,439,882); and 3) modified bovine Hb solution polymerized with glutaraldehyde (GLUT-Hb). The HCAECs were also incubated with EBM (negative control) and EBM containing bacterial endotoxins in a concentration of 50 EU/ml (positive control). After treatment, cells were exposed to primary antibodies; anti-human ICAM-1, anti-human VCAM-1 or anti-human vWF, and consequently to the secondary antibody (fluorescein isothiocyanate-conjugated F(ab)2). Immunofluorescence analysis revealed different expressions of ICAM-1 and VCAM-1 on the surface membranes of variously treated cells. Although negative control cells had an undetectable level of adhesion molecules, the positive control cells, activated by endotoxin, exhibited high immunoreactivity for ICAM-1 and VCAM-1. The Hb's treated cells demonstrated differing degrees of activation. An insignificant expression of ICAM-1 was observed in HCAEC, following treatment with a 0.1 or 0.2 mmol of Hb-PP-GSH and 0.1 mmol of UHb. Cell treated with 0.2 mmol of UHb and both concentrations of GLUT-Hb demonstrated a massive expression of this adhesion molecule. A similar effects was observed during induction of VCAM-1. While a lack of expression was noted with both concentrations of Hb-PP-GSH and 0.1 mmol of UHb, the GLUT-Hb stimulated significant VCAM-1 induction at all tested concentrations. Immunofluorescence analysis confirmed the expression of vWF uniformly in HCAEC from the different experimental groups. The data suggest, vWF expression was unaffected by all but the GLUT-Hb treatment. In conclusion, the Hb stimulatory activity toward ICAM-1 and VCAM-1 inductions were related with the type of Hb chemical modification method. Although modification of Hb with glutaraldehyde potentiates adhesion molecules expression, our novel Hb modification procedure, which comprises intramolecular cross-linking with o-adenosine triphosphate and intermolecular with o-adenosine, and combined with reduced glutathione, apparently prevents these inflammatory events.

The effects of propranolol hydrochloride singularly or in conjunction with ethanol on clotting activity in the rat.

1. This study was designed to see if propranolol hydrochloride alone or in conjunction with ethanol had any marked effect on blood coagulation. 2. Rats were given the compounds for 7 days and clotting activity measured. 3. Propranolol induced changes in coagulation, both alone and in conjunction with ethanol. 4. The data suggest that propranolol plus ethanol induce changes that could be detrimental to hemostasis to a greater degree than propranolol alone.

Biochemical effects in women following one year's exposure to a new triphasic contraceptive--I. chemistry profiles.

1. Thirty-nine nonsmoking women, 14 who had never used oral contraceptives and 25 who had a prior history of contraceptive use were placed on a 1-year regimen of oral triphasic contraception containing a new progestin. 2. Biochemical determinations of 21 different variables were made at baseline, 3 months, 6 months, and 12 months of exposure. 3. Most of the significant changes were in those women with no prior exposure to contraceptives. 4. Thyroxine increased and T3 decreased, as did urinary cortisol. No changes were noted in the CBC, hematocrit, or platelet count. Slight increases in cholesterol and triglycerides resulted, with small nonsignificant increases in LDL also occurring; this increase was also noted for HDL. 5. The experimental contraceptive seems to have a very minimal influence on chemistry profiles, suggesting a favorable biochemical response to the progestin.

Biochemical effects in women following one year's exposure to a new triphasic contraceptive--II. coagulation profiles.

1. Coagulation variables were determined in 14 contraceptive nonusers and 25 prior contraceptive users at 3, 6, and 12 months' exposure to a new oral form of progestin. 2. Overall hemostasis was unaffected, as was the intrinsic pathway. 3. Changes were noted in several vitamin-K dependent factors along with a marked decrease in fibrinogen. 4. Protein C antigen (a coagulation inhibitor), was elevated. 5. This new triphasic contraceptive appears to have a minimal influence on thrombosis while activating antithrombotic protein C. The data suggest a favorable hematologic response to this contraceptive.

