Volume-regulatory Cl- channel currents in cultured human epithelial cells. 1. During osmotic swelling, cultured human small intestinal epithelial cells (Intestine 407) exhibited activation of large Cl- currents under the patch-clamp whole-cell configuration. The volume-sensitive Cl- conductance was independent of intracellular Ca2+ and cyclic AMP. 2. The anion permeability sequence of the current was SCN- > I- > Br- > Cl- > F- > gluconate-, corresponding to Eisenman's sequence I. 3. Cl- currents were instantaneously activated by command pulses in a range of -120 to +45 mV. At potentials more positive than +50 mV the current showed a time-dependent inactivation. This inactivation was accelerated by increased depolarization. The instantaneous current-voltage relationship rectified in the outward direction. 4. A stilbene-derivative Cl- channel blocker, 4-acetamido-4'-isothiocyanostilbene (SITS), inhibited the Cl- current at micromolar concentrations. SITS facilitated inactivation at positive potentials. Outward currents were more prominently suppressed by SITS than inward currents. The concentrations required for 50% inhibition (IC50) of outward and inward currents were 1.5 and 6 microM, respectively. The outward and inward currents were equally inhibited by a carboxylate analogue Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) or diphenylamine-2-carboxylate (DPC) at higher doses (IC50 = 25 for NPPB or 350 microM for DPC). Inactivation kinetics at large depolarizations was not affected by NPPB or DPC. 5. The Cl- current was blocked by an unsaturated fatty acid, arachidonic acid (IC50 = 8 microM). Arachidonic acid was still effective in the presence of inhibitors of lipoxygenase (nordihydroguaiaretic acid, 10 microM), cyclo-oxygenase (indomethacin, 10 microM) and protein kinase C (polymyxin B, 30 microM). The Cl- current was also sensitive to another cis unsaturated fatty acid, oleic acid, which is not a substrate for oxygenases. A trans isomer of oleate, elaidic acid, and a saturated fatty acid, palmitic acid, were ineffective. 6. Single Intestine 407 cells exposed to a hypotonic solution showed a regulatory volume decrease after initial osmotic swelling. The volume regulation was abolished by SITS, NPPB, arachidonate and oleate, but not by elaidate and palmitate. 7. It is concluded that outwardly rectifying Cl- channels, which are sensitive to arachidonic acid, are activated upon osmotic swelling and involved in the subsequent cell volume regulation.


Resting membrane potential and potassium currents in cultured parasympathetic neurones from rat intracardiac ganglia. 1. Whole-cell K+ currents contributing to the resting membrane potential and repolarization of the action potential were studied in voltage-clamped parasympathetic neurones dissociated from neonatal rat intracardiac ganglia and maintained in tissue culture. 2. Rat intracardiac neurones had a mean resting membrane potential of -52 mV and mean input resistance of 850 M omega. The current-voltage relationship recorded during slow voltage ramps indicated the presence of both leakage and voltage-dependent currents. The contribution of Na+, K+ and Cl- to the resting membrane potential was examined and relative ionic permeabilities PNa/PK = 0.12 and PCl/PK < 0.001 were calculated using the Goldman-Hodgkin-Katz voltage equation. Bath application of the potassium channel blockers, tetraethylammonium ions (TEA; 1 mM) or Ba2+ (1 mM) depolarized the neurone by approximately 10 mV. Inhibition of the Na(+)-K+ pump by exposure to K(+)-free medium or by the addition of 0.1 mM ouabain to the bath solution depolarized the neurone by 3-5 mV. 3. In most neurones, depolarizing current pulses (0.5-1 s duration) elicited a single action potential of 85-100 mV, followed by an after-hyperpolarization of 200-500 ms. In 10-15% of the neurones, sustained current injection produced repetitive firing at maximal frequency of 5-8 Hz. 4. Tetrodotoxin (TTX; 300 nM) reduced, but failed to abolish, the action potential. The magnitude and duration of the TTX-insensitive action potential increased with the extracellular Ca2+ concentration, and was inhibited by bath application of 0.1 mM Cd2+. The repolarization rate of the TTX-insensitive action potential was reduced, and after-hyperpolarization was replaced by after-depolarization upon substitution of internal K+ by Cs+. The after-hyperpolarization of the action potential was reduced by bath application of Cd2+ (0.1 mM) and abolished by the addition of Cd2+ and TEA (10 mM). 5. Depolarization-activated outward K+ currents were isolated by adding 300 nM TTX and 0.1 mM Cd2+ to the external solution. The outward currents evoked by step depolarizations increased to a steady-state plateau which was maintained for > 5 s. The instantaneous current-voltage relationship, examined under varying external K+ concentrations, was linear, and the reversal (zero current) potential shifted in accordance with that predicted by the Nernst equation for a K(+)-selective electrode. The shift in reversal potential of the tail currents as a function of the extracellular K+ concentration gave a relative permeability, PNa/PK = 0.02 for the delayed outward K+ channel(s).(ABSTRACT TRUNCATED AT 400 WORDS)


Intracellular cations modulate noradrenaline-stimulated calcium entry into smooth muscle cells of rat portal vein. 1. The action of noradrenaline (NA, 10 microM) was studied in single patch-clamped smooth muscle cells of rat portal vein where free internal Ca2+ concentration in the cell (Ca2+i) was estimated using the emission from the dye Indo-1. 2. In the presence of 50 microM D600, a blocker of voltage-dependent Ca2+ channels, NA applied to the cell evoked an initial peak in Ca2+i followed by a smaller sustained rise. The initial rise in Ca2+i was associated with the activation of Ca(2+)-dependent Cl- channels. 3. The maintained rise in Ca2+i induced by NA was enhanced by increasing the external Ca2+ concentration and was abolished in Ca(2+)-free solution. The transient rise was resistant to the absence of external Ca2+. 4. Following the transient rise in Ca2+i induced by NA, the mechanisms extruding and/or sequestering cytoplasmic Ca2+ were stimulated. This stimulation was measured during the sustained rise in Cai and was maintained for a few seconds after NA was removed. 5. Unlike the transient rise in Ca2+i, the sustained rise in Ca2+i produced by NA was affected by changing the cell membrane potential. 6. Changing the Na+ gradient showed that the Na(+)-Ca2+ exchange was not involved in the sustained rise in Ca2+i. 7. The sustained increase in Ca2+i produced by NA was modulated by intracellular cations. This phase could be observed with 130 mM Na+ or 130 mM K+ in the pipette solution, but was severely reduced when the only cation in the intracellular solution was Cs+ and abolished with NMDG (N-methyl-D-glucamine) or TEA. However, inclusion of only 10 mM Na+ or 50 mM K+ in the pipette solution was sufficient to obtain a sustained rise in Ca2+i of maximal amplitude, similar to that obtained with 130 mM Na+i or 130 mM K+i during NA application. 8. In portal vein smooth muscle cells, NA induced a two-phase increase in Ca2+i similar to the two phases which have been previously observed upon muscarinic receptor activation by carbachol in intestinal smooth muscle. The transient rise was due to Ca2+ store release whereas the sustained rise was due to an increased Ca2+ entry into the cell down its electrochemical gradient, but not through voltage-dependent Ca2+ channels. The Ca2+ permeability pathway involved in the sustained rise in Ca2+i induced by NA was modulated by the intracellular cations.


The antiatherogenic effects of amlodipine: promise of preclinical data. Atherosclerosis is a complex and multifactorial disease, the endpoint of which is the formation of a calcified plaque. Intermediate events include intimal injury, smooth muscle cell proliferation and migration, macrophage infiltration, lipid accumulation and excess formation of ground substance. To determine whether the newly developed, long-acting calcium antagonist, amlodipine, slows the development of atherosclerotic lesions under experimental conditions, young New Zealand white rabbits were fed on a diet of 2% cholesterol plus 1% peanut oil for up to 12 weeks. Half the rabbits received 1 or 5 mg amlodipine/kg body weight/day. Amlodipine caused a significant and dose-dependent reduction in lesion formation in the thoracic aorta. At the same time thoracic aorta Ca2+ and cholesterol content were maintained at near normal levels, despite the raised plasma cholesterol levels. The protective effect of amlodipine persisted throughout a treatment period of 12 weeks, indicating the absence of tachyphylaxis.


Effects of niacin on bleomycin-induced increases in myeloperoxidase, prolyl hydroxylase, and superoxide dismutase activities and collagen accumulation in the lungs of hamsters. It has been shown that lung nicotinamide adenine dinucleotide (NAD) depletion accompanies bleomycin (BL)-induced lung fibrosis in the hamster and that treatment with niacin (NA), a precursor of NAD, was found to attenuate lung fibrosis caused by this agent. Niacin was used in the present study to investigate changes in some biochemical parameters and enzymes involved in the development of BL-induced lung fibrosis in the hamster. Niacin (500 mg/kg, IP), or an equivalent volume of saline (SA, IP), was given daily 2 days prior to intratracheal instillation of BL (7.5 U/5 mL/kg) or SA and everyday thereafter throughout the study. Hamsters were killed at 1, 4, 7, 10, and 14 days after the BL or SA instillation and their lungs processed for various biochemical assays. Hydroxyproline content and superoxide dismutase (SOD) activity in SABL treated animals were significantly (P < or = 0.05) elevated at 7 and 10 days, peaking at 14 days to 161 +/- 11% and 159 +/- 11% of the SASA treated animals, respectively. Although the hydroxyproline level of NABL treated animals was significantly elevated at 7 and 10 days and peaked at 14 days to 123 +/- 8% of the NASA control, these values were significantly lower than the SABL treated animals at the corresponding times. The lung SOD activity of NABL groups was significantly higher at 4 days but significantly lower at 10 and 14 days than the SABL groups at the corresponding times. Prolyl hydroxylase (PH) activity and total lung calcium in SABL treated groups were significantly elevated compared to SASA treated groups starting at 4 days, with PH peaking at 10 days to 163 +/- 13% and calcium peaking at 7 days to 148 +/- 8% of SASA treated groups. The NABL treated animals displayed a significant elevation in PH activity at 4 days only (132 +/- 15%), while the calcium content in this group was significantly increased at 4 and 14 days compared to NASA treated animals. However, the activity of PH in the NABL treated animals was significantly lower than the SABL treated animals at 7, 10, and 14 days. The calcium content of the NABL group was significantly lower than the SABL group at 7 and 10 days. The thiobarbituric acid reactive substance equivalents (TBARS) content and myeloperoxidase (MPO) activity were significantly elevated at all time points in SABL groups as compared to SASA groups, with peak elevation of TBARS to 160 +/- 9% at 4 days and MPO to 268 +/- 40% at 1 day.(ABSTRACT TRUNCATED AT 400 WORDS)


Tryptophan and serotonin metabolism in familial erythrophagocytic lymphohistiocytosis. Reduced concentrations of tryptophan and 5-hydroxyindoleacetic acid (the major CSF metabolite of serotonin) were found in the cerebrospinal fluid of two children with familial erythrophagocytic lymphohistiocytosis. This was associated with elevated cerebrospinal fluid neopterin concentrations indicating increased macrophage activity within the central nervous system. In one child, cytotoxic therapy induced a complete clinical remission and an increase of tryptophan and 5-hydroxyindoleacetic acid concentrations to normal; during a subsequent relapse, concentrations of these analytes again fell below normal. In the other child, in whom therapy produced only a transient improvement, tryptophan and 5-hydroxyindoleacetic acid concentrations remained low and the child died. It is likely that increased activity of indoleamine 2,3-dioxygenase induced by the activation of macrophages was responsible for the disturbance in serotonin and tryptophan homeostasis within the brain. Excessive tryptophan catabolism and the disturbance of serotonin turnover may play a role in the aetiology of the neurological symptoms seen in familial erythrophagocytic lymphohistiocytosis.


