Vascular endothelial growth factor and basic fibroblast growth factor urine levels as predictors of outcome in hormone-refractory prostate cancer patients: a cancer and leukemia group B study.

Better prognostic markers are needed for hormone-refractory prostate cancer (HRPC) patients. No single biochemical or clinical parameter can reliably predict patient response to therapy or rapidity of disease progression. Peptide factors involved in major cancer growth pathways, such as tumor angiogenesis, are attractive candidates as markers of low- and high-risk HRPC patients. We analyzed prospectively collected urine specimens from 100 of 390 HRPC patients undergoing therapy with the growth factor antagonist suramin as part of CALGB 9480. Levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were assessed from day 1 of therapy (D1) and day 29 (D29) urine samples from this subset of 100 randomly selected patients. Growth factor levels were determined by standardized ELISA microtiter plate assays from a commercial (bFGF) or proprietary (VEGF) source. Pretreatment urine VEGF levels were predictive of survival. In univariate analysis, patients whose baseline urine VEGF level was < or =28 pg/ml (the median level) had an average survival of 17 months; those with baseline VEGF >28 pg/ml had a significantly shorter survival of 10 months (P = 0.024). This difference corresponded to a 60% increased risk of dying for the higher urine VEGF patients (hazard ratio, 1.62; P = 0.03) and remained significant in multivariate analysis (hazard ratio, 1.72, P = 0.02). No significant correlations between urine bFGF level or change in bFGF levels and survival were found. These results support the notion that certain peptide growth factor-mediated, mitogenic pathways are important in HRPC and that their levels can predict outcome.

Comparisons of the intraocular tissue distribution, pharmacokinetics, and safety of 125I-labeled full-length and Fab antibodies in rhesus monkeys following intravitreal administration.

Access of recombinant proteins to the retina following intravitreal administration is poorly understood. A study was conducted in male Rhesus monkeys (15 to 28 mo of age; 2.8-3.3 kg) in order to compare the intraocular tissue distribution, pharmacokinetics, and safety of 125Iodine (I)-labeled full-length humanized rhuMAb HER2 antibody (148 kD) and of 125I-labeled humanized rhuMAb vascular endothelial growth factor Fab antibody (48.3 kD) following bilateral bolus intravitreal injection on day 0 (5 animals/group). The dose administered to each eye was 25 microg (9-10 microCi) in 50 microl. Animals were euthanatized on day 0 (1 hr postdose) and on days 1, 4, 7, and 14. Safety assessment included direct ophthalmoscopy, intraocular pressure measurements, clinical observations, body weight, and hematology and clinical chemistry panels. Blood and vitreous samples were collected daily (blood only) and at necropsy for pharmacokinetics and analysis for antibodies to the test materials; the ocular tissue distribution of the test material was evaluated by microautoradiography. All animals completed the study. Microautoradiography demonstrated that the full-length antibody did not penetrate the inner limiting membrane of the retina at any of the time points examined. In contrast, the Fab antibody fragment diffused through the neural retina to the retinal pigment epithelial layer at the 1-hr time point and persisted in this location for up to 7 days. Systemic exposure to test material was low but variable: the highest plasma concentration of the full-length antibody was 20.3 ng/ml, whereas plasma concentrations for the Fab antibody remained below the limit of quantitation (i.e., <7.8 ng/ml). An immune response to the test material was not evident in either treatment group. The half-life in vitreous was 5.6 days for the full-length antibody and 3.2 days for the Fab antibody. The shorter intravitreal half-life of the Fab antibody is related to its smaller size and its significant diffusion through the retinal layers. The differences in pharmacokinetics and tissue distribution that are noted between the full-length and Fab antibodies in this study identify potential therapeutic approaches that may be exploited in specific disease conditions.

A sensitive fluorometric enzyme-linked immunosorbent assay that measures vascular endothelial growth factor165 in human plasma.

A specific and sensitive fluorometric enzyme-linked immunosorbent assay (ELISA) was developed to measure endogenous levels of vascular endothelial growth factor (VEGF165) in human plasma. The ELISA can be performed in 10% human EDTA plasma, yielding a neat plasma sensitivity of 10 pg/ml or 0.2 pM. The recovery of recombinant human VEGF (rhVEGF) added to human plasma ranges from 89 to 100%. The capture antibody depletes the endogenous signal in normal human plasma, suggesting that the signal is specific for VEGF. The inter-assay and intra-assay coefficients of variation (CV) for the ELISA ranges from 5 to 14% and 8 to 18%, respectively. Characterization of the ELISA using plasmin derived VEGF variants suggests the assay is specific for the VEGF165 isoform. The heterodimer, VEGF(165/110) quantitates similar to that of the intact VEGF165 homodimer, however, the homodimers VEGF121, VEGF110 and the carboxy terminal domain (residues 111-165) are not detected in the assay. Circulating endogenous VEGF levels measured in 50 normal healthy individuals range from 20 to 141 pg/ml, with a mean of 42 +/- 22 pg/ml. There were no significant differences in VEGF levels between males and females. Circulating endogenous VEGF levels in cancer patients ranged from 32 to 418 pg/ml, averaging 129 +/- 17 pg/ml.

