Natural killer cell activity against cultured melanoma cells: a dye-reduction technique with studies on augmented activity by interferon subtypes. We assessed natural killer (NK) cell activity against cultured melanoma cells using a novel method, with observations on the comparative effects of interferons (IFNs) in NK-stimulating and anti-proliferative assays. Since the tumour cells tested were adherent, a semi-automated colorimetric MTT dye reduction assay was developed to assess NK activity. The three adherent human melanoma cell lines Sk-Mel-28, MM418 and MM96 were shown to be suitable targets for determining NK activity. Also, these were representative of the range of sensitivities of melanoma cells to the anti-proliferative action of type 1 IFNs. The dose-dependent stimulation of NK activity by type 1 IFNs was confirmed in this alternative assay system. IFN-alpha 2b and IFN-beta ser had equivalent stimulatory activities, and IFN-alpha 4 proved less effective, as with assays using the classical K562 system. Augmented NK cytotoxicity did not correlate with anti-proliferative effects of IFN. In anti-proliferative assays, the hierarchy of activity is IFN-beta greater than IFN-alpha 2 greater than IFN-alpha 4, whereas, in the NK augmentation assay IFN-beta and IFN-alpha 2 were of equivalent activity. Interestingly, MM96 was the cell line most resistant to the direct anti-proliferative action of IFN, yet it was the most susceptible of the melanoma cell lines to cytotoxicity by NK cells, whether stimulated or unstimulated by IFN.


A brief review of Croatian medical history until the 19 century--Part 1. The purpose of this study is to remind the contemporary readers of the role and contribution of Croatian medical heritage in the development of medicine in Europe and the World, as well as of the development of medical concepts and medical practice on the territory of what is Croatia today. Selected examples are presented of the famous events and notable persons from the ancient time until the middle of the 19th century.


Protective effect of MDP-Lys(L18), a synthetic derivative of muramyldipeptide, on murine cytomegalovirus infection. The host-mediated antiviral effect of N alpha-(N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N epsilon-stearoyl-L-lysine [MDP-Lys(L18)] was evaluated in mice against murine cytomegalovirus (MCMV) infection. Mice treated with 800 micrograms of MDP-Lys(L18) on day 2 before the virus challenge survived systemic lethal infection. The protective effect of MDP-Lys(L18) was evidenced by an increase in plaque-forming units per LD50 and a decrease in virus titers in the target organs. No significant difference in the serum interferon level and 2',5'-oligoadenylate synthetase activity was observed among the mice treated with test compounds or phosphate-buffered saline, thought they were higher in MCMV-infected mice than in mock-infected mice. The natural killer (NK) activity was augmented remarkably in the mice treated with MDP-Lys(L18) or its original component, muramyldipeptide (MDP). MDP-Lys(L18) showed neither virocidal nor virostatic activity on MCMV in vitro. Thus, MDP-Lys(L18)-induced resistance against MCMV infection seems to be host-mediated. The MDP-Lys(L18)-induced resistance was not abrogated by the treatment with anti-asialo GM1 serum. The NK activity augmented by MDP-Lys(L18) may contribute to some part of the protective effect, though the augmentation of the NK activity alone did not correlate completely with the protective effect of MDP-Lys(L18). In addition, no difference was observed in anti-MCMV activity among peritoneal exudate cells from mice treated with MDP-Lys(L18), MDP or phosphate-buffered saline. Therefore, another host-mediated factor(s) may also participate in the antiviral effect of MDP-Lys(L18).


The natural history of mild idiopathic scoliosis. The aim of the research was to monitor the natural history of mild idiopathic scoliosis in school children, primarily those with a scoliotic curve lesser than 20 degrees according to Cobb who had been treated by regular observation and sports activities. A total of 97 children, 73 girls and 24 boys, aged between 9 and 14 years were enrolled. All the children were monitored for three years and in this period they were subjected to regular examinations and to at least three X-ray examinations. There were 51 thoracal, 39 thoracolumbar and 7 lumbar cases of scoliosis. After the three year observation period thoracic scoliosis improved in 56.9% of patients, 17.7% showed no change while 25.5% of the cases deteriorated. Nonprogressive thoracolumbar scoliosis was found in 76.9% of the cases, 12.9% showed no change from baseline while in 10.3% curve progression was noticed. Nonprogressive lumbar scoliosis was observed in 4 cases, in 2 cases the condition was unaltered while in 1 case curve progression was noticed. In the whole group there were 18.6% cases of progressive scoliosis. The results of this research have confirmed that regular observation and sports activities are the best methods of treating idiopathic scoliosis with a curve lesser than 20 degrees according to Cobb.