Immune proteins in peripheral tissue fluid-lymph in patients with filarial lymphedema of the lower limbs. Immune proteins and cytokine concentrations and activity were measured in skin tissue fluid-lymph and blood serum of patients with lower leg filarial lymphedema. High levels of lymph gamma-globulins, alpha-1-acid glycoprotein and IgG lymph/serum ratio were found. Lymph from filarial patients had an elevated lymph stimulatory effect on blood mononuclear cell culture with phytohemagglutinin. There was also a high concentration and activity of IL-1 beta but gamma-interferon was not detected. The deranged pattern of immune proteins and high activity of IL-1 suggest persistence of an ongoing local inflammatory process despite the absence of overt dermatitis. The high tissue-lymph IL-1 concentration and activity may signify a cytokine network promoting keratinocyte and fibroblast proliferation commonly seen on skin histology in patients with filariasis.


Ventricular beat classifier using fractal number clustering. A two-stage ventricular beat 'associative' classification procedure is described. The first stage separates typical beats from extrasystoles on the basis of area and polarity rules. At the second stage, the extrasystoles are classified in self-organised cluster formations of adjacent shape parameter values. This approach avoids the use of threshold values for discrimination between ectopic beats of different shapes, which could be critical in borderline cases. A pattern shape feature conventionally called a 'fractal number', in combination with a polarity attribute, was found to be a good criterion for waveform evaluation. An additional advantage of this pattern classification method is its good computational efficiency, which affords the opportunity to implement it in real-time systems.


[The effect of occupational environment on the development of skin lesions in workers at a tableware porcelain plant]. This work reports on the skin diseases which occur among the workers of the ceramic industry. The most frequently observed diseases were: dermatomycosis, eczema as a result of wear, and allergic diseases. Nickel, and also cobalt, turpentine, paraphenyldiamine and detergents were the main allergens. Mycotic infection was observed primarily among the workers using common bathrooms.


[The isolation of ribosomes from Acholeplasma and the study of their physicochemical characteristics]. Ribosomes are produced from Acholeplasma laidlawii PG-8 and A. laidlawii var. granulum str. 118. It is proved as possible to use the standard method of differential centrifugation for isolation of mollicute ribosomes. The physico-chemical properties of acholesplasma ribosomes are studied. The protein content for A. laidlawii PG-8 amounted to 39.6%, rRNA content--60.4% and for A. laidlawii var. granulum--39.1 and 60.8%, respectively. The RNA: protein ratio equals 1.5:1, the ratio of optic density indicates at 260 and 280 nm--2, that is peculiar to treated preparations of ribosomes. Sedimentation coefficient of acholeplasma ribosomes is 70S. The produced preparations of acholeplasma ribosomes can be used subsequently for creating the system of translation in vitro to study the biosynthesis processes of mollicute protein.


[The agglutinability of cholera O-monoclonal immunoglobulins]. A set of 10 monoclonal antibodies specific for vibrio species and of 5 monoclonal antibodies specific for serovar (Ogawa) was studied. These monoclonal antibodies were directed toward V. cholerae O1 antigen in agglutination reaction and on slide plates. Monoclonal antibodies agglutinating typical strains of cholera vibrios with titration range from 1: 6000 to 1: 25,000 were selected. MA were revealed to interact with cholera vibrio strains with reduced agglutinability. The set of agglutinating O monoclonal immunoglobulins was developed for laboratory identification of cholera O1 vibrios.


[The effect of the acid exopolysaccharide produced by the causative agent of melioidosis on macrophage function]. Acid exopolysaccharide of the melioidosis agent, isolated from the slime of alysogenic strain of Pseudomonas pseudomallei SVBDO-141 inhibits the capture of sheep erythrocytes--55Fe by mouse peritoneal macrophages. Administration of the sheep erythrocytes--55Fe, sensibilized by acid exopolysaccharide was proved not to have an antiphagocytic effect. Inhibition of phagocytic macrophagal activity depends on the conditions of isolation of acid exopolysaccharide.


Identification and characterization of a parasite antigen in the circulating immune complexes of Onchocerca volvulus infected patients. A PEG-ELISA was used to demonstrate parasite specific immune complexes in a significant proportion (25/26) of Onchocerca volvulus infection sera from Sierra Leone. The parasite antigen was detected using a peroxidase-conjugated rabbit serum raised to the bovine parasite O. gibsoni. Controls including European control serum, endemic control serum and Rh+ sera gave consistently low readings. Characterization of the parasite component in the immune complexes by Western blotting demonstrated a heat stable antigen of M(r) 46,000. This antigen was not present in the circulating immune complexes (CIC) prepared from patients with Wuchereria bancrofti infection, but a cross-reactive molecule of the same size was weakly recognized in the CIC of Loa loa and Mansonella perstans infected patients. No association between the level of parasite specific CIC and clinical disease was observed in the O. volvulus patients.


[Plasma histamine levels after the administration of atracurium or pancuronium in patients undergoing general anesthesia]. To compare the release of histamine induced by atracurium and pancuronium. We studied 20 patients ASA III undergoing vascular surgery under etomidate anesthesia. Patients were randomly treated with either 0.5 mg/kg of atracurium or 0.1 mg/kg of pancuronium as muscle relaxant agents. Plasma histamine concentration, heart rate, arterial blood pressure, PaO2, and PaCO2 were measured at the basal state and 1.2 and 5 min after administration of the muscle relaxant drug. Plasma histamine concentration at baseline were 0.691 +/- 0.6 ng/ml in the atracurium group and 0.756 +/- 0.612 ng/ml in the pancuronium group. These levels raised up to 2.748 +/- 6.278 ng/ml (atracurium) and 2.553 +/- 5.454 ng/ml (pancuronium). These differences were not statistically significant. The course of the remaining parameters studied in these patients was also comparable between the two groups. There were no clinical manifestations associated with the release of histamine. Plasma levels of histamine after administration of atracurium were not significantly different from those induced by pancuronium.


[Comparative sensitivity of different solid-phase immunoassays for detecting HBsAg and anti-HCV in blood]. The sensitivities of seven methods of enzyme-immunoassay for HBsAg detection, as well as of twelve immunoassays for detecting antibody to HCV, were compared, in an attempt to evaluate the need for an official technical validation of such methods prior to its commercialization in Spain. Significant differences were seen for the sensitivities of these methods, which may influence the proficiency of blood bank screening for HBsAg and anti-HCV for preventing cases of post-transfusional viral hepatitis. As a conclusion, it is recommended to establish legal regulations for pre-marketing validation of such assays in Spain, as well as to take the results obtained in these evaluation studies as the basis for selecting those tests which may be more convenient for screening purposes at the blood banks.


[A cytophotometric study of the RNA and glycogen content of human hepatocytes at different stages in the development of traumatic disease]. Absorption and fluorescent cytophotometry techniques were applied to studies of RNA, glycogen and its fractions in hepatocytes of patients with hard mechanic trauma, both with and without endointoxication. For measuring RNA and glycogen contents, slides were stained by gallocyanin-chromalum or underwent fluorescent PAS-reaction, respectively. The repeated aspiration biopsy material was used for investigation of RNA and glycogen contents in dynamics. A quick increase in RNA content took place at the first stage (within the first 3 days) of traumatic illness of both the groups of patients. At the second stage of illness the hepatocyte RNA content in patients without endointoxication was seen to decrease to the initial level, whereas that in patients with endointoxication increased from the initial level by 36%. At different stages of illness the total glycogen content is changed insignificantly in the course of illness, but its labile fraction decreases to 70% of the total glycogen in patients with endointoxication. The increase of hepatocyte synthetic activity and the maintenance of the normal glycogen level may suggest a sufficient compensatory possibility of the liver, in spite of a high functional load under endointoxication.


[The level of chromosomal DNA methylation in mice in the early stages of embryogenesis studied by the action of restriction endonucleases on the chromosomes]. The degree of genome methylation was estimated on chromosomal slides of mouse zygotes, morulae and embryos of 10 day of gestation using in situ digestion with restriction endonucleases MspI and HpaII. The chromosome preparations of all the embryo stages were made by the same method. The degree of methylation was evaluated by the appearance of chromosomes after digestion and by staining with the Giemsa stain. At the zygote stage, both maternal and paternal genomes are more methylated than chromosomes of the next stages of development. The paternal genome is more methylated than maternal. The homologous chromosomes of morulae and 10 day embryos were identical and the pattern of G-banding was formed.


Clinical chemistry profile data for Hispanics, 1982-84. This report presents descriptive data for clinical chemistry profiles by age and sex. This information is from the Hispanic Health and Nutrition Examination Survey, a sample survey of selected groups of civilian noninstitutionalized Hispanic persons residing in the United States. The survey was conducted during 1982-84.


RNA delivery in Saccharomyces cerevisiae using electroporation. An efficient delivery method for introducing in vitro synthesized RNA into yeast has been developed using electroporation. Spheroplast preparation, electroporation, and subsequent expression analysis can be accomplished within a single day. The use of introduced mRNA constructs avoids any complications due to nuclear regulation and is particularly suited for cytoplasmic regulatory studies. Moreover, this technique is useful for introducing those RNAs that cannot be made in vivo, such as poly(A)- mRNAs or RNAs with base modifications. We demonstrate that the Escherichia coli GUS gene and the firefly Luc gene are both excellent reporter genes for RNA electroporation.


