Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product.

Vascular smooth muscle cell (SMC) proliferation in response to injury is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis after balloon angioplasty. The retinoblastoma gene product (Rb) is present in the unphosphorylated and active form in quiescent primary arterial SMCs, but is rapidly inactivated by phosphorylation in response to growth factor stimulation in vitro. A replication-defective adenovirus encoding a nonphosphorylatable, constitutively active form of Rb was constructed. Infection of cultured primary rat aortic SMCs with this virus inhibited growth factor-stimulated cell proliferation in vitro. Localized arterial infection with the virus at the time of balloon angioplasty significantly reduced SMC proliferation and neointima formation in both the rat carotid and porcine femoral artery models of restenosis. These results demonstrate the role of Rb in regulating vascular SMC proliferation and suggest a gene therapy approach for vascular proliferative disorders associated with arterial injury.

Comparison of cleavage site specificity of gelatinases A and B using collagenous peptides.

The gelatinases (type IV collagenases) are members of the matrix metalloproteinase family that not only have a high degree of structural homology but are known to be nearly identical in their digestion profile against macromolecular substrates. We have shown previously that the preferred cleavage sites in the hydrolysis of type I gelatin, catalyzed by gelatinase A (72 kDa type IV collagenase), are bracketed by hydroxyproline in the P5 and P5' positions. In this report, a kinetic investigation using a series of collagenous dodecylpeptides in which the P5 and P5' hydroxyprolines were systematically varied and used as substrates for recombinant human gelatinase A, we show that replacement with either proline or alanine always resulted in increased Km. In contrast, substitution of the hydroxylated amino acids tyrosine and serine at P5 and P5' reduced the Km significantly, indicating that the hydroxyl moiety of the hydroxyproline is the functional group responsible for favorable enzyme-substrate affinity. This was shown by the kcat/Km ratio, which was doubled by the substitution of serine in that site. Cleavage of the same series of dodecylpeptides by recombinant human gelatinase B (92 kDa type IV collagenase) showed a very different kinetic profile for which no patterns were discernible. In subsequent comparisons of the two enzymes, it was found that gelatinase B cleaved the thiopeptolide substrate AcProLeuGly-S-LeuGly-OC2H5 at double the velocity of gelatinase A. In contrast, gelatinase A digested type I gelatin about 2.5-times faster than gelatinase B. SDS-PAGE analysis of gelatin cleavage products showed different patterns of product peptides for each enzyme. Further comparisons of the proteinases using synthetic peptide substrates with variations in size and in substituents at the P2' site again showed marked kinetic differences. Although these two matrix metalloproteinases seem similar in that they are both gelatinolytic and can degrade a nearly identical battery of macromolecular matrix components including type IV collagen, it is clear from these results that they are very different enzymatically. Since the regulatory portions of gelatinases A and B differ markedly, it has been assumed that the enzymes serve the same function, but respond to different stimuli. The differences in substrate specificity described herein suggest that their proposed physiological roles may require reevaluation.

Protein tyrosine phosphorylation in signalling pathways leading to the activation of gelatinase A: activation of gelatinase A by treatment with the protein tyrosine phosphatase inhibitor sodium orthovanadate.

Fibroblasts in monolayer culture secrete gelatinase A (MMP2; 72 kDa type IV collagenase) only in its proenzyme form. Unlike other secreted matrix metalloproteinases, progelatinase A is refractory to activation by serine proteinases. Disparate agents, including monensin, cytochalasin D, and concanavalin A, have been found to mediate the activation of gelatinase A zymogen secreted by fibroblast monolayers. Our finding that monensin-mediated activation can be reversed by the protein tyrosine kinase inhibitor genistein (Li et al., Experimental Cell Research 232 (1997) 332) prompted us to investigate the effect of the specific inhibitor of protein tyrosine phosphatases, sodium orthovanadate, on progelatinase A activation. Treatment of fibroblast monolayers with orthovanadate also results in the secretion of activated gelatinase A. This activation is dose- and time-dependent, requires protein synthesis, and is associated with cell membranes. Vanadate-mediated activation does not occur in the presence of herbimycin A, a protein tyrosine kinase inhibitor. As with progelatinase activation mediated by monensin, concanavalin A, and cytochalasin D, orthovanadate treatment results in increased synthesis of the membrane proteinase MT1-MMP, that can catalyze the activation of progelatinase A. Protein tyrosine kinase inhibitors are able to prevent the increase of MT1-MMP mRNA, as shown by Northern blot and RT-PCR. In addition, orthovanadate potentiates the effects of monensin and concanavalin A. While treatment with monensin or concanavalin A result only in an increase of the putative activator MT1-MMP, orthovanadate also reduces the production of the specific inhibitor TIMP-2. These experiments implicate protein tyrosine phosphorylation in the signal transduction pathways which lead to the activation of progelatinase A.