Allergen-induced histamine release in rat mast cells transfected with the alpha subunits of Fc epsilon RI.

A rat mast cell histamine assay (RMCHA) has been developed to quantitate the biological activity of a recombinant humanized, monoclonal anti-IgE antibody (rhuMAbE25). Rat mast cells (RBL 48), transfected with the alpha subunit of the high affinity human IgE receptor (Fc epsilon RI), were presensitized for 2 h with human plasma containing IgE specific for ragweed and challenged with ragweed allergen in the presence of 50% D2O. Histamine release plateaus at 0.1 micrograms/ml of ragweed. The release of histamine was time, temperature and Ca2+ dependent. This ragweed-induced histamine release could be inhibited by rhuMAbE25 in a dose-dependent fashion with an IC50 of 1.19 +/- 0.31 micrograms/ml (n = 25). Other humanized MAbs and recombinant human growth factors neither trigger histamine release nor inhibit ragweed-induced histamine release. This RMCHA correlates well with the human basophil histamine assay (HBHA) (Fei et al., 1994) with a correlation coefficient of 0.93 (n = 59, p < 0.0001). Histamine was also released when the cells were presensitized with human plasma containing the respective allergen-specific IgE and then challenged with standardized mite, D. farinae, house dust mix, standardized cat pelt, or Alternaria tenuis. Comparison of allergen-induced histamine release showed a good correlation between RMCHA and HBHA with a correlation coefficient of 0.69 (n = 37, p = 0.0001). We conclude that RMCHA provides a useful tool to confirm allergen-specific IgE in allergic patients and can be used to evaluate the biological activity of any anti-IgE monoclonal antibody. Moreover, RMCHA provides an unique opportunity to study the mechanism of IgE-mediated histamine release in the absence of interfering proteins and growth factors normally present in whole blood.

A novel bioactivity assay for monoclonal antibodies directed against IgE.

A novel bioactivity assay has been developed to quantitate the biological activity of a humanized, monoclonal anti-IgE antibody (rhuMAbE25) in human whole blood. Heparinized blood specimens from prescreened healthy donors were sensitized for 2 h with a constant amount of human plasma containing IgE specific for ragweed and then challenged with ragweed allergen. Histamine was released in a dose-dependent fashion and reached plateau levels after 30 min. As expected, the release of histamine by ragweed allergen was time, temperature and Ca2+ dependent, and could be enhanced by the presence of 33% deuterium oxide. Allergen-triggered release could be inhibited by rhuMAbE25 with an effective dose range from 0.1 to 1 microgram/ml. Preincubation with other humanized MAbs, which exhibit 95% homology to rhuMAbE25 but differ in epitope specificity, failed to inhibit the ragweed-induced histamine release. Overall, this bioactivity assay has a low interassay variability (%CV) of 17% (n = 23) and can be readily modified to determine if rhuMAbE25 or other anti-allergy therapeutics are capable of blocking histamine release elicited by other allergens. Moreover, the assay can be used to confirm IgE-mediated allergic responses and to provide early information regarding safety and potential efficacy of therapeutics aimed at blocking IgE dependent responses.

Difficulties in precise quantitation of CD4+ T lymphocytes for clinical trials: a review.