High-performance capillary electrophoresis of SDS-protein complexes using UV-transparent polymer networks. This paper demonstrates the use of UV-transparent replaceable polymer networks for the separation of SDS-protein complexes on the basis of molecular weight. First, the use of linear (i.e. non-cross-linked) polyacrylamide is shown to provide molecular separation of SDS-protein complexes. A study reveals such columns to yield significantly greater lifetime than cross-linked gels because of the flexibility of the noncovalently attached polymer chains. However, column lifetime was still found to be limited (approximately 20-40 injections), and detection at 214 nm was problematical because of the absorbance of polyacrylamide. UV-transparent polymer networks of dextran and PEG were substituted for polyacrylamide with successful molecular weight sieving of SDS-protein complexes at 214 nm. Due to their low to moderate viscosities, these networks could be routinely replaced leading to the possibility of hundreds of injections with a single column. Migration time reproducibilities of 0.5% RSD or less were found with replacement of the network. Using dextran, calibration plots of peak area vs concentration of standard protein were linear over the range of 0.5 microgram/mL up to at least 0.25 mg/mL. Furthermore, plasma samples could be directly utilized because of the strong solvating power of SDS. Rapid separation of protein mixtures are demonstrated with these UV-transparent polymer networks.


Soluble CD14 but not interleukin-6 is a new marker for clinical activity in atopic dermatitis. Levels of soluble IL-2 receptors, IL-6, soluble CD23, soluble CD14 and ECP (eosinophilic cationic protein) were measured as markers of T-cell, B-cell, monocyte and eosinophilic leucocyte activation in 26 patients with atopic dermatitis (AD) on admission to (A) and at discharge from (D) the Department of Dermatology in Zurich. The serum levels of sIL-2R, IL-6, sCD23, sCD14 and ECP were significantly elevated in AD patients in comparison with the normal values of healthy donors. A significant decrease in sIL-2R (p = 0.0093) and in sCD14 (p = 0.0134) levels was demonstrated between A and D, correlating with the improvement in the skin intensity score (SIS). In addition, a significant correlation of the sCD14 levels and the SIS at A was demonstrated (p = 0.0415). These results also incriminate monocytes in the pathogenesis of AD, indicating that, besides sIL-2R and ECP, SCD14 could also be a possible marker for the disease activity.


Epidermal growth in the skin equivalent. The skin equivalent (SE) has been validated as a model for studies on proliferation of keratinocytes. SEs were prepared from normal skin by implanting punch biopsies on dermal equivalents consisting of fibroblasts in a collagen matrix. The outgrowths were measured by planimetry. An immunohistochemical investigation with antibodies against markers associated with proliferation was performed on frozen sections from SEs during outgrowth at 3-6 days (SE6) as well as after completion of outgrowth at 21 days (SE21). Biopsies from normal controls and from uninvolved and involved skin in psoriatic patients were also studied. The antibodies used were Ki-67, cytokeratin 8.12, and antibodies against the receptors for epidermal growth factor (EGFr) and transferrin (TFr). The increase in area was linear during the first 7 days of culture and usually reached the edges of the dermal equivalent at this time. In SE6 TFr was expressed in the basal part of the outgrowth while the other markers were not observed. In SE21 and in psoriasis there was abundant epidermal staining of Ki-67-positive nuclei and cytokeratin 8.12 was detected in the suprabasal part of the epidermis. EGFr and TFr were seen in the basal layer in SE21. In the psoriatic lesions these receptors were found both in the basal and suprabasal layers. The lack of proliferation markers in SE6 indicates that the initial increase in the area of keratinocytes is due to migration from the punch biopsies. Increased cell proliferation is present in SE21, a finding in common with psoriasis and wound healing. The skin equivalents should therefore be an appropriate model for studies on these phenomena.


[Transurethral thermotherapy in prostatic adenoma]. Microwave thermotherapy is considered to be an alternative to conventional surgical treatment of benign prostatic hyperplasia (BPH). Our initial experience of the first six months shows this technique has achieved objective improvement of the symptoms and uroflowmetry, as well as a significant reduction of postmicturition residual urine. It carries a minimal morbidity, no mortality and has permitted recovery of micturition in over 90% of the patients with urinary retention.


Congenital sensory neuropathy with anhidrosis (hereditary sensory neuropathy type IV). Hereditary sensory neuropathies comprise a group of rare childhood diseases which are classified into four types. We present a Greek boy 11 years old with hereditary sensory neuropathy type IV (congenital sensory neuropathy with anhidrosis) whom we have followed up and studied during the last seven years. Our patient presented for the first time with recurrent hyperthermic episodes without sweating, and lack of pain sensation from the first months of life. Insensitivity to pain and thermal stimuli had resulted in burns on the extremities and self-mutilation of the tongue, lips and fingertips. When he was five and seven years old respectively he had two painless fractures of the ankles which led to insoluble orthopedic problems. He also suffered from mental retardation, which was obvious from his first years of life. Sweat gland investigations showed significant hypohidrosis or anhidrosis although the sweat glands were normal microscopically. Hereditary sensory neuropathy type IV, although rare, is important for dermatologists because it must be differentiated from other anhidrotic syndromes, and in view of the poor prognosis of the condition.


[Histochemical detection of hepatic iron. A comparative study of four stains]. Due to its specificity and easiness, Perls' stain is widely used in the histochemical assessment of liver iron content. However, it can underestimate slight iron overload, which can hamper screening for genetic hemochromatosis, especially in young people. The aim for the present study was to compare Perls' stain to three other specific iron stains (Tirmann-Schmeltzer (TRM); Hukill and Putt (HPT); Perls with Diaminobenzidine (DAB)), biochemical liver iron concentration (LIC) being used as the reference. 1) There is a significant difference between number of stained cells with TRM or DAB, compare with Perls' stain (p < 0.05). 2) Correlation (r) between histological assessment (T) and LIC was 0.39 for Perls' stain, 0.64 for TRM 0.53 for HPT and 0.64 for DAB. These data suggest that Perls' stain is not the most sensitive method for the assessment of slight iron overload. Tirmann-Schmeltzer's stain and Perls plus dAB should be preferred, especially in the screening of early liver siderosis.


Modulation of gastric mucosal calcium channels activity by platelet-derived growth factor. A dihydropyridine-sensitive gastric mucosal calcium channels were isolated from the solubilized epithelial cell membranes by affinity chromatography on wheat germ agglutinin. The channels following labeling the calcium antagonist receptor site with [3H]PN200-100 were reconstituted into phospholipid vesicles which exhibited active 45Ca2+ uptake as evidenced by La3+ displacement assays. The uptake of calcium was independent of sodium and potassium gradients indicating the electroneutral nature of the process. The channels responded in a dose dependent manner to dihydropyridine calcium antagonist, PN200-110, which at 0.5 microns exerted maximal inhibitory affect of 66% on 45Ca2+ uptake, while a 52% enhacement in 45Ca2+ uptake occurred with a specific calcium channel activator, BAY K8644. On platelet-derived growth factor (PDGF) binding in the presence of ATP, channel protein showed an increase in tyrosine phosphorylation of 55 and 170 kDa calcium channel proteins. Such phosphorylated channels following reconstitution into vesicles displayed a 78% greater 45Ca2+ uptake. The results demonstrate the importance of PDGF in the regulation of gastric mucosal calcium uptake.


Identification of novel adhesion proteins in mouse peritoneal macrophages. When mouse peritoneal macrophages were made to adhere firmly on glass surface and then removed by sequential treatment with hypotonic triethanolamine and Nonidet P-40, a set of proteins were found to be left behind at the sites of adherent cells. Such glass-adherent proteins were detected as round or ellipsoidal patches of autofluorescence under a confocal laser microscope, and visualized ultrastructurally as aggregates of narrow threads of unique loop structures which were composed of linearly aligned particles of 22 +/- 2 nm in diameter. Lithium dodecylsulfate-polyacrylamide gel electrophoresis of the glass-adherent proteins showed two major bands, 12 kDa and 14 kDa, which always co-existed in any different sample. The polyclonal antibody raised against these two proteins specifically stained the glass-adherent proteins in situ. The adhesion of macrophages to glass was significantly blocked with Fab fragments of the antibody. The in situ cross-linking experiment suggested that these two proteins might be closely associated with each other to form complexes. Hence, these proteins can be reasonably considered to be responsible for non-specific adhesion of macrophages to glass.


Computer-assisted analysis of the structure of regular branched polysaccharides containing 2,3-disubstituted rhamnopyranose and mannopyranose residues on the basis of 13C NMR data. A computer-assisted approach to the analysis of the structure of branched polysaccharides that contain 2,3-di-O-glycosylated alpha-rhamnopyranose and alpha-mannopyranose residues is based on evaluation of the 13C NMR spectra, using glycosylation effects and their deviations from additivity (delta delta values) at the branch points. This approach, in combination with monosaccharide and methylation analysis data, has been verified on a series of bacterial polysaccharides of known structure.


Structure of the O-antigen of Escherichia coli 0119 lipopolysaccharide. The structure of the O-polysaccharide component of the lipopolysaccharide produced by Escherichia coli 0119 was determined by the use of methylation analysis, periodate oxidation, 1D and 2D nuclear magnetic resonance spectroscopy, and mass spectrometric methods. The O-polysaccharide was found to be a high molecular weight polymer of a repeating pentasaccharide unit composed of D-mannose, D-galactose, L-rhamnose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2,3-dideoxy-3-formamido-D-rhamnose residues (1:1:1:1:1) and had the structure: [formula: see text]


Structures of the glycopeptidolipid antigens of serovars 25 and 26 of the Mycobacterium avium serocomplex, synthesis of allyl glycosides of the outer disaccharide units and serology of the derived neoglycoproteins. The pentasaccharide hapten released from the glycopeptidolipid (GPL) antigen of M. avium serovar 26 has been characterized as O-(2,4-di-O-methyl-alpha-L-fucopyranosyl)-(1-->4)- O-beta-D-glucopyranosyluronic acid-(1-->4)-O-(2-O-methyl-alpha-L-fucopyranosyl)-(1-->3)-alpha-L- rhamnopyranosyl-(1-->2)-6-deoxy-L-talose. The allyl glycosides of the outer glycosyl and glycobiosyl units of this hapten have been synthesized, the latter by a route involving oxidation of the corresponding D-glucopyranose derivative. Conjugation of allyl glycosides to protein by ozonolysis and reductive coupling afforded neoantigens (neo 26-1 and 26-2), both of which interacted with antibodies to M. avium serovar 26. The terminal sugar residue of the pentasaccharide hapten of the serovar 25 GPL had been shown to have the galacto configuration on the basis of 1H-13C NMR correlation spectroscopy, but absolute configurational assignment for the sugar awaited the synthesis, as for neo 26, of two glycobiosyl NGPs bearing the terminal sugar in the D and L enantiomeric forms, respectively. Only the glycobiosyl NGP bearing the terminal sugar as the D-enantiomer interacted with antibodies to M. avium serovar 25, thus providing evidence for the absolute configuration of the sugar, and showing that the complete oligosaccharide hapten has the structure, O-(4-acetamido-4,6-dideoxy-2-O-methyl-alpha-D- galactopyranosyl)-(1-->4)-O-beta-D-glucopyranosyluronic acid-(1-->4)-O-(2-O-methyl-alpha-L-fucopyranosyl)-(1-->3)-O-alpha-L- rhamnopyranosyl-(1-->2)-6-deoxy-L-talose.