Evidence for mammalian collagenases as zinc ion metalloenzymes.

Collagenases (EC 3.4.24.3) from human skin, rat skin and rat uterus were inhibited by the chelating agents EDTA, 1,10-phenanthroline and tetraethylene pentamine in the presence of excess Ca2+, suggesting that a second metal ion participates in the activity of the enzyme. Collagenase inhibition by 1,10-phenanthroline could be both prevented and reversed by a number of transition metal ions, specifically Zn2+, Co2+, Fe2+ and Cu2+. However, Zn2+ is effective in five-fold lower molar concentrations (1-10(-4) M) than the other ions. Furthermore, Zn2+ was the only ion tested able to prevent and reverse the inhibition of collagenase by EDTA in the presence of excess Ca2+. Atomic absorption analysis of purified collagenase for Zn2+ showed that Zn2+ was present in the enzyme preparations, and that the metal co-purifies with collagenase during column chromatography.

Regulation of gelatinase in human skin organ cultures by glucocorticoids.

Hydrocortisone and dexamethasone prevent the appearance of gelatinase in serum-free explant cultures of normal human skin. Hydrocortisone inhibits maximally at 10(-6) M and dexamethasone at 10(-8) M in culture medium. Glucocorticoids at these concentrations do not cause a generalized decrease in protein synthesis; thus the effect on gelatinase shows specificity. The reduction in gelatinase activity caused by dexamethasone can be overcome in the presence of dexamethasone 21-mesylate, a glucocorticoid antagonist that binds irreversibly to the cytoplasmic steroid receptor. These data suggest that the enzymes of collagen degradation, collagenase and gelatinase, may be coregulated.

Regulation of gelatinase in human skin organ cultures by glucocorticoids.

Hydrocortisone and dexamethasone prevent the appearance of gelatinase in serum-free explant cultures of normal human skin. Hydrocortisone inhibits maximally at 10(-6) M and dexamethasone at 10(-8) M in culture medium. Glucocorticoids at these concentrations do not cause a generalized decrease in protein synthesis; thus the effect on gelatinase shows specificity. The reduction in gelatinase activity caused by dexamethasone can be overcome in the presence of dexamethasone 21-mesylate, a glucocorticoid antagonist that binds irreversibly to the cytoplasmic steroid receptor. These data suggest that the enzymes of collagen degradation, collagenase and gelatinase, may be coregulated.

Retinoic acid inhibition of collagenase and gelatinase expression in human skin fibroblast cultures. Evidence for a dual mechanism.

Human skin fibroblast cultures have been employed to study the effects of a variety of vitamin A analogues (retinoids) on the expression of two enzymes involved in collagen degradation in the skin, collagenase and a gelatinolytic protease. In normal and recessive dystrophic epidermolysis bullosa fibroblast cultures, retinoic acid compounds were effective inhibitors of the accumulation of both enzymes in the culture medium with half-maximal inhibitions occurring at 0.25-1 microM for collagenase and at 3-6 microM for the gelatinolytic protease. Various retinoids exhibited differing degrees of inhibitory actions, so that at a 1 microM concentration, relative inhibitions were: 13-cis-retinoic acid greater than all-trans-retinoic acid greater than aromatic retinoid (Ro 10-9359) much greater than retinol. The retinoic acid-mediated decrease in collagenase activity was accompanied by a parallel decrease in immunoreactive collagenase protein, suggesting that the retinoic acids were acting to inhibit synthesis of the enzyme. However, an additional effect of these agents was encountered. Although the retinoids themselves had no direct collagenase inhibitory action, medium derived from cultures maintained in these retinoids showed direct inhibitory capacity which was dependent both on the concentration of retinoic acid and on the length of time in culture. The results suggest that the retinoic acids modulate collagenase in vitro by two mechanisms: by decreasing the synthesis of enzyme protein and by modulating the expression of an inhibitory molecule.