Maintenance of CD4+ T helper lymphocyte counts has been used as a surrogate marker of efficacy for drugs in the treatment of AIDS. In a multicenter clinical trial, subtle improvement of CD4+ T cell counts may be masked and misinterpreted if care is not paid to likely sources that can contribute to the variability of measurement of CD4+ T lymphocytes. This review addresses major areas that can contribute to the variability of measurement of CD4+ T lymphocytes, with emphasis on applications to multicenter clinical trials, and proposes areas of improvement that may not be well recognized by the medical community. Whereas there are excellent guidelines for immunophenotyping, equal attention is needed in hematologic enumeration of WBC and absolute lymphocytes. In particular, allowing the margin of error acceptable to blood cell standards for HIV-infected specimens is unsatisfactory. Special attention should also be given to the stability of lymphocytes in the anticoagulant during storage, the lysing method, the quality assurance programs as well as intrasubject fluctuations which may be derived from exercise, medications and diurnal variations. Awareness of these contributing factors by physicians and technical analysts will expedite the discovery of potential therapy in the treatment of AIDS. For a multicenter clinical trial, it is advisable to select a centralized laboratory adopting a uniform protocol with regard to sample preparation and handling, using more stringent quality controls for hematologic analysers, calibration of instruments and immunophenotyping. Pending a true reference standard that can monitor the variation of the entire analytical procedure, we anticipate that future interlaboratory quality assurance programs will include absolute T lymphocyte count, an important parameter for assessing the accuracy and consistency of CD4+ T helper cell counts generated from a laboratory.

RGD-containing peptides inhibit adhesion of 293 cells transfected with GpIIb/IIIa to fibrinogen: comparison to inhibition of platelet aggregation.

Cyclic RGD-containing peptides caused a dose-dependent inhibition of binding of human embryonic kidney cells transfected with recombinant GpIIb/IIIa (r293 clone B) to human fibrinogen coated on to non-tissue culture plates. The inhibitory activity, IC50, of a panel of seventeen RGD-containing peptides ranged from 0.12 to 89.2 microM. These IC50 values correlated with those determined by the inhibition of platelet aggregation (r = 0.99). Even though there was a correlation, there were differences between the platelet aggregation and the bioadhesion assay. The binding of r293 clone B to fibrinogen was not increased by ADP suggesting that GpIIb/IIIa expressed on the surface of r293 clone B cells may be in the 'activated' form. Moreover, preincubation of r293 clone B cells with a monoclonal antibody (mAb) specific for GpIIIa (4B12) resulted in a dose-dependent decrease of binding to fibrinogen while a mAb specific for GPIIb (2D2) had no effect. Neither of these mAbs inhibited platelet aggregation. The binding of r293 clone B cells to fibrinogen required Ca2+ or Mg2+. This cell-based bioadhesion method can provide a tool for screening potential GpIIb/IIIa antagonists and investigating the interaction of GpIIb/IIIa and fibrinogen not possible with platelet aggregation.

Recombinant porcine prorelaxin produced in Chinese hamster ovary cells is biologically active.

Although prorelaxin has a similar structure as proinsulin, the posttranslational processing of prorelaxin seems to be quite different from that of proinsulin. There are no pairs of basic residues flanking the relaxin moiety in most prorelaxins studied so far. Instead, the prorelaxins of many species contains a tetrabasic sequence (Arg-Lys-Lys-Arg) between the connecting peptide and the A-chain. This is the recognition sequence of furin. In order to study this possible processing by furin, we express the recombinant porcine prorelaxin in Chinese hamster ovary cells. The expected 19 kDa recombinant porcine prorelaxin was found to be constitutively secreted into the medium at a level of approximately 250 ng/ml. No conversion of the 19 kDa prorelaxin into the 6 kDa relaxin was observed. Unlike most prohormones which are biologically inactive, the recombinant prorelaxin was found to be biologically active in an in vitro bioassay.

Purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli.

In this report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, pOTS, and the recombinant plasmid was transformed into the E. coli host (AR120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg137 residues of the recombinant prorelaxin, producing the des-ArgA1-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin.

Use of recombinant DNA derived human relaxin to probe the structure of the native protein.

This report describes the physical, chemical, and biological characterization of recombinant human relaxin (rhRlx) used as a probe to establish the disulfide pairing in native human relaxin. This strategy is necessary since native human relaxin is only available in the nanogram range. The relaxin molecule is composed of two nonidentical peptide chains, an A-chain 24 amino acids in length and a B-chain of 29 amino acids, linked by two disulfide bridges with an additional disulfide linkage in the A-chain. Native relaxin isolated from human corpora lutea was compared to rhRlx by reversed-phase chromatography, partial sequence analysis, mass spectroscopy, and bioassay. The potency of rhRlx was established by its ability to stimulate cAMP from primary human uterine endometrial cells. Native relaxin isolated from human corpora lutea was equipotent to chemically synthesized relaxin, which in turn was equipotent to rhRlx. A tryptic map was developed for rhRlx to confirm the complete amino acid sequence and assignment of the disulfide bonds. The three disulfide bonds (CysA10-CysA15, CysA11-CysB11, and CysA24-CysB23) were assigned by mass spectrometric analysis of the tryptic peptides and by comparison to chemically synthesized peptides disulfide linked in the two most probable configurations. In addition, the observed amino acid composition and sequence of rhRlx was in agreement with that predicted from the cDNA sequence with the exception that the A-chain amino terminal was pyroglutamic acid. The migration of rhRlx upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis was consistent with a monomeric structure, and the identity of the band was demonstrated by immunoblotting.