Influence of corticosterone acetate on the spleen in intact and ovariectomized rats. Increasing evidence of the interaction of glucocorticoids and ovarian steroids prompted the current study. Effects of exogenously administered corticosterone acetate (3.5 mg/100 g b.w/day for one week) were examined on splenic nucleic acids, protein, lactate, and on lactate dehydrogenase (LDH) specific activity and its isozymes in ovariectomized and ovary-intact Wistar rats (65-75 days old). Ovariectomy resulted in no significant change in the parameters studied except DNA which increased significantly. The administration of corticosterone to these rats did not produce any remarkable change in the ovariectomy caused increase in splenic DNA content. Nevertheless, it decreased the ratio of heart type subunits (H)/muscle type subunits (M) [H/M] of LDH isozymes. In the case of ovary-intact rats, corticosterone produced an increase in the concentration of splenic lactate but a decrease in the H/M ratio. Exogenously administered corticosterone exerts selective synergistic interaction with ovarian hormones on splenic lactate. The specific activity of LDH and the concentrations of RNA and protein remained unchanged during the interaction between ovarian hormones and corticosterone.


Usefulness and limitation of measurement of insulin-like growth factor binding protein-3 (IGFBP-3) for diagnosis of growth hormone deficiency. To analyze the utility of insulin-like growth factor binding protein-3 (IGFBP-3) radioimmunoassay for diagnosis of growth hormone deficiency (GHD) we measured IGFBP-3 in sera from normal children, short children and patients with GHD. The sensitivity (true positive ratio) of IGFBP-3 for complete GHD (cGHD) was 93%, while the specificity (true negative ratio) for normal short children (NS) was 88%. In contrast, the sensitivity of IGFBP-3 for partial GHD (pGHD) was only 43%. The poor discrimination between patients with pGHD and NS may be the result of their relatively similar GH level, as compared to cGHD, or due to the limitations of GH stimulation tests. The specificity of IGFBP-3 for NS was excellent in children of all ages: less than 10 years old (87%) and older than 10 (88%). However, sensitivity for GHD was good for children less than 10 years old (84%) but poor for children older than 10 (64%). IGFBP-3 may be less sensitive for diagnosing GHD in older children because IGFBP-3 levels may also increase during puberty due to mechanisms independent of the GH-IGF-I axis.


Anti-hepatitis C virus antibodies amongst Italian homo-bisexual males. The authors report on an anti-hepatitis C virus antibody (HCV Ab) prevalence (6.9%) in 622 homo-bisexual males from Northern Italy, voluntarily attending an HIV and STDs screening program in the period 1984-89. The anti-HCV antibody prevalence shows a significant correlation with: i) presence of serological markers for HBV (O.R. = 3.12, 95% C.I. = 1.53-6.52) and HIV (O.R. = 12.09; C.I. = 6.52-22.52) infection; ii) a stable relationship with an anti-HCV antibody positive partner (O.R. = 7.79; 95% C.I. = 2.50-23.90); iii) more than twenty different male partners per year (O.R. = 2.55; 95% C.I. = 1.17-5.66). These data demonstrate the existence of a sexual transmission of HCV among homosexuals. This route might contribute in maintaining endemic levels of HCV infection in the homo-bisexual population and it might represent an important way of spreading the virus in the general population too.


[Hemodynamic and neuroendocrine profile of 2 different cardiovascular responses in vasodepressor syncope induced by the head-up tilt test]. Syncope in apparently healthy subjects is usually attributed to a vasovagal reaction. However, a vagal cardio-inhibitory component is not always associated with a vasodepressor component in causing syncope: in fact, increases in heart rate, arterial pressure and plasmatic levels of catecholamines frequently precede loss of consciousness. Prolonged 60 degrees head-up tilt table test (HUTT) was performed in 50 healthy subjects (27 male, 23 female - mean age 37.2 years) with recurrent syncope of vasodepressor or unknown origin. The upright-tilt test lasted 45 minutes: every minute of HUTT we measured heart rate (HR) and systolic (SBP) and diastolic blood pressure (DBP); at set intervals we took a blood sample to determine epinephrine (EP) and norepinephrine (NEP) levels. In patients with positive HUTT (42%) we observed a vaso-vagal response (10 patients) characterized by a sharp drop in SBP and DBP (> 50% of the basal values) and bradycardia (< 40 bpm) and/or sinus node arrests, and a hyperchronotropic-vasodepressor response (11 patients) characterized by a considerable increase in HR (> 60%) and simultaneous drop in SBP and DBP (> 30% of the basal values), and a large increase in plasmal EP (+881.9%). According to the Authors, vasovagal response is mainly due to a reflex reaction originating from the cardiac stretch-receptors, whereas hyperchronotropic-vasodepressor response is mainly due to psychic stress and anxiety provoked by prolonged and forced posture during HUTT. The high levels of adrenergic activity and plasmal EP cause the excessive chronotropic response and the vasal effects of the syndrome. Due to the induction of a state of anxiety and its postural effects, HUTT is a useful provocative tool for complete evaluation of young patients with syncope of vasodepressor origin. We treated the patients differently, depending on how they responded to HUTT. Those with a vaso-vagal response were treated with alpha-sympathomimetic agents (ethylephrine or mydodrine) and those with a hyperchronotropic-vasodepressor response received non-selective beta-blockers. None of our patients had syncope recurrences during a mean follow-up of 12.3 months. Only two patients complained of dizziness; in one of them, symptomatology was abolished by an alpha-sympathomimetic beta-blocker association.


Bactericidal activity of two IgG2a murine monoclonal antibodies with distinct fine specificities for group B Neisseria meningitidis capsular polysaccharide. To analyze the fine specificity of the protective IgG response for the capsule of group B Neisseria meningitidis (Men B) induced after immunization with live bacteria, two specific IgG2a monoclonal antibodies (mAb) have been generated from hyperimmunized Balb/c and NZB mice (101C11 and 30H12). They specifically recognize in direct and competitive binding assays the capsular polysaccharides of Men B and Escherichia coli k1 on condition that the length of the polysaccharidic chain is sufficient to make a conformational structure (more than 15 monomers of alpha (2-->8) linked N-acetyl neuraminic acid). They do not interact with group A and group C Neisseria meningitidis polysaccharides in ELISA. A chemical derivative of the Men B polysaccharide, the N-propionylated Men B polysaccharide, considered as mimicking a unique bactericidal epitope on the surface of Men B is recognized by 101C11 but not by 30H12. The two mAb have, in vitro, a specific bactericidal activity against live Men B which do not seem serotype specific. Moreover, the killing of Men B mediated by 30H12 can be neutralized by an anti-idiotypic mAb (216F11) generated from A/J mice, immunized with polymerized 30H12. These data show that at least two distinct bactericidal epitopes exist on the surface of the Men B capsule.


Enhanced production of monoclonal antibodies against zona pellucida-free, unfertilized mouse oocytes by intrasplenic immunization. Monoclonal antibodies were obtained by intrasplenic insertion of a total of 40-50 zona pellucida-free, unfertilized mouse oocytes into each mouse. The oocytes were prepared by a combined enzyme-mechanical method without impairing the fertility of the oocyte or inducing parthenogenesis. Of a total of 1063 hybrids obtained, 15 yielded supernatants with positive reactions to unfertilized oocytes. Tests for preimplantation stage specificity demonstrated that 9 of the supernatants detected antigen epitopes in unfertilized oocytes only. The other supernatants showed positive reaction patterns to various embryonal stages of the preimplantation period. Two supernatants recognized materials in the culture medium shedded by zona pellucida-free oocytes. Thus, by applying intrasplenic immunization, the production of antibodies against zona pellucida-free mouse oocytes is enhanced. The procedures described outline a rational way of selecting antibodies of interest for research in the mechanism of fertilization.


Generation of monoclonal antibodies against a human T cell receptor beta chain expressed in transgenic mice. The generation of a panel of monoclonal antibodies specific for different variable (V) regions of human T cell receptors will be of great importance in the study of T cell-mediated diseases. However, relatively few such reagents exist, due in part to the poor immunogenicity of TcRs on the surface of human T cells. We have employed a strategy in which T cells from a transgenic mouse line expressing a human V beta 3 C beta 1 TcR were used to immunise syngeneic conventional mice to generate two monoclonal antibodies specific for human T cell receptors. Binding of antibody JOVI.3, which stained approximately 5% of human peripheral blood CD3 positive T cells, correlated with the expression of the human TcR V beta 3 gene segment. Antibody JOVI.1 recognised a determinant on the majority of TcRs, staining 50-75% of peripheral blood T cells and T cell lines expressing different V beta regions. Some TcRs, however, failed to react with this antibody. Both antibodies immunoprecipitated detergent-solubilised TcR molecules and were capable of inducing proliferation of peripheral blood T cells.


Equine tumor necrosis factor alpha: cloning and expression in Escherichia coli, generation of monoclonal antibodies, and development of a sensitive enzyme-linked immunosorbent assay. We describe the production and purification of recombinant equine tumor necrosis factor alpha (rETNF alpha), generation and characterization of murine monoclonal antibodies (Mabs) and rabbit polyclonal antibodies (Pabs) against ETNF alpha, and development of a sensitive enzyme-linked immunosorbent assay (ELISA). Genomic-derived DNA sequences encoding mature ETNF alpha were reconstructed by the polymerase chain reaction (PCR) and oligonucleotide-directed mutagenesis and were cloned into the vector pFLAG-1 for expression in Escherichia coli. rETNF alpha was purified by anti-FLAG immunoaffinity chromatography and then used as immunogen for production of murine Mabs and rabbit Pabs. Three Mabs (6H4, 9B10, and 12F6) were obtained from one fusion. All three Mabs recognized rETNF alpha on western blots. Mabs 6H4 and 9B10 recognized similar epitopes on rENTF alpha and neutralized both rETNF alpha and native ETNF alpha (nETNF alpha) in a WEHI cell cytotoxicity assay. A sensitive ELISA was developed using Mab 6H4 and biotin-labeled rabbit Pabs. The ELISA was shown to detect levels of ENTF alpha as low as 100 pg/ml and was used to demonstrate the induction of ETNF alpha in horses with experimental endotoxemia. The rETNF alpha, antibodies, and ELISA developed in this report should be useful tools for studies of TNF-mediated diseases in horses.


A monoclonal antibody (IID510g52) for the determination of functional domains within integrin cell surface receptors. Monoclonal antibodies against cell surface receptors can be useful for the study of structural and biochemical features involved in protein interactions underlying platelet adhesion and aggregation. We report here the characterization of a monoclonal antibody, IID510g52 (hereafter referred to as IID5), which has been selected based on its specific binding properties against the platelet membrane glycoprotein IIIa. Characterization of the reactive epitope, including evolutionary conservation and identification of related IID5 target antigens in tumor cells, suggest that the IID5 epitope is implicated in the ligand-binding function of integrin receptors. Indeed, we show that this MoAb acts as a potent inhibitor of platelet aggregation and cell adhesion. Taken together, these results indicate that such a monoclonal may be a strategic tool for better understanding multiple integrin-mediated adhesive reactions, as well as the determination of interacting recognition sites.