Generalized dominant epidermolysis bullosa simplex: decreased activity of a gelatinolytic protease in cultured fibroblasts as a phenotypic marker.

To characterize biochemical traits associated with various forms of epidermolysis bullosa (EB), we used skin fibroblast cultures to measure a gelatin-specific neutral metalloprotease. Compared to normal cultures, levels of this gelatinase were 7-fold decreased in cell cultures from 3 patients from 3 kindreds with generalized dominant EB simplex of the Koebner type (DEBS-K) (p less than 0.001). The specificity of this trait was shown in several ways. The growth kinetics and total protein synthesis of the cells were unaltered. The activity of lactic dehydrogenase, a cytoplasmic enzyme, was also unaltered, indicating the integrity of the cells was not compromised. The decrease in gelatinase activity is specific for generalized DEBS-K, since cultures from recessive dystrophic EB, recessive junctional EB, dominant dystrophic EB, and a second genetic type of DEBS all fail to show this defect. However, since it has been suggested that DEBS-K and the localized form of DEBS, the Weber-Cockayne type (DEBS-WC), may represent allelic mutations of varying severity, we measured gelatinase activity in cell cultures of 13 patients of this type. The levels displayed a biphasic segregation in which 7 of 13 values were equal to, or greater than, the mean activity of control cells. Cultures from the remaining 6 patients were greater than 1 SD below the mean control value and approximated those seen in the generalized DEBS-K patients. These studies suggest that the decrease in gelatinolytic activity is a marker for DEBS-K and that DEBS-K and DEBS-WC may be closely related genetic disorders in which the defect in gelatinolytic protease represents a pleiotropic effect of the gene for DEBS or is genetically linked to the DEBS gene.

Fluorometric determination of pyruvate and alpha-ketoglutarate in cerebrospinal fluid and plasma of infants and children. A simple test that screen for metabolic disorders.

Levels of pyruvate and alpha-ketoglutarate in the cerebrospinal fluid (CSF) of 26 children, aged 4 months to 5 1/2 years, with febrile seizures and of 19 children, aged 4 months to 14 years, with the diagnosis of epilepsy were not different from values seen in 119 "normal" children 8 days to 14 years of age. The CSF samples from 24 adults, 24 to 81 years of age, suspected of having a herniated disk were also examined. In the pediatric age group, the data showed a highly significant downward trend of CSF and plasma alpha-ketoglutarate values with age; pyruvate values did not change. A correlation of the values of the two keto acids in the blood and CSF of 42 other children without apparent neurologic disease was also made. Findings in a child with thiamine deficiency suggest that CSF alpha-ketoglutarate may be a more sensitive indicator of deficiency than plasma alpha-ketoglutarate or pyruvate. Measurements of these keto acids in plasma and CSF may be diagnostically useful in a variety of metabolic disorders. Findings in 155 children from birth (20 minutes) to 17 years of age without neurologic disease are submitted as a standard of reference.

Histamine contamination of pokeweed mitogen.

The histamine content of commercial preparations of pokeweed mitogen was measured by amino acid analysis technique, automated fluorometry, and bioassay employing the guinea pig ileum. Ten samples from 5 companies were examined and found to contain between 0.026 micrograms and 167.5 micrograms of histamine per ml of solution. The protein content of 9 of these putative 5 mg samples measured by folin assay and by amino acid analysis varied from 0.56 to 4.4 mg. Their amino acid compositions were similar, except for notable variations in 3 of the 16 residues quantitated.