Evidence for the direct inhibition of endothelin-1 secretion by hemoglobin in human endothelial cells.

The actual hemoglobin (Hb) contribution to endothelin-1 (ET-1) production in human umbilical vein endothelial cells (EC) was investigated. Cells were incubated with 0.1 mmol or 0.3 mmol of bovine: 1) unmodified (U) ferrous-Hb; 2) U-ferric-Hb; 3) U-ferryl-Hb; 4) polymerized low molecular weight (m.w.) Hb with chemically modified surface (< 400 kDa); and 5) glutaraldehyde polymerized, high m.w. Hb (< 1020 kDa). The incubation medium was tested at 6 and 24 hr for lactate dehydrogenase (index of cellular injury), and for ET-1 release by the cells. Before radioimmunoassay, the ET-1 was extracted from cell culture medium by a two-step purification procedure: 1) ultrafiltration, and 2) column extraction with C18 cartridges. The data suggested that the oxidation status of Hb and its concentration play an important role in causing EC injury. The highest toxicity was observed when EC were incubated with 0.1 mmol of ferryl-Hb, and there was no toxicity with 0.3 mmol of ferric-Hb. These results indicate that the ferric-Hb and low m.w. polymerized Hb at a concentration of 0.1 mmol did not alter ET-1 synthesis and produced a level similar to that of the control. However, it was found that ferryl-Hb and ferrous-Hb in a concentration of 0.1 mmol significantly reduced ET-1 release. All Hbs at a concentration of 0.3 mmol markedly inhibited the production of ET-1. The greatest decrease in ET-1 levels was produced by ferryl-Hb, and the lowest by ferric-Hb and low m.w. polymerized Hb. The Hb's inhibitory effect was more pronounced at 24 hr of incubation. It was also found that although Hb molecules showed a high degree of cross-reactivity with polyclonal anti ET-1 antibodies, the presence of different Hb solutions in the EC culture medium did not change the immunologic properties of ET-1 peptide. In conclusion, Hb inhibitory activity toward ET-1 production might be related to Hb mediated endothelial oxidative injury.

Cytokines and PAF release from human monocytes and macrophages: effect of hemoglobin and contaminants.

Monocytes [M] were isolated from venous blood of healthy volunteers and activated macrophage-leukocytes (Mø-L] were obtained from peritoneal fluid of patients with mild endometriosis. The M were incubated with pyrogen free CELLGRO culture medium [Control], and with 0.2 mM of [A] unmodified bovine hemoglobin (UHb), [B] Hb crosslinked to form polymers with M.W. < 400 kDa (LMWHb), [C] Hb crosslinked to form large polymers (< 1,020 kDa) (HMWHb), and Mø-L additionally with [D] UHb contaminated with endotoxin (Hb+E) (2.5 EU/mL), and [E] UHb contaminated with phospholipids (Hb+PLs). The Mø-L medium of incubation was tested for TNF alpha, IL-1 alpha, IL-6, GM-CSF and PAF after 6 and 24 hours, but M for TNF alpha and GM-CSF at 12, 24 and 36 hours. Mø-L were found more responsive than M colonies. The strongest reaction of Mø-L was to Hb+E, which produced levels of cytokines and PAF higher than Controls (p < 0.001). Hb+PLs induced smaller increases of TNF and IL-6, and a decrease in the levels of IL-1 and GM-CSF. However, the release of PAF was much greater with this Hb than with Hb+E. UHb caused an increase in TNF, as compared to control (p < 0.01). LMWHb generated a similar increase in TNF, but also a decrease in IL-1. Both polymerized Hb forms inhibited expression of GM-CSF. HMWHb induced high levels of TNF, IL-1 and PAF. UHb, LMWHb and HMWHb significantly increase levels of TNF in M cultures after 36 hours of incubation.