Antigen specificity and cross-reactivity of fifteen monoclonal antibodies against porcine pancreatic alpha-amylase II and its AB and C domains. Highly productive hybridoma secreting mabs specific for porcine alpha-pancreatic amylase II were established. Fifteen clones were selected. The mabs produced (KD = 1.68-11.2 nM) were checked for cross-reactivity with six heterologous antigens, namely porcine pancreatic alpha-amylase I, barley amylase, human pancreatic alpha-amylase, Taka amylase and triose phosphate isomerase, using direct ELISA assay; mabs were classified within seven groups: in a few groups mabs cross-reacted with a single heterologous antigen either porcine pancreatic amylase I (6 mabs) or barley amylase (2 mabs) or human pancreatic amylase (3 mabs). Two other groups cross-reacted with two heterologous antigen either porcine I and human or porcine I and barley. Only one mab out of fifteen cross-reacted in direct ELISA binding to all amylases and triose phosphate isomerase. Using sandwich ELISA test only three mabs were found to bind porcine amylase II present at high concentration. Results consistent with direct porcine amylase binding were obtained from binding inhibition assays. Analysis by the additivity test allowed to find that 3 mabs, B10.10, B1.11, C6.4 recognize distinct epitopes while the epitopes for the other pairs tested are either overlapping or at least close to each other. Finally mabs binding specifically either to the AB or to the C domain fragment or to both fragments have been obtained.


Monoclonal antibodies to human thyroglobulin: evaluation of immunoreactivity. We have earlier reported production and characterization of monoclonal antibodies (MAbs) to human thyroglobulin (h-tg). In the present study H10 I MAb was evaluated for its immunoreactivity towards different forms of tg and various human thyroid tumours. The specificity of H10 I MAb was validated by the absence of cross reaction with tri-iodothyronine (T3) Thyroxine (T4) and human gamma globulins. Sodium-dodicyl-sulphate polyacrylamide gel electrophoresed (SDS-PAGE) immunoblot of h-tg on the nitrocellulose membrane revealed multiple immunoreactive bands on reaction with polyclonal antibody (PAb) in comparison with total lack of reactivity with H10 I MAb. The absence of immunoreactivity of H10 I MAb was demonstrated with SDS treated, Dithiothreitol (DT) treated and heat denatured tg using dot immunobinding technique. However, the H10 I MAb was able to react with tg treated with unfolding agents such as urea and guanidine hydrochloride. All the treated forms of tg were equally recognized by PAb. The immunoreactivity of the oxidized/reduced tg towards H10 I MAb was markedly reduced (60.0%) as compared to that obtained with native tg. It appears that H10 I MAb is directed towards conformational epitope involving sulphydryl bonds. Immunohistochemically, a comparable immunoreactivity between PAb and MAb was observed with normal thyroid tissues, follicular thyroid tissues, Hurthle cell carcinoma tissues and poorly differentiated thyroid tumor tissues using immunoperoxidase staining. The sections from papillary carcinoma tissue (thyroid as well as metastatic lymph node) exhibited intense immunoreactivity with PAb. Thyroglobulin present on these sections was not recognized by H10 I MAb. Nonetheless, H10 I MAb was able to detect tg in follicular differentiation wherever present. The absence of immunoreactivity of H10 I MAb in papillary carcinoma strongly suggests that this neoplasm produces tg which is antigenically different from the protein present in the normal tissue. The reactivity of H10 I MAb with metastatic lymph node of an unknown primary origin suggests its usefulness in the identification of prevalent metastasis of differentiated thyroid carcinoma other than papillary type.


Epitope specificity of monoclonal antibodies against Newcastle disease virus: competitive fluorogenic enzyme immunoassay. A test to determine the epitope specificity of monoclonal antibodies (MCA) was developed for hybridoma clones producing antibodies against Newcastle disease virus (NDV). The virus was first immobilized on nitrocellulose membranes of Millititer HA plates. Dilutions of MCA were then added, singly, or simultaneously in pairs, and bound antibody was quantitated with alkaline phosphatase-labelled detector antibody and a fluorogenic substrate, 4-methylumbelliferyl phosphate (4-MUP). Fluorescence count was measured fluorometrically. Additivity indices were calculated and plotted against dilutions of paired MCA. Antibodies that recognized identical epitopes displayed non-additivity at saturating antibody dilutions, followed by partial additivity and by total additivity at low, non-saturating dilutions. In contrast, MCA that recognized distinct epitopes exhibited total additivity throughout the curve. MCA that exhibited partial additivity were interpreted as competing for overlapping shared epitopes, or, distinct epitopes in close proximity, resulting in steric hinderance.


Effect of phorbol ester and cytokines on matrix metalloproteinase and tissue inhibitor of metalloproteinase expression in tumor and normal cell lines. The effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) on matrix metalloproteinases (MMP) and metalloproteinase inhibitors was studied in a variety of human cell lines. Expression of the mammalian collagenase (MMP-1), 72-kD gelatinase/type IV collagenase (MMP-2), stromelysin (MMP-3), 92-kD gelatinase/type IV collagenase (MMP-9), and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) was assessed by zymography and Northern blot analysis. MMP-2 and TIMP-2 activities were refractory to TPA, IL-1 and TNF-alpha treatment in most of the cell lines. In contrast, MMP-3, MMP-9 and TIMP-1 activities were markedly stimulated by TPA in most of the tumor cell lines and human umbilical vein endothelial cells (HUVEC), whereas the fibroblast lines were minimally stimulated or unresponsive to TPA. The MMP-3, MMP-9 and TIMP-1 stimulation in response to IL-1 and TNF-alpha treatment was detected in some of the tumor cell lines and HUVEC. The increase in activity was less marked than in TPA. A breast carcinoma cell line, MDA-MB-231, which did not express MMP-2, had high expression of MMP-3 and MMP-9 which were unaffected by TPA and cytokine treatment. Northern blot analysis of MMP and TIMP mRNA expression reflected the zymogram findings for most of the cell lines. TPA-mediated stimulation of MMP-1 was similar to that of MMP-3 and MMP-9. Exceptions were the fibroblast cell lines which showed either a much more marked mRNA response of MMP-9 to TPA than observed at protein level, or a high constitutive MMP-9 mRNA when MMP-9 activity was not detectable by zymography. TPA-mediated stimulation of MMP-9 and TIMP-1 activity was blocked by staurosporine, an inhibitor of protein kinase C (PKC). A non-PKC-activating phorbol ester, 4 alpha-phorbol-12,13-didecanoate, did not stimulate MMP-9 and TIMP-1 activity. TPA treatment caused the increased expression of c-fos containing AP-1-specific binding activity in selected tumor cell lines. This activity was maximal at 6 h. An association was observed between AP-1 binding activity and increased expression of MMP-1, MMP-3 and MMP-9, which possess TPA-responsive elements (TRE). TPA-sensitive MMPs and TIMP-1 were variably stimulated by biologically relevant cytokines, such as IL-1 and TNF-alpha.(ABSTRACT TRUNCATED AT 400 WORDS)


Variability of muscle fibre composition and fibre size in the horse gluteus medius: an enzyme-histochemical and morphometric study. To determine the variability in fibre types and fibre sizes in the equine gluteus medius muscle, biopsy specimens were removed from 5 sites, at 4 different depths, within the right and left muscles of 3 Andalusian stallions. The percentage, lesser fibre diameter and cross-sectional area of the various fibre types were measured systematically in myosin ATPase and NADH-tetrazolium reductase-stained, serial cryostat sections of these multiple samples. Significant differences in muscle fibre type composition were recorded, with a lower percentage of type I fibres (high myosin ATPase activity at pH 4.5) being observed towards superficial regions of the muscle and a greater percentage towards the deep areas. Type II B fibres (moderate myosin ATPase activity at pH 4.5), including both II B nonoxidative (low NADH-TR activity) and II B oxidative (moderate NADH-TR activity), displayed the opposite tendency, and the percentage of type II A fibres (low myosin ATPase activity at pH 4.5) did not change with depth. Types I and II A fibres in the deep regions were larger than superficially, whereas the II B fibres in the deep regions were smaller than in the superficial parts of the muscle. The results also imply that type I fibres tend to be larger than type II fibres in the deep regions. The size of type I fibres is more homogeneous in the deep parts than in the superficial regions of the muscle, while II B fibres vary more in size in the peripheral portions than in deep regions. A single biopsy taken from the gluteus medius muscle of the horse is therefore a poor representative of the whole muscle and care should be exercised in sampling and interpreting data obtained from limited biopsy of this muscle. The pattern of variation in fibre types and fibre sizes between the different depths of the muscle probably reflect different functional demands on the gluteus medius muscle.


Immunocytochemical localisation of substance P in vagal ganglion cells and pericellular arborisations in the monkey. The quantitative cell count showed the presence of about 20,000 ganglion cells with associated satellite elements in the nodose ganglion in the monkey. Among these closely packed cells, at least one-third were substance P (SP) immunoreactive, ranging from faint or moderate to intense staining. Substance P immunoreactivity (SP-IR) was localised in the cell bodies and their long extending neurites. Ultrastructural study showed that SP-IR was not associated with any particular organelles or inclusions. A striking feature of the nodose ganglion was the occurrence of SP-positive pericellular arborisations associated with about 0.5% of the ganglion cells which were almost exclusively SP-negative. The pericellular arborisation displayed diverse morphological forms from a simple tortuous fibre to complex glomerular networks or plexuses encircling the soma of SP-negative ganglion cells. The varicose nerve fibres forming the pericellular arborisations appeared to terminate as 'boutons' contacting the soma of the ganglion cells. Electron microscopic study demonstrated the close spatial relation between the SP-IR profiles and the ganglion cell but there was no direct synaptic contact. In some instances, the SP-IR profiles containing agranular and dense-cored vesicles penetrated the cytoplasm of satellite cells, almost reaching the surface of the soma of the ganglion cell. The sources of origin of the nerve plexuses in the pericellular arborisation were either from the small and sparsely distributed jugular ganglion cells which were intensely SP-IR or from the intrinsic SP-IR nodose ganglion cells. The possibility that the efferent neurons in the dorsal motor nucleus of the vagus could also contribute to the pericellular arborisation was also considered. The function of the pericellular arborisations may be related to the modulation of the SP-deficient ganglion cells with which they associate through the release of SP and probably by way of the satellite cells.