Psychoneuroimmunology.

This paper develops hypotheses regarding the interactions among stress, immunity, and chemical sensitivities and gives an overview of the questions and hypotheses generated by a working group exploring the application of psychoneuroimmunology to chemical sensitivities. Consideration is given to prospective longitudinal studies designed to find cases among at-risk exposed populations. Relevant immune parameters to be measured longitudinally and in challenge studies for patients with MCS are discussed. Immune system changes in response to the chronic stress of having MCS and as primary responses to chemical exposure also are considered.

Symptoms in pediatric asthmatics and air pollution: differences in effects by symptom severity, anti-inflammatory medication use and particulate averaging time.

Experimental research in humans and animals points to the importance of adverse respiratory effects from short-term particle exposures and to the importance of proinflammatory effects of air pollutants, particularly O(subscript)3. However, particle averaging time has not been subjected to direct scientific evaluation, and there is a lack of epidemiological research examining both this issue and whether modification of air pollutant effects occurs with differences in asthma severity and anti-inflammatory medication use. The present study examined the relationship of adverse asthma symptoms (bothersome or interfered with daily activities or sleep) to O(3) and particles (less than or equal to)10 micrometer (PM10) in a Southern California community in the air inversion zone (1200-2100 ft) with high O(3) and low PM (R = 0.3). A panel of 25 asthmatics 9-17 years of age were followed daily, August through October 1995 (n = 1,759 person-days excluding one subject without symptoms). Exposures included stationary outdoor hourly PM10 (highest 24-hr mean, 54 microgram/m(3), versus median of 1-hr maximums, 56 microgram/m(3) and O(3) (mean of 1-hr maximums, 90 ppb, 5 days (greater than or equal to)120 ppb). Longitudinal regression analyses utilized the generalized estimating equations (GEE) model controlling for autocorrelation, day of week, outdoor fungi, and weather. Asthma symptoms were significantly associated with both outdoor O(3) and PM(10) in single pollutant- and co-regressions, with 1-hr and 8-hr maximum PM(10) having larger effects than the 24-hr mean. Subgroup analyses showed effects of current day PM(10) maximums were strongest in 10 more frequently symptomatic (MS) children: the odds ratios (ORs) for adverse symptoms from 90th percentile increases were 2.24 [95% confidence interval (CI), 1.46-3.46] for 1-hr PM10 (47 microgram/m(3); 1.82 (CI, 1.18-2.81) for 8-hr PM10 (36 microgram/m(3); and 1.50 (CI, 0.80-2.80) for 24-hr PM10 (25 microgram/m(3). Subgroup analyses also showed the effect of current day O(subscript)3 was strongest in 14 less frequently symptomatic (LS) children: the ORs were 2.15 (CI, 1.04-4.44) for 1-hr O(3) (58 ppb) and 1.92 (CI, 0.97-3.80) for 8-hr O(3) (46 ppb). Effects of 24-hr PM10 were seen in both groups, particularly with 5-day moving averages (ORs were 1.95 for MS and 4. 03 for LS; p(less than or equal to)0.05). The largest effects were in 7 LS children not on anti-inflammatory medications [5-day, 8-hr PM10, 9.66 (CI, 2.80-33.21); current day, 1-hr O(3), 4.14 (CI, 1.71-11.85)]. Results suggest that examination of short-term particle excursions, medication use, and symptom severity in longitudinal studies of asthma yields sensitive measures of adverse respiratory effects of air pollution.

The effect of outdoor fungal spore concentrations on daily asthma severity.