Reaction of human endothelial cells to bovine hemoglobin solutions and tumor necrosis factor.

Human umbilical vein endothelial cells (HUVEC) were incubated for 24 hours with 0.1 mM or 0.3 mM of: [A] unmodified (U) Hb-FeIIO2; [B] UHb-FeIII; [C] UHb-FeIV-OH; [D] polymerized low molecular weight Hb (< 400 kDa); [E] polymerized high molecular weight Hb (< 1,020 kDa); [F] polymerized low molecular weight Hb + Endotoxin (2.5 EU/mL); [G] rTNF alpha 100 pg/mL; [H] rTNF alpha 400 pg/mL; [I] rTNF alpha 800 pg/mL. The medium of the incubation was tested for LDH (index of cell injury), and for cytokines GM-CSF and IL-1 alpha released by the cells. The data suggests that oxidation status of the iron in the Hb molecule and concentration of Hb play an important role in causing EC injury. The highest toxicity was observed when EC were incubated with 0.1 mM of UHb-FeIV-OH (ferryl-Hb) and no toxicity with 0.3 mM of Hb-FeIII (ferric-Hb). The direct stimulation of EC by Hb for the production of IL-1 was limited, related only to high molecular weight Hb polymers or to Hb+E, however GM-CSF expression was increased by almost all Hb forms. TNF induced dose-related injury (R2 = 0.986), and dose-related release of IL-1 (R2 = 0.977). A different EC reaction was observed on the release of GM-CSF. Intermediate levels of TNF (400 pg/mL) increased the expression of this cytokine, while high levels (800 pg/mL) blocked its release.

Beta-endorphin levels in women with elevated prolactin and following bromocriptine therapy.

1. Plasma levels of beta-endorphin were not significantly different in women with normal plasma prolactin or women with hyperprolactinemia. 2. A bromocriptine induced decrease in plasma prolactin was not accompanied by a decrease in beta-endorphin. 3. This study suggests that no direct link exists between plasma prolactin levels and endogenous beta-endorphin.

Effects of danazol on coagulant activity in the rat.

Coagulation factor activity was evaluated in female rats treated with danazol. Following 30 days of treatment, slight decreases in factor VII and X activities were noted. After 60 days, a prolongation of the prothrombin time, as well as a decrease in factor VII, was noted. The data suggest that in the rodent, danazol has minimal effects on coagulation activity.

Seminal concentssrations of total and ionized calcium from men with normal and decreased motility.

In this study, sperm motility, velocity, and progression were compared with the total and Ca++ concentrations in the SF from men with normal and decreased motility (less than 60%). No significant difference in SF total calcium content was observed in men with normal and hypomotility. However, a statistically significant decrease in seminal Ca++ was observed in those men with decreased motility, when compared with that of men with normal motility.

Effects of marine fish oil (omega-3 fatty acids) on lipid profiles in women.

1. Cycling women both taking or not taking oral contraceptives and menopausal women on replacement estrogen ingested 3 g daily of marine fish oil for 30 days. 2. Triglycerides decreased in the contraceptive users, cholesterol and LDL increased in the non-contraceptive user; while LDL decreased in the menopausal women. 3. After 14 days removal of the fish oil, lipid profiles generally returned to a pattern generally thought to be harmful. 4. Fish oil appears to alter lipids favorably in women receiving exogenous estrogens compared to natural circulating estrogen.

PIP: Cycling women who used oral contraceptives (OCs) and those who did not and menopausal women taking replacement estrogen ingested 3 g daily of marine fish oil for 30 days. Triglycerides decreased in the contraceptive users, cholesterol, and LDL increased in the noncontraceptive user, while LDL decreased in the menopausal women. After 14 days removal of the fish oil, lipid profiles generally returned to a pattern generally though to be harmful. Fish oil appears to favorably alter lipids in women receiving exogenous estrogens compared to natural circulating estrogen.