Morphological cephalometric differences between two European populations. Earlier research in craniofacial morphology of the inhabitants of Mainz (Germany) and Zagreb (Croatia), who represent the populations of wider areas, have shown some roentgen-cephalometric differences. The aim of this study is to determine the craniofacial morphologic differences of these populations in three dimensions. The sample consists of 200 adult examinees, male and female respectively, inhabiting the mentioned areas. The sample has been chosen so that the examinees with prominent craniofacial anomalies have been excluded. Twenty three craniofacial parameters with the usual anthropometric instruments have been measured. The data have been processed by using elementary descriptive statistics and discriminant analysis. The results have shown that in Mainz examinees longer and narrower heads and higher faces in comparison with Zagreb examinees, predominate. Significant differences in forehead widths and bipupilar distances have been found.


Changes in coagulability of blood in patients with cerebrovascular disease in various meteorological situations. The authors are dealing with the influence of meteorological factors on vascular diseases, primarily on patients with cerebro-vascular disturbances. The parameters mentioned in some studies are also presented in the introduction. There are still few studies by means of which it can be enlightened what happens in the human body affected by atmospheric disturbances. Therefore the basic intention of this study was to come up with answers to at least some segments of this complex problem. A group of patients with cerebrovascular symptoms and signs was chosen for examination. Tests were carried out during cold and warm front passages in anticyclonal situations and low gradient pressure fields within 47 days. In the first stage the tests were carried out only in one day and later for three consecutive days. Meteorological reports are given too. As for medical parameters, the following were controlled: the neurology status, blood pressure, blood test, ECG, REG. The coagulum was regularly checked. The examination was carried out in 12 male and 14 female patients, the age range 43-78. On the basis of the tests, the authors concluded that the most significant changes were noticed in the coagulum and thromboelastogram. According to the results, these changes are manifested during the cold front and during the cold spell caused by it. The findings point to a significant increasing of the thrombocyte aggregation and partly the aggregation index, which can be clinically manifested as the patient's health aggravation. Although other parameters of coagulation did not show significant changes, it cannot be deduced whether they are susceptible to the circumstances in the atmosphere or not.(ABSTRACT TRUNCATED AT 250 WORDS)


An approximation of stenotic aortic valve area by phonomechanocardiographic method: the comparative study of noninvasive methods. Phonomechanocardiographic and ultrasonocardiographic parameters were compared in the multiplex manner in order to assess a degree of narrowing of the stenotic aortic valve areas. Adult patients with aortic valve stenosis were included in the study. The main condition for admission in the research sample was that the mean rate of circumferential fibre shortening be greater than 1 s-1 i.e. "compensated" preejection period/ejection time ratio (PEP/LVET). The control group were persons as sample stratified from healthy population. A possibility of approximate assessment of valve areas in patients with aortic stenosis is rendered by inserting the phonomechanocardiographic parameters in the modified Gorlin and Gorlin formula, provided that values of the normalised ejection function index (PEP/LVET2) and the ejection-isovolumetric coefficient corrected for pulse transmission time (LVET/IVCT+PTT) are known. The phonomechanocardiographic indexes of the transvalvular aortic pressure gradient and normalised stroke volume correlate curvilinear. The value of the LVET/IVCT + PTT equal or greater than that extrapolated for the given PEP/LVET2 in our formula means critically stenotic aortic valve area below 0.8 cm2. The given approximation could be used as a noninvasive and nongeometric polycardiographic or phonomechanocardiographic pattern for assessing the degree of narrowing of aortic valve area. The aortic valve stenosis is an illness in which a lot is expected from noninvasive cardiologic parameters when a surgical indication is in question. A severe or tight aortic valve stenosis, which required a surgical treatment according to current views, existed when valve area is less than 0.8 cm2 or when the transvalvular aortic systolic pressure gradient is greater than 50 mm Hg or 6.67 kPa, but with normal cardiac index in the same time.(ABSTRACT TRUNCATED AT 250 WORDS)


[The effect of 6% HES 200/0.6-0.66 on plasma volume and blood coagulation]. The main goal of the recent study was to evaluate changes in plasma volume due to the application of 6% HES 200/0.6-0.66. 12 patients according to the ASA physical status classification (I, II) undergoing minor surgical interventions received 500 ml of this artificial plasma substitute within 30 min. In a control group (n = 12), 500 ml of lactated Ringer's solution was given within the same period. A further question of the present investigation was the possible influence of 6% HES on coagulation during the following period (1st-3rd postoperative days). 6% HES 200/0.6-0.66 led to an additional augmentation of plasma volume measured via the mechanical oscillator technique of 200 ml (40% of the volume given) immediately at the end of infusion. A second increase in plasma volume of 100 ml (20% of the volume infused) could be observed 1 h later. With exception of the activity of factor VIII, the coagulation parameters had not been altered by infusion of 6% HES. The activity of factor VIII decreased to about 50% of the control level but showed a tendency to normalization within the following observation period. 6% HES 200/0.6-0.66 has a marked volume-expanding effect and exerts no influence on coagulation except a temporary decrease of factor VIII activity.