The relationship between day-to-day changes in asthma severity and combined exposures to community air pollutants and aeroallergens remains to be clearly defined. We examined the effects of outdoor air pollutants, fungi, and pollen on asthma. Twenty-two asthmatics ages 9-46 years were followed for 8 weeks (9 May-3 July 1994) in a semirural Southern California community around the air inversion base elevation (1,200 ft). Daily diary responses included asthma symptom severity (6 levels), morning and evening peak expiratory flow rates (PEFR), and as-needed beta-agonist inhaler use. Exposures included 24-hr outdoor concentrations of fungi, pollen, and particulate matter with a diameter < 10 microns (PM10; maximum = 51 micrograms/m3) and 12-hour day-time personal ozone (O3) measurements (90th percentile = 38 ppb). Random effects longitudinal regression models controlled for autocorrelation and weather. Higher temperatures were strongly protective, probably due to air conditioning use and diminished indoor allergens during hot, dry periods. Controlling for weather, total fungal spore concentrations were associated with all outcomes: per minimum to 90th percentile increase of nearly 4,000 spores/m3, asthma symptom scores increased 0.36 (95% CI, 0.16-0.56), inhaler use increased 0.33 puffs (95% CI, -0.02-0.69), and evening PEFR decreased 12.1 l/min (95% CI, -1.8-22.3). These associations were greatly enhanced by examining certain fungal types (e.g., Alternaria, basidiospores, and hyphal fragments) and stratifying on 16 asthmatics allergic to tested deuteromycete fungi. There were no significant associations to low levels of pollen or O3, but inhaler use was associated with PM10 (0.15 inhaler puffs/10 micrograms/m3; p < 0.02). These findings suggest that exposure to fungal spores can adversely effect the daily respiratory status of some asthmatics.

Neuropsychological profiles in schizophrenia: paranoid versus undifferentiated distinctions.

Research on schizophrenia has searched for subtype-specific cognitive dysfunction to elucidate the neurodevelopmental underpinnings of the disorder. The neuropsychological distinctions between 21 paranoid and 15 undifferentiated schizophrenics were studied. The paranoid group had significantly better Verbal IQ, executive functioning on the Wisconsin Card Sorting Test, and memory for spoken language on the Sentence Repetition Test compared to undifferentiated schizophrenics. While the paranoid subtype showed trends toward better performance on a wide variety of tasks, both subtypes showed impairments on tasks requiring continuous auditory attention and fine motor speed and coordination. Despite the paranoid subjects' more intact verbal skills, both groups showed significant deficits in verbal learning on the Rey Auditory Verbal Learning Test. This suggests that temporal-hippocampal system dysfunction may be a common denominator in both schizophrenic subtypes. These findings were not accounted for by subtype differences in the level of education, depression or severity of illness.

Effect of tidal volume and anesthetic agent on airway responsiveness to histamine.

Dose-response relationships for bronchoconstriction in response to aerosal histamine were assessed before and after vagotomy in 11 dogs anesthetized with barbiturates and in 9 dogs anesthetized with alpha-chloralose-urethan. The dose-response relationships following vagotomy were assessed during spontaneous ventilation and during muscular paralysis and mechanical ventilation with tidal volume (VT) similar to each animal's VT prior to vagotomy. After vagotomy the spontaneous VT of both groups increased but the VT of the alpha-chloralose-urethan group was significantly less than that of the barbiturate group. The histamine responsiveness of the animals anesthetized with barbiturates was significantly greater during mechanical ventilation when VT was reduced to prevagotomy levels compared with during spontaneous ventilation. In contrast, the histamine responsiveness of the alpha-chloralose-urethan group was not significantly changed by reducing VT to prevagotomy levels. In six other dogs anesthetized with pentobarbital sodium and studied after vagotomy, responsiveness to histamine aerosol during controlled ventilation with breaths of prevagotomy VT was greater than responsiveness during mechanical ventilation with large volume breaths given immediately afterward. Thus the magnitude of VT of dogs after vagotomy may influence airway responsiveness, and the influence of anesthetic agents on airway responsiveness after vagotomy may in part be due to their effects on VT. Furthermore, bronchodilation accompanying large volume ventilation persists after vagotomy, suggesting that it is not exclusively mediated by changes in parasympathetic activity.

O3-induced change in bronchial reactivity to methacholine and airway inflammation in humans.