On the physiological significance of tumor necrosis factor-alpha in the human amniotic fluid. Tumor Necrosis Factor (TNF)-alpha and Interleukin (IL)-1 beta levels have been measured in 16 weeks gestation amniotic fluids and mother's sera. Detectable levels of TNF-alpha were found in amniotic fluids, while IL-1 beta was absent. No cytokines were detected in mother's sera. The possible role of TNF-alpha as growth factor for fetal hematopoietic cells is discussed.


Simultaneous appearance of a unique common epitope in fetal colon, skin, and biliary epithelial cells. A possible link for extracolonic manifestations in ulcerative colitis. The tissue distribution of a previously identified M(r) 40 K epithelial autoantigen in ulcerative colitis was examined in situ in the fetal tissue by immunocytochemical method, using an immunoglobulin M (IgM) monoclonal antibody (7E12H12). Fetal-autopsy tissue from 9 to 19 weeks of development (11-21 gestational weeks) gestations, including specimens of colon (20 specimens), skin (19), small intestine (12), gallbladder (15), liver (20), spleen (20), kidney (3), urinary bladder (3), and umbilical cord/placenta (25) were examined. 7E12H12 reactive epitope appeared first in a few colonic epithelial cells at 10 weeks of gestation. By 11 weeks, 7E12H12 reactivity was clearly evident in most of the colonic epithelial cells. Goblet-cell reactivity was evident from 15 weeks of gestation. Enterocytes lining the small intestine from all the specimens were negative. In the skin, earliest immunoreactivity was evident in the basal layer of epidermis at 10 weeks of development, and by 11 weeks, the staining was intense and localized exclusively to squamous epithelial cells of the epidermis. The immunoreactivity in the fetal gallbladder mucosa was also evident earliest at 11 weeks of development. The staining was distinct in the mucosal epithelial cells, and localized along the periphery of the cells and in the apical areas. None of the other tissue specimens reacted with 7E12H12. These results demonstrate that the 7E12H12 reactive epitope is novel, is shared by colon, skin, and biliary epithelium, and appears more or less simultaneously in the three organs at 10 to 11 weeks of development of the fetus. A possible link of this crossreactive protein or proteins in the immunopathogenesis of ulcerative colitis, sclerosing cholangitis, and pyoderma gangrenosum is discussed.


Synergistic effect of 1,25-dihydroxyvitamin D3 and retinoic acid in inducing U937 cell differentiation. We examined the effects of retinoic acid (RA), 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), and its synthetic analogue, 22-oxa-1,25-(OH)2D3, on differentiation of U937 cells by studying the cellular growth, surface marker expression and cytosolic free Ca2+ concentration ([Ca2+]i). RA inhibited cellular growth but did not induce expression of Mo2 (CD14), a monocyte/macrophage specific surface marker. To the contrary, 1,25-(OH)2D3 did not inhibit cellular growth, but increased CD 14-positive cells. Simultaneous addition of 1,25-(OH)2D3 and RA had no additive effect on cellular growth inhibition or CD14 expression. With regard to [Ca2+]i, however, 5 days' incubation with either of them increased the basal [Ca2+]i level and induced U937 cells to respond to formyl-methionyl-leucyl-phenylalanine (FMLP). When the cells were incubated with both 10(-6) M RA and 10(-8) M 1,25-(OH)2D3, basal [Ca2+]i was higher and FMLP caused a greater increase in [Ca2+]i than when only RA or 1,25-(OH)2D3 was added. These data suggest that RA and 1,25-(OH)2D3 induce monocytoid differentiation in U937 cells through different pathways and act synergistically in the differentiation process. The 22-oxa-1,25-(OH)2D3 induced CD14 expression, basal [Ca2+]i increase and [Ca2+]i response to FMLP, but did not cause cellular growth inhibition in U937 cells, and in these points, 22-oxa-1,25-(OH)2D3 exhibited no significantly different effects from 1,25-(OH)2D3. Thus, 22-oxa-1,25-(OH)2D3 has the same potent activity as 1,25-(OH)2D3 in inducing differentiation of U937 cells.


Abnormal glycosylation of hemopexin in arthritic rats can be blocked by bindarit. Induction of arthritis in rats with Freund's complete adjuvant was accompanied by a distinctive alteration of concanavalin A (Con-A) reactivity in their serum proteins in which the concentrations of selected Con-A reactive proteins were significantly higher when compared to healthy rats. To assess if the observed increase in Con-A reactivity of specific serum proteins reflects an increase in carbohydrate moieties in these proteins in addition to an increase in their protein concentrations, a heme binding serum glycoprotein, hemopexin, also an acute phase reactant, was selected as a marker protein. Hemopexin was purified to apparent homogeneity from pools of serum samples derived from rats with yeast induced inflammation, a monospecific polyclonal antibody was prepared and was used for immunoblot analysis. It was noted that the concentration of hemopexin increased in rats with adjuvant induced arthritis; however, its concentration fell to normal levels after administration with a newly synthesized drug, bindarit, (2-[(1-benzyl-indazol-3-yl)methoxy]-2-methyl propionic acid, C19H20N2O3. Hemopexin was micropurified individually from healthy rats, adjuvant induced arthritic rats, and adjuvant arthritic rats treated with bindarit, cleaved with a Glu-C endopeptidase, Staphylococcus aureus protease V8, and the resultant peptide fragments resolved by SDS-PAGE and examined by silver staining, Coomassie blue staining, and lectin blots using Con-A. It was subsequently noted that hemopexin isolated from adjuvant induced arthritic rats showed a significant increase in Con-A reactivity in selected peptide fragments and that such an increase in glycosylation could be reversed to a pattern similar to healthy rats following treatment with bindarit.(ABSTRACT TRUNCATED AT 250 WORDS)


Immunologic effects in patients with psoriatic arthritis treated with cyclosporine A. Twelve patients with psoriatic arthritis (PsA) and very active articular disease resistant to conventional second line therapy entered into a 6-month open study of cyclosporine A (CsA) at a starting dosage of 3 mg/kg/day. Comparisons of phenotypic characteristics of lymphocytes and response to mitogens of peripheral blood mononuclear cells (PBMC) were made between these patients with PsA before CsA therapy, 7 patients without prior 2nd line therapy, 14 untreated patients with psoriasis alone, and 61 healthy controls. We confirmed a significant reduction of the basal percentage of CD8+ cells and an increase in the CD4/CD8 ratio in patients with PsA before CsA therapy compared to controls. These abnormalities were not present in patients with PsA without prior 2nd line therapy and in patients with psoriasis alone. Peripheral blood activated T cells (CD3+, HLA-DR+), natural killer (NK) (CD3-, CD16+ and/or CD56+), total B and CD5+ B cells were decreased only in patients with PsA before CsA therapy. The reduction of non-MHC restricted cytotoxicity T (CD3+, CD16+ and/or CD56+) was observed in all the 3 groups of patients compared to controls. After the 6 months of CsA therapy we observed a significant increase of CD3+, HLA-DR+, CD3+, CD16+ and/or CD56+, total B, and CD20+, CD5+ cells in the 11 patients with PsA compared to pretreatment values. Contrary to azathioprine, CsA does not impair the NK cell population which has a protective role against cancer and viral infections.


[Reconstruction of glomerular tufts--observation of mesangiolysis induced by anti-thymocyte serum]. The reconstruction process of the glomerular capillary structure following mesangiolysis induced in rats by anti-thymocyte serum (ATS) was investigated for three months. Cystic ballooning change of the glomerular tufts was remarkable four days after the administration of ATS, which was followed by inflammatory cell infiltration, and hypercellularity in mesangial area was observed one week later. Glomerular tufts were gradually reconstructed thereafter, leaving focal and segmental sclerotic or adhesive lesions. In the periphery of the ballooning area, a large vascular lumen was subdivided into smaller capillary lumina by endothelial and mesangial bridging, which resembled to the process in the fetal glomerulogenesis. The endothelial cells in mesangiolysis connected one another by junctional complexes and formed new capillary structure. The mesangial cells got in touch with the endothelial cells by mesangial bridging. In the proliferative lesion, endothelium-like cells could be recognized by immunostaining and had fenestrae with diaphragmatoid structure without mature basement membrane in the initial stage on electron microscopic observation. Interstitial-typed collagen fibers were found in the proliferative lesion, and they still existed in the focal segmental sclerotic lesion in the late stage of the experiment. The glomerular basement membrane (GBM) was seen to be reformed by fusing the original GBM with the newly synthesized one covering subendothelial space, where mesangial cells were interposed. Endothelial cells, endothelial-mesangial interaction and extracellular matrix formation seemed to play an important role in reconstruction of the glomerular tufts after mesangiolysis induced by ATS.


[The use of kontrikal in treating unstable stenocardia by specialized first-aid teams]. The paper presents the results of unstable angina pectoris treatment in 121 patients who received or did not receive (65 and 56 patients, respectively) contrykal in a dose of 20,000 units as an adjuvant to standard treatment (analgetics, tranquilizers, vasodilators). All the patients were admitted to cardiological departments. The comparative assessment of the data favours contrykal infusions in emergency prehospital care of developing unstable angina pectoris. The drug produces a cardioprotective effect reducing the risk of macrofocal myocardial infarction.


Insulin resistance, hyperinsulinemia, and coronary artery disease: a complex metabolic web. Diabetes mellitus is commonly associated with systolic and diastolic hypertension, and a wealth of epidemiological data suggest that this association is independent of age and obesity. Much evidence indicates that the link between diabetes and essential hypertension is hyperinsulinemia. Thus, when hypertensive patients, whether obese or of normal body weight, are compared with age- and weight-matched normotensive controls, a heightened plasma insulin response to a glucose challenge is found consistently. A state of cellular resistance to insulin action subtends the observed hyperinsulinism. Using the insulin/glucose clamp technique in combination with tracer glucose infusion and indirect calorimetry, it has been demonstrated that the insulin resistance of essential hypertension is located in peripheral tissues (muscle), is limited to nonoxidative pathways of glucose disposal (glycogen synthesis), and correlates directly with the severity of hypertension. The reasons for the association of insulin resistance and essential hypertension can be sought in at least four general types of mechanisms: sodium retention, sympathetic nervous system overactivity, disturbed membrane ion transport, and proliferation of vascular smooth-muscle cells. Physiological maneuvers, such as caloric restriction (in the overweight patient) and regular physical exercise, can improve tissue sensitivity to insulin; good evidence indicates that these maneuvers also can lower blood pressure in both normotensive and hypertensive individuals. Insulin resistance and hyperinsulinemia also are associated with an atherogenic plasma lipid profile. Elevated plasma insulin concentrations enhance very-low-density lipoprotein (VLDL) synthesis, leading to hypertriglyceridemia. Progressive elimination of lipid and apolipoproteins from the VLDL particle leads to an increased formation of intermediate density and low-density lipoproteins, both of which are atherogenic. Last, insulin per se, independent of its effects on blood pressure and plasma lipids, is known to be atherogenic. The hormone enhances cholesterol transport into arteriolar smooth-muscle cells and increases endogenous lipid synthesis by these cells. Insulin also stimulates the proliferation of arteriolar smooth-muscle cells, augments collagen synthesis in the vascular wall, increases the formation of and decreases the regression of lipid plaques, and stimulates the production of a variety of growth factors. In summary, insulin resistance appears to be a syndrome that is associated with a clustering of metabolic disorders, including type II diabetes mellitus, obesity, hypertension, lipid abnormalities, and atherosclerotic cardiovascular disease.