Incorporation of radiophosphorus from labeled oligodeoxynucleotides into RNA of mycoplasma in cell cultures. We have found that various mycoplasma species quickly and efficiently incorporate radiophosphorus into their RNA from labeled oligonucleotides added to the medium. The label can be in any of several positions in an oligodeoxynucleotide, and incorporation also occurs efficiently from labeled RNA. Mycoplasmas also incorporate the radiolabel when they infect a mammalian cell culture; the host cells do not. This incorporation presumably involves uptake of the oligodeoxynucleotide followed by digestion to mononucleotides, conversion to ribonucleotides, and incorporation in new RNA. We believe that the processing of oligodeoxynucleotides by mycoplasma could be a source of artifacts in antisense work in cell culture and could have implications for the development of antisense therapeutics. We also suggest ways to exploit the incorporation phenomenon in mycoplasma testing.


Motors for fast axonal transport.

Motors for fast axonal transport. In neurons and other animal cells, membrane-bound vesicles course rapidly along cytoskeletal filaments to reach their destinations. Based on a variety of in vivo studies it is becoming clear that the microtubule-based motor, kinesin (and its relatives), drive vesicle movements in axons. Surprisingly, some axonal membranes have the capacity to move on both microtubules and actin filaments.


Identification of solvent-exposed regions of an FK-506 analog, ascomycin, bound to FKBP using a paramagnetic probe. The solvent-exposed regions of (U-13C)ascomycin when bound to its putative target protein, FKBP, have been identified based on the different proton longitudinal relaxation rates (R1 = 1/T1) measured in the absence and presence of the paramagnetic relaxation reagent, 4-hydroxy-2,2,6,6-tetramethyl-piperidinyl-1-oxy (HyTEMPO). The proton T1S of bound ascomycin were determined using a pulse sequence (T1-HMQC) which consists of a 180 degree proton pulse and a variable delay (tau) followed by a heteronuclear multiple quantum correlation (HMQC) experiment. The solvent-exposed regions of ascomycin determined by these experiments are compared to NOE data in which ascomycin/FKBP contacts were identified and to the X-ray structure of the FK-506/FKBP complex.


NMR spectroscopy of hydroxyl protons in aqueous solutions of peptides and proteins. Hydroxyl groups of serine and threonine, and to some extent also tyrosine are usually located on or near the surface of proteins. NMR observations of the hydroxyl protons is therefore of interest to support investigations of the protein surface in solution, and knowledge of the hydroxyl NMR lines is indispensable as a reference for studies of protein hydration in solution. In this paper, solvent suppression schemes recently developed for observation of hydration water resonances were used to observe hydroxyl protons of serine, threonine and tyrosine in aqueous solutions of small model peptides and the protein basic pancreatic trypsin inhibitor (BPTI). The chemical shifts of the hydroxyl protons of serine and threonine were found to be between 5.4 and 6.2 ppm, with random-coil shifts at 4 degrees C of 5.92 ppm and 5.88 ppm, respectively, and those of tyrosine between 9.6 and 10.1 ppm, with a random-coil shift of 9.78 ppm. Since these spectral regions are virtually free of other polypeptide 1H NMR signals, cross peaks with the hydroxyl protons are usually well separated even in homonuclear two-dimensional 1H NMR spectra. To illustrate the practical use of hydroxyl proton NMR in polypeptides, the conformations of the side-chain hydroxyl groups in BPTI were characterized by measurements of nuclear Overhauser effects and scalar coupling constants involving the hydroxyl protons. In addition, hydroxyl proton exchange rates were measured as a function of pH, where simple first-order rate processes were observed for both acid- and base-catalysed exchange of all but one of the hydroxyl-bearing residues in BPTI. For the conformations of the individual Ser, Thr and Tyr side chains characterized in the solution structure with the use of hydroxyl proton NMR, both exact coincidence and significant differences relative to the corresponding BPTI crystal structure data were observed.


Reduction of CSF monoamine metabolites in poststroke depression: a preliminary report. Monoamine metabolites were measured in the cerebrospinal fluid (CSF) of depressed and nondepressed patients with acute stroke lesions and in nondepressed patients without stroke lesions. Depressed stroke patients had a significantly lower concentration of CSF 5-hydroxyindoleacetic acid (5-HIAA; a serotonin metabolite) than the other two groups. These findings suggest that poststroke depression may be mediated by serotonergic mechanisms.


Effects of elevated zinc intake on the copper metabolism and