The increase in airway responsiveness induced by O3 exposure in dogs is associated with airway epithelial inflammation, as evidenced by an increase in the number of neutrophils (polymorphonuclear leukocytes) found in epithelial biopsies and in bronchoalveolar lavage fluid. We investigated in 10 healthy, human subjects whether O3-induced hyperresponsiveness was similarly associated with airway inflammation by examining changes in the types of cells recovered in bronchoalveolar lavage fluid obtained after exposure to air or to O3 (0.4 or 0.6 ppm). We also measured the concentrations of cyclooxygenase and lipoxygenase metabolites of arachidonic acid in lavage fluid. We measured airway responsiveness to inhaled methacholine aerosol before and after each exposure and performed bronchoalveolar lavage 3 h later. We found more neutrophils in the lavage fluid from O3-exposed subjects, especially in those in whom O3 exposure produced an increase in airway responsiveness. We also found significant increases in the concentrations of prostaglandins E2, F2 alpha, and thromboxane B2 in lavage fluid from O3-exposed subjects. These results show that in human subjects O3-induced hyperresponsiveness to methacholine is associated with an influx of neutrophils into the airways and with changes in the levels of some cyclooxygenase metabolites of arachidonic acid.

A dose-response evaluation of pipecuronium bromide in elderly patients under balanced anesthesia.

Pipecuronium bromide is a new steroidal non-depolarizing muscle relaxant currently under investigation. It is similar to pancuronium with respect to the duration of action, but lacking its cardiovascular side effects. We examined the dose-response relation of pipecuronium in 27 patients, ages 66-79 years, utilizing the incremental dose method under balanced anesthesia. The ED50, ED90 and ED95 were 22.42 (5.2) mcg/kg, 31.81 (6.9) mcg/kg and 35.12 (7.8) mcg/kg, respectively (log probit method). Our recovery data also demonstrate that residual neuromuscular blockade due to pipecuronium can easily be antagonized with neostigmine as long as spontaneous recovery of T1- at the time of reversal administration is greater than 13%. The authors conclude that under balanced anesthesia the cumulative dose-response of pipecuronium in the elderly patients is consistent with those previously described for younger population. Therefore, no dose adjustment appears necessary for the elderly. However, as with all medications, careful administration is appropriate.

A review of neuropsychological differences between paranoid and nonparanoid schizophrenia patients.

This review examines the literature on neuropsychological differences between paranoid and nonparanoid schizophrenia subjects. Thirty-two studies related to intellectual functioning, attention, memory, language, visual-spatial, and motor functions are discussed. Subjects with paranoid schizophrenia did not demonstrate higher intellectual functioning than those with nonparanoid schizophrenia, and both groups performed similarly on tests of verbal ability and visual-spatial functions. Several studies suggest that the paranoid subtype is associated with higher performance on tests of executive functions, attention, memory, and motor skills. However, the findings are inconsistent. Methodological issues in the literature are examined, including varying degrees of participants' chronicity and severity of illness among studies, criteria for diagnostic group membership, medication effects, reliability and validity of the neuropsychological measures, and statistical power.

Pain control for the urologist.

Pain states can be divided into three categories: acute, chronic not resulting from malignancy, and chronic malignant pain. A Pain Clinic can provide in-depth evaluation and treatment of difficult pain problems. A variety of oral and parenteral medications, ranging from nonsteroidal analgesics to narcotics, are available to control pain. Local anesthetics can be used for local infiltration, and peripheral and central nerve blocks can also be used as indications warrant.

Thyroid function in chronic renal failure after successful renal transplantation.

Chronic renal failure is associated with a variety of thyroid function abnormalities. Information on thyroid function in patients with chronic renal failure after successful renal transplantation is limited. We studied thyroid function in 13 such patients and found that serum TT3 in the transplant group (136 +/- 30 ng/ml) was significantly (P less than 0.01) higher than the TT3 in the control group (112 +/- 29 ng/ml). All patients were clinically euthyroid and had normal serum TT4, FT4, FT3, TSH, and TBG levels and a normal T3 resin uptake. The pathogenesis of the observed increased TT3 in these patients is not clear.