A solid phase extraction technique for the isolation and identification of opiates in urine. A quantitative method was developed for the simultaneous analysis of morphine, codeine, hydromorphone, hydrocodone, and oxycodone in urine by gas chromatography/mass spectrometry. Samples were hydrolyzed with beta-glucuronidase and then extracted by solid phase extraction on Bond Elute Certify cartridges at pH 6.8. Nalorphine was used as the internal standard. The opiates were analyzed by full-scan electron impact GC/MS after derivatization with acetic anhydride-pyridine. The standard curves for all five drugs were linear between 50 and 1000 ng/mL, with correlation coefficients exceeding 0.99. Coefficients of variation were less than 7%. The method was applied to the analysis of postmortem urines positive by EMIT opiate assay, and the effect of the hydrolysis procedure on recovery of each drug was measured. The results indicate that the hydrolysis procedure is effective in increasing the recovery of all five drugs from urine. The described method enables the laboratory to identify the five opiates most commonly encountered in forensic and clinical laboratories. Its sensitivity for all five drugs is well below GC/MS cutoffs for codeine and morphine employed in NIDA laboratories, and it provides for conclusive full-scan drug identification.


Determination of insulin sensitivity: methodological considerations. During the past decade, it became obvious that in contrast to defective insulin secretion in type I diabetes, defective insulin action (insulin resistance) is the most pertinent feature of type II diabetes. In addition, it has been known for a long time that obesity and insulin resistance are closely linked. Recently, hypertension also has been shown to often coincide with insulin resistance, although any causal relationships are still hypothetical. Last, several widely used pharmacological drugs such as diuretics, adrenergic blockers, and angiotensin-converting enzyme inhibitors may influence insulin sensitivity. Therefore, growing interest has emerged to most accurately measure insulin sensitivity. Although considerable knowledge has accumulated as to the actual mechanisms of insulin-dependent glucose transport, the signal transduction pathway of insulin remains poorly understood. When insulin sensitivity is measured, it is the overall glucose uptake that is quantified under controlled conditions. Other actions of insulin, such as the transport of ions, (e.g., sodium and potassium), synthesis of insulin-like growth factor-binding proteins, translocation of transporter proteins, and regulation of enzyme activities, are much more difficult to quantify. Of the many approaches used to quantify insulin action, the euglycemic hyperinsulinemic clamp technique has emerged as the most reliable tool, fulfilling clinical and scientific demands equally. In combination with tracer methodology and calorimetry, a detailed view into the quantitative aspects of insulin action at different target cells is possible. Whether insulin resistance extends to other known actions of insulin in addition to those on glucose metabolism remains open to debate.


What is the clinical significance of insulin resistance? Insulin resistance is a frequent phenomenon and a marker of increased risk for non-insulin-dependent diabetes mellitus (NIDDM) and atherosclerosis. According to recent estimations, not only individuals with obesity, NIDDM, and impaired glucose tolerance (IGT) but also one fourth of the "healthy" glucose tolerant and the majority of the hypertensive population are insulin resistant. Insulin resistance describes a tissue- and pathway-specific defect of glucose metabolism that is compensated for by hyperinsulinemia, leading to a cluster of undesirable hypertensiogenic, diabetogenic, and atherogenic processes. The initial defect can be directly measured by glucose clamp and other sophisticated techniques; the clinical syndrome may be derived from a network of related variables known to be associated with reduced insulin action. Because neither clamps nor serum insulin screenings will be available on a widespread basis, early diagnosis based on clinical criteria is crucial. A new interpretation of the "thrifty" genotype hypothesis may explain why insulin resistance, which formerly apparently represented an advantage in the evolutionary selection process, is such a frequent phenomenon. Improvement of impaired insulin action as a therapeutic principle may play a future central role in an integrated lifestyle approach of primary prevention of noncommunicable diseases such as NIDDM, hypertension, and atherosclerosis.


Effects of nonpharmacological intervention on insulin sensitivity. Hyperinsulinemia and insulin resistance have been implicated to play a role in the development of hypertension and to contribute to the increased risk for cardiovascular disease in diabetic, obese, hypertensive, and normotensive salt-sensitive humans. Reviewed herein are the effects of nonpharmacological measures, including exercise, weight loss, diet, and changes in lifestyle, on insulin resistance. Based on the evidence from both experimental and clinical studies, regular exercise, moderate weight reduction, and a low-fat, high-carbohydrate, high-fiber diet can markedly improve insulin sensitivity. The possible mechanisms involved are discussed. Because these nonpharmacological measures have also been shown to lower blood pressure and correct dyslipidemia, they can contribute substantially to the reduction of major cardiovascular risk factors and should be implemented in all patients who may be at risk for cardiovascular disease.


Angiotensin-converting enzyme inhibitors and insulin sensitivity: metabolic effects in hypertension, diabetes, and heart failure. Angiotensin-converting enzyme (ACE) inhibitors are established in the treatment of hypertension and heart failure; both conditions are complicated by resistance to insulin-mediated glucose disposal. The defect in essential hypertension is both tissue and pathway specific, i.e., confined to nonoxidative (glycogen synthetic) routes of intracellular glucose utilization in skeletal muscle, whereas heart failure and non-insulin-dependent diabetes mellitus (NIDDM) are associated with more widespread abnormalities of carbohydrate and lipid metabolism. Thus, the mechanisms of the insulin resistance in hypertension, NIDDM, and heart failure are fundamentally different, so metabolic responses to drug therapy may not be the same in all insulin-resistant states. There have been conflicting reports about the effects of ACE inhibitors on insulin sensitivity and glycemic control. A number of studies, both with captopril and with enalapril, have shown small increases in insulin sensitivity, and there is evidence that this is due to enhanced glucose uptake into skeletal muscle. The interpretation of these studies, however, is often compromised by poor trial design, lack of full placebo data, various indirect measurements of insulin sensitivity, and heterogeneous patient populations in whom the biochemical mechanisms of insulin resistance (and drug responses) may not be the same. Overall, there probably is a modest class effect of ACE inhibitors that enhances insulin-mediated glucose disposal; the mechanism of this effect is likely to be a combination of increased muscle blood flow, local renin-angiotensin system blockade, and elevated kinin levels.


Effects of antihypertensive treatment with beta-blockers on glucose metabolism. The beneficial effects of beta-blockers on morbidity and mortality in both essential hypertension and diabetes mellitus have been demonstrated in several antihypertensive trial. Recently, negative side effects of beta-blockers on glucose metabolism have been suggested that could limit their wide-spread use. However, on detailed analysis, any side effects of beta 1-selective beta-blockers on glucose metabolism do not appear to be relevant for clinical practice and hence do not restrict the use of these agents as first-line antihypertensive drugs in diabetic and nondiabetic patients.


Influence of diuretics, calcium antagonists, and alpha-blockers on insulin sensitivity and glucose tolerance in hypertensive patients. Treatment with thiazide diuretics causes impaired glucose tolerance, biochemical diabetes, and insulin resistance. The effect of diuretics on glucose tolerance is clearly dose-related. Spironolactone does not impair glucose tolerance, even at high dosage, but differences among other diuretics could be due to comparisons at doses that are not equal. Diuretic-induced changes in glucose metabolism are not conclusively related to altered potassium homeostasis, and impaired glucose tolerance occurs even when relatively low doses of thiazide are combined with potassium-sparing agents. The effects of diuretics on glucose homeostasis are in large part and probably entirely reversible. These disturbances of glucose metabolism have been detected only by detailed biochemical testing, and their clinical relevance is uncertain. In established diabetes, diuretics have a rapid and substantial adverse effect on metabolic control. In nondiabetic subjects, diuretics rarely cause or trigger a serious hyperosmolar nonketotic diabetic syndrome. Otherwise, it is not known whether the metabolic changes cause clinical diabetes or lead to microvascular complications in the long term. Evidence from large outcome trials suggests that biochemical diabetes, glucose intolerance, and insulin resistance do not increase the risk of coronary heart disease in treated hypertensive patients. Diuretics should be avoided in patients with diabetes unless their use is essential. Otherwise, a low dose of thiazide remains as excellent choice for first-line antihypertensive therapy. Dihydropyridine calcium antagonists, diltiazem, and verapamil appear to have no important effects on glucose homeostasis. There is very limited evidence that selective alpha-antagonists increase insulin sensitivity. The importance of metabolic differences between drug classes will be established only by comparative outcome trials with coronary events as the end point.


Beta-blockers: primary and secondary prevention. Coronary heart disease is the most frequent cause of death in Western, industrialized countries. Coronary risk factors are prevalent in such countries and sometimes combine to constitute the so-called syndrome X--hypertension, central obesity, serum lipid and clotting disturbances, and insulin resistance. beta-Blockers, unlike calcium antagonists, have proved highly effective in secondary prevention of myocardial infarction. If present at the time of the myocardial infarction, beta-blockers (unlike calcium antagonists and diuretics) probably decrease mortality 1 month later. Early intervention (within 12 h) of chest pain with intravenous beta-blockers results in a 15% reduction in cardiovascular mortality at 1 week. Later intervention (3-28 days) with oral non-ISA beta-blockers results in a 30% reduction in mortality after 1 year; ISA-containing beta-blockers are probably less effective (less decrease in heart rate). Hydrophilicity/lipophilicity of beta-blockers is unimportant in terms of decreased mortality. Primary prevention of myocardial infarction, unlike stroke, in hypertensive patients has been disappointing, possibly due to treatment-induced biochemical/lipid changes or inappropriate lowering of diastolic blood pressure in high-risk subjects (J-curve effect). beta-Blockers should be first-line therapy for hypertensive patients up to the age of 65 years, particularly men (and nonsmokers) as Q-wave myocardial infarction is significantly decreased by beta-blockers and significantly increased by diuretics. However, in elderly hypertensive subjects, beta-blockers have not significantly decreased myocardial infarction (unlike stroke), whereas diuretics have. The effects of beta-blockers and diuretics on heart size (and thus coronary flow reserve) in the elderly may be important. Thus, beta-blockers should be second-line therapy for the elderly hypertensive individual but first-line if overt ischemia (e.g., angina or recent myocardial infarction) also is present. In patients with angina but normal blood pressure, beta-blockers tend to decrease and calcium antagonists increase cardiovascular events. Thus, beta-blockers are highly effective agents in the secondary prevention of myocardial infarction and are moderately effective in primary prevention of myocardial infarction in hypertensive patients (particularly men) under the age of 65 years.