Cyclic AMP response to recombinant human relaxin by cultured human endometrial cells--a specific and high throughput in vitro bioassay.

A specific and high throughput 96-well format bioassay for recombinant human relaxin (rhRLX) has been developed using human endometrial cells (NHE cells). rhRLX caused a time- and dose-dependent stimulation of cyclic AMP (cAMP) with 1/2 maximal activity of 3.56 +/- 0.65 ng/ml (n = 30). The range of the standard curve was 0.39 to 25 ng/ml with interplate precision of 17 and 22% CV for high and low controls respectively. The cAMP response requires forskolin and 3-isobutyl-1-methylxanthine, and is enhanced by prostaglandin E2 and F2 alpha. The NHE cells do not respond to A or B chains of rhRLX, or a whole array of hormones. Preincubation of rhRLX with specific monoclonal antibody completely abolished the cAMP response. This bioassay has been used to determine the biological activity of several manufactured lots of recombinant human relaxin.

Augmented plasma renin levels in dehydrated sheep with periventricular lesions.

Ablation of tissue in the midline anterior wall of the third ventricle (AV3V) of sheep did not consistently alter baseline plasma renin concentration (PRC) in water replete animals, but caused a greatly augmented increase in PRC in response to water deprivation. PRC might increase in these sheep in order to maintain blood pressure, however it is possible that a central inhibitory influence on renal renin release, operative during dehydration, is disrupted by AV3V-lesions.

Active and inactive renin in critically ill patients.

The relationship between active (A) and inactive (I) plasma renin concentrations (PRC) was examined in critically ill patients to test for intravascular renin activation in states of shock and tissue damage. Critically ill patients had significantly elevated APRC and lowered IPRC:APRC ratio compared with age and sex matched healthy subjects. IPRC in the critically ill was similar to the control group. During blood donation normal volunteers showed a twofold increase in APRC. The rise in APRC was proportionately greater than for IPRC, with a subsequent fall in IPRC:APRC ratio. In both critically ill patients and blood donors elevated APRC was associated with decreased IPRC:APRC ratio, consistent with either consumption of the inactive renin zymogen or preferential secretion of the active form. Individual critically ill patients displayed markedly depressed ratios but with only moderately elevated APRC, a pattern suggestive of intravascular renin activation. Consistent evidence for intravascular or extravascular activation of renin was not apparent.

Prostacyclin infusion does not prevent ACTH-induced hypertension in sheep.

The present experiments examine the hemodynamic effects of an intravenous infusion of prostacyclin on the development of ACTH-induced hypertension in conscious sheep. Prostacyclin was infused at either 0.01 microgram/kg min-1 for 9 days or 0.25 microgram/kg min-1 for 4 days. At 0.01 microgram/kg min-1 prostacyclin had no effect on blood pressure in normotensive sheep or on the development of ACTH hypertension. Infusion at 0.25 microgram/kg min-1 increased heart rate, cardiac output and plasma renin concentration and decreased stroke volume and peripheral resistance in normotensive sheep. Despite these effects it did not prevent development of ACTH-induced hypertension. It is unlikely on the basis of these results that glucocorticoid-induced inhibition of vasodepressor prostacyclin and resulting increase in pressor responsiveness to circulating agonists is the primary cause of ACTH induced hypertension in sheep.

Onset and dose relationships of ACTH effects on blood pressure in sheep.

The threshold and dose-response relationships for the blood pressure and metabolic effects of adrenocorticotropic hormone (corticotropin, ACTH) were examined in conscious sheep. Corticotropin was infused at five rates (0.5 micrograms/kg/day, n = 4; 1 micrograms/kg/day, n = 4;2 micrograms/kg/day, n = 6; 5 micrograms/kg/day, n = 5; and 10 micrograms/kg/day, n = 5) for 3 days, and the time of onset of the rise in blood pressure was assessed with a computer-based system. The effects of equimolar infusion of beta-endorphin and ACTH at 5 micrograms/kg/hour also were examined. Corticotropin infusion at 0.5 microgram/kg/day had no effect on mean arterial pressure. An ACTH infusion of 1.0 microgram/kg/day significantly increased mean arterial pressure (p less than 0.001), but the rise was less than that at the three higher doses, all of which produced similar effects. Changes in heart rate were significant at the 10 micrograms/kg/day level only (p less than 0.01). Initial urinary sodium retention was present at the three higher but not the two lower rates of infusion. Corticotropin infusion had no effect on urinary potassium excretion at any rate but produced hypokalemia at rates of 1.0 microgram/kg/day and above, which appeared to be dose related. Plasma sodium concentration was increased significantly only at the three higher rates (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin converting enzyme inhibition does not prevent development of ACTH-induced hypertension in sheep.