The effects of dietary marine fish oils (omega-3 fatty acids) on coagulation profiles in men.

1. This study was undertaken to evaluate the effects of low dose ingestion of omega-3 fatty acids on clotting profiles in healthy men ingesting 3 g of MaxEPA (900 mg omega-3 fatty acids) daily for 30 days. 2. No effect was noted on either platelet aggregation or circulating prostaglandin levels. 3. Significant decreases were noted for total cholesterol and low density lipoprotein. 4. Clotting factor decreases were noted for factors primarily of the intrinsic pathway and several factors which promote fibrinolysis. 5. The data suggests that low level ingestion of marine fish oil has a beneficial effect on lipids and possibly the clotting profiles in healthy men.

Newborn's fibrinolytic mechanism: components and plasmin generation.

Plasminogen activity and antigen, tissue-type plasminogen activator (tPA) activity and antigen, plasminogen activator inhibitor (PAI) activity, and plasmin generation rates were determined in 32 normal newborn plasmas and 25 normal adult plasmas. The newborns showed reduced levels of plasminogen activity and antigen and tPA antigen, and activity, normal levels of PAI activity, and slower plasmin generation rates. The slower generation was shown to be due to the hypoplasminogenemia. The in vitro plasmin generation studies also showed that the newborn needed 11 times the usual concentration of urokinase and 5 times the usual concentration of tPA to achieve the minimal activation rate of the adult.

Toxic factors in the red blood cell membrane.

The toxic effects of hemolysed RBCs have been studied for more than 100 years, but the specific factors involved have not been identified. This study focused on phosphatidylethanolamine (PE) and phosphatidylserine (PS), two aminophospholipids that normally reside on the cytoplasmic side of the red cell membrane. An in vitro experiment with murine peritoneal exudate macrophages showed that PE and PS: a) stimulated the production of H2O2, complement factor C3a, prostacyclin, and thromboxane at a dose of 5 micrograms/ml; b) produced cell injury, evidenced by release of lipid peroxides, LDH, and by morphologic changes on phase-contrast and electron microscopy at a dose of 50 micrograms/ml; and c) caused cell death in 50-66% of cells at a dose of 100 micrograms/ml. An in vivo experiment showed that PE and PS injected intravenously into various groups of rabbits: a) caused only transient hypotension at a dose of 0.05 mg/kg body weight; b) caused significant hypotension, cardiac arrhythmias, bronchospasm, activation of intravascular coagulation, complement, platelets, and leukocytes with release of histamine, serotonin, and thromboxane at a dose of 0.10 mg/kg; and c) caused cardiac arrest and death at a dose of 0.30 mg/kg. In contrast, the phospholipids of the outer cell membrane (phosphatidylcholine and phosphatidylinositol) caused minimal toxicity in vitro and none in vivo.

The effects of short term diazinon exposure on blood clotting activity in the rat.

Female rats were exposed to a drinking solution containing 157 parts per million (ppm) of the organophosphate insecticide diazinon for 14 days. Plasma samples were assayed for prothrombin time (PT), partial thromboplastin time (APTT), hematocrit (HCT), platelet count (PLT) and clotting times for coagulation factors I (Fibrinogen), II, V, VII, VIII, X and XII. Significant change following ingestion of diazinon were noted for the PT, APTT, Fibrinogen, factors II, VII, VIII, X, XII and hematocrit. The data suggest that in the rat, diazinon ingestion has a direct effect on the clotting activity of various blood coagulation parameters.

Elevated testosterone level in response to clomiphene in male Anolis carolinensis.

Anolis carolinensis were used as experimental models to study the short-term effects of the fertility drug, clomiphene citrate, on male squamate reproductive function. Daily injections of 10.0 micrograms of clomiphene induced an increase in plasma testosterone over a 5 day period. No change in testis mass or morphology was elicited over this time.