New antihypertensive agents: benefit of treatment. Large-scale end-point trials have demonstrated that antihypertensive treatment reverses the risk of stroke attributable to high blood pressure and probably reduces the incidence of myocardial infarction. Despite this major achievement in therapeutics, substantial goals still need to be achieved. Thus, there is a need for improvement in existing antihypertensive agents in terms of greater blood pressure-lowering efficacy and improved impact on coronary heart disease in younger subjects. There is also a pressing need for a reduction in adverse effects. The incidence of these far exceeds the potential benefit of treatment. Last, the costs of therapy are assuming growing importance in the face of tight financial constraints on health care systems throughout the world.


Hypertension and insulin resistance.

Hypertension and insulin resistance. Insulin resistance and hyperinsulinemia is now recognized in non-insulin-dependent diabetes, essential hypertension, obesity, atherosclerotic heart disease, dyslipidemia, heart failure, and in heavy smokers. Several mechanisms have been proposed to explain hyperinsulinemia, insulin resistance and its relationship to hypertension; reduced sodium excretion, activation of the sympathetic nervous system, increased activity of the sodium/hydrogen pump, and stimulation of cellular growth. Some of the nonpharmacological methods to control hyperinsulinemia are of benefit in the management of hypertension, most notably weight loss, exercise program, and reduced salt intake. High-fiber and reduced-protein diets also reduce hyperinsulinemia. Thiazide diuretics can result in insulin resistance, and insulin secretion may be inhibited, possibly associated with concomitant hypokalemia. beta-Blockers result in some reduction of glucose tolerance and mask some of the features of hypoglycemia. Angiotensin-converting enzyme (ACE) inhibitors and alpha-receptor blockers do not effect insulin resistance; probably the same is true for calcium antagonists. Although the effect on risk factors should not be discounted, it is the effect of treatment on hard end points, cerebrovascular accidents, myocardial infarction, or death that is most important. Evidence in hypertension is at present restricted to diuretics and beta-blocking drugs.


Gallopamil: cardiovascular scope of action of a highly specific calcium antagonist. According to Fleckenstein's original classification, gallopamil represents a prototype of highly specific calcium antagonists of Group A. Its large scope of cardiovascular effects is lastly based on one and the same mechanism of action: inhibition of transmembrane calcium influx into myocardial, cardiac pacemaker, and vascular smooth muscle cells. Thereby, in experimental studies that use electrophysiological, biochemical, isotopic, and histological techniques, gallopamil reduced calcium-dependent myocardial contractility and oxygen consumption; dampened nomotopic and ectopic cardiac pacemaker activity; exerted pronounced vasodilator effects; and protected myocardial cells from calcium overload-induced necrotization. The effects of gallopamil on myocardium, cardiac pacemakers, and vasculature are quantitatively comparable, surpassing those of verapamil by approximately one order of magnitude. Further clinical studies must demonstrate to what extent humans will benefit from the experimentally proven cardiovascular potential of gallopamil.


Effect of gallopamil on cardiac sarcoplasmic reticulum. We investigated the effect of gallopamil on cardiac sarcoplasmic reticulum (SR) function. Heavy SR was prepared from bovine ventricular muscle. Oxalate-supported calcium uptake was stimulated by gallopamil at concentrations ranging from 10 to 300 nM, whereas higher concentrations were ineffective. Peak stimulation averaged 25-30% of control calcium uptake and was observed at free calcium concentrations ranging from 1 to 6 microM. Calcium uptake is actually the difference between active calcium transport by SR calcium-adenosine triphosphate (calcium-ATPase), and passive efflux through SR calcium-release channels. In the presence of 300 microM of ryanodine, a blocker of SR channels, calcium uptake increased by 43% under control conditions, but not further stimulation was produced by gallopamil. SR calcium-ATPase was not affected by gallopamil. Similar results were obtained when oxalate-supported calcium uptake was determined with use of unfractionated homogenate obtained from rat hearts. We conclude that gallopamil acts on SR calcium-release channels and reduces the probability of channel opening and/or channel conductivity. The dose-response curve is bell shaped, and the maximum effect, which corresponds to 65% of the maximum effect of ryanodine, is achieved at therapeutic concentrations. Such action might contribute to the beneficial effect of gallopamil in the treatment of myocardial ischemia.


Calcium antagonism and norepinephrine release in myocardial ischemia. In myocardial infarction, adrenergic stimulation of the heart is thought to cause cell damage and malignant arrhythmias. In rat hearts as well as in human cardiac tissue, ischemia induces norepinephrine (NE) release, which results in micromolar catecholamine concentrations in the interstitial space of the ischemic myocardium. It has been found that local metabolic, rather than centrally evoked NE release, plays the crucial role in excess adrenergic activation of the ischemic myocardium. NE release in ischemia is nonexocytotic and has been characterized as a two-step process. (a) Induced by energy deficiency, NE escapes from its storage vesicles and accumulates in the axoplasm. (b) NE is transported across the plasma membrane into the extracellular space via the neuronal NE carrier (uptake1), which has reversed its normal transport direction because of increased intracellular sodium concentration. NE release induced by ischemia is independent of the presence of calcium in the extracellular space and is not altered by blockade of N-type (neuronal) calcium channels. Furthermore, modulation of protein kinase C does not interfere with NE liberation in the ischemic myocardium. This independence of extracellular calcium, calcium entry into the neuron, and protein kinase C activity is in contrast to the strong calcium dependence of exocytotic transmitter release, which is found under physiological conditions. On the basis of these findings, it was unexpected that calcium antagonists such as gallopamil, verapamil, diltiazem, felodipine, and nifedipine suppress ischemia-induced NE release. The most potent effect was found for gallopamil with a concentration of 50% inhibition (IC50) of 300 nmol/L.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of gallopamil on neutrophil function: experimental and clinical studies. The purpose of the study was to investigate whether gallopamil interferes with neutrophil activation. In vitro, gallopamil caused a dose-dependent reduction in phorbol myristate acetate-stimulated superoxide anion production by neutrophils as measured by the superoxide dismutase inhibited reduction of cytochrome C [concentration of 50% inhibition (IC50) = 9.5 x 10(-6) mol/L]. Furthermore, gallopamil reduced the platelet-activating factor-induced loss of neutrophil deformation, which was assessed by filtrometry (IC50 = 4.3 x 10(-6) mol/L). The effect of gallopamil was assessed in 24 patients during elective balloon angioplasty. Gallopamil (0.4 mg) or placebo (double-blind conditions) was administered by intracoronary application during the 10-min interval between the first two balloon inflations. In the placebo group, blood samples obtained simultaneously from the coronary sinus and from the femoral artery revealed an increase in the proportion of activated neutrophils in the coronary sinus blood after the second balloon inflation [nitro blue tetrazolium (NBT) score, NBT test: 15 +/- 9% relative coronary sinus and arterial blood difference, p < 0.05]; these changes in NBT score were similar to those after the first balloon inflation. In the gallopamil-treated group, however, significant arterial and coronary sinus blood differences in NBT scores were not found after the second balloon inflation. Gallopamil may, thus, attenuate the local neutrophil activation during balloon angioplasty.


Calcium antagonists and experimental myocardial ischemia reperfusion injury. A number of previous studies have reported that administration of calcium antagonists reduces acute ischemic injury in a variety of experimental models. More recently, however, it has been suggested that the beneficial effects of reperfusing the ischemic myocardium might be blunted by the paradoxical occurrence of a specific form of reperfusion-mediated injury. Although the ultimate mechanisms responsible for this phenomenon have not been completely elucidated, it has been suggested that oxygen radical generation, neutrophil activation, and calcium overload, may all contribute to the development of myocardial damage in postischemic hearts. Several experimental studies suggest that, in addition to their well-known effects on ischemic injury, calcium antagonists may variably affect these mechanisms of reperfusion-mediated cell damage. In particular, evidence has been provided that suggests these drugs may inhibit oxygen radical-mediated peroxidation of membrane lipids and may also reduce activation of stimulated neutrophils. Furthermore, calcium-channel blockers might also prevent calcium overload in reperfused hearts, and they might interfere with reperfusion injury indirectly, secondary to a reduction in the severity of ischemia. From the experimental data available it can be speculated that calcium antagonists might contribute to reducing oxygen radical damage following reflow. At the same time, these drugs may allow the extension of the time window of reperfusion therapy, thus further expanding the benefits of thrombolysis.


Abnormal coronary vasomotion in ischemic heart disease. Myocardial ischemia has long been thought to be caused by only critical arteriosclerotic plaques, which are the most obvious single denominators of ischemic cardiac syndromes. The author argues that just because other transient and dynamic mechanisms are more elusive than atherosclerotic plaques, we should not presume them to be unimportant. He reviews the work of his group and others, which suggests that dilation and constriction can occur at the site of coronary artery stenoses and in distal coronary vessels. Clinical and angiographic findings and the possible pathophysiological causes of vasomotor dysfunction that may modulate residual coronary flow in patients with chronic stable, unstable, and variant angina, and syndrome X are reviewed. Only when these mechanisms are more fully understood will it be possible to develop specific therapy.


Intracoronary gallopamil during percutaneous transluminal coronary angioplasty. The present study was designed to investigate the effect of the calcium-channel antagonist gallopamil on myocardial ischemia during percutaneous transluminal coronary angioplasty (PTCA). Twenty-four adult patients with coronary artery disease and significant proximal stenosis of the left anterior descending coronary artery (LAD) were randomly assigned to receive gallopamil or placebo under double-blind conditions. Patients with recent myocardial infarction, apparent collateralization of the LAD, myocardial failure, sinoatrial or atrioventricular block, severe hepatic disease, or renal failure were excluded from the study. PTCA was performed with use of at least two balloon inflations, each of 2 min in duration. Gallopamil (0.4 mg) or placebo (0.9% sodium chloride) was administered during the 10-min interval between the two inflations. For determination of myocardial lactate and hypoxanthine release, blood samples were taken simultaneously from the great cardiac vein and the femoral artery before and immediately after each inflation. Electrocardiogram changes were analyzed by measuring ST-segment deviations (80 ms after the J point) and maximal T-wave deviations of the leads I, II, III, and V2, V4, and V6. The most sensitive leads for identification of myocardial ischemia in the LAD area were V2 and V4. If compared to the first balloon inflation, the degree of ST-segment/T-wave changes induced by the second inflation was significantly reduced only in the presence of gallopamil. Furthermore, if compared to placebo, ischemia-induced lactate and hypoxanthine release was decreased in the presence of gallopamil. These results suggest that intracoronary application of gallopamil attenuates myocardial ischemia during PTCA.