The role of the renin-angiotensin system in the onset of ACTH-induced hypertension was examined in five conscious sheep. Captopril infusion alone (15 mg/kg per day) for 2 days produced a small fall in blood pressure. After 2 days of captopril ACTH was infused (20 micrograms/kg per day) for 3 days together with captopril. The blood pressure and electrolyte effects of ACTH administration were not modified by captopril pretreatment. These experiments establish that angiotensin II is not important in the onset of ACTH-induced hypertension in sheep.

The hemodynamic effects of prostacyclin infusions in normotensive and ACTH induced hypertensive conscious sheep.

The aim of this study was to determine the effect of ACTH-induced hypertension on the hemodynamic dose-response curves to intravenous infusion of prostacyclin (PGI2) in conscious sheep. PGI2 was infused for 10 minutes at doses of 0.05-0.50 micrograms/kg per min and hemodynamic dose-response curves were performed before, during and after ACTH-induced hypertension. Prior to ACTH administration prostacyclin infusions produced dose dependent decreases in mean arterial pressure (MAP), calculated total peripheral resistance (CTPR) and stroke volume (SV). These changes were accompanied by an increase in cardiac rate (CR) and cardiac output (CO). After five days of ACTH treatment MAP had risen from 72 +/- 1 to 91 +/- 2 mm Hg and infusions of PGI2 produced similar effects on MAP to those seen prior to ACTH. However the effects on CTPR, CO, SV and CR were all potentiated relative to normotensive animals. Three days after ACTH administration had ceased and basal pressure had returned to normotensive levels, the responses of CR, CO and SV to PGI2 infusions were similar to those seen prior to ACTH. However the exaggerated fall in CTPR seen during ACTH treatment was still present and this resulted in a very large decrease in MAP. These studies suggest that in this model of steroid-induced hypertension the resistance vessels are more sensitive to PGI2 and that the blood pressure response to PGI2 is regulated by different mechanisms to those seen prior to ACTH.

Stress, ACTH, salt intake and high blood pressure.

Epidemiological evidence supports the thesis that high salt intake is involved in the aetiology of hypertension. If sodium intake is not causal, it appears other factors do not cause high blood pressure in unacculturated societies with low sodium intake. In this context, it is potentially important that stress causing ACTH release, as well as other neurohumoral effects, causes increased salt appetite and can impair renal sodium excretion.

Synthetic ovine corticotropin-releasing factor stimulates adrenocorticotropin release in the ovine fetus over the last fifth of gestation.

Synthetic ovine corticotropin-releasing factor (0.1-1.0 micrograms) was injected intravenously to 6 chronically cannulated ovine fetuses between 104 and 149 days of gestation (term 142-152 days). Arterial plasma immunoreactive adrenocorticotropin was first elevated by this procedure if the fetuses were between 118 and 130 days of gestation, and an increased response occurred later in gestation. This suggests that a progressive reset of the hypothalamic-pituitary-adrenal feedback relationship occurs over the last 2-3 weeks of gestation in the ovine fetus.

On the importance of CSF Na in the regulation of renal sodium excretion and renin release.

Infusions (20 microliters/min) of isotonic (0.27 M) mannitol dissolved in Na-free artificial cerebrospinal fluid (CSF) were made for 2 h into the lateral cerebral ventricle (IVT) of conscious 68 h dehydrated sheep. The IVT infusion induced a conspicuous drop in renal sodium excretion and marked rise in plasma renin concentration (PRC). The antinatriuretic response to the IVT infusion was not altered by the intravenous administration of ADH or te converting enzyme blocker (SQ 14225, Captopril). Surgical bilateral renal denervation did not change the antinatriuretic response while the increase in PRC was extinguished. Samples of CSF were collected prior to, and 15 min after the end of the infusion. These showed a reduction in CSF [Na], while CSF osmolality remained unchanged. The study supports the view that sodium sensitive receptors close to the cerebral ventricular system participate in the regulation of renal sodium excretion and renin release, it also suggests that renal sodium excretion is affected by an unknown hormonal factor of cerebral origin, while the release of renin seen in response to a reduction in CSF [Na] is mediated by the renal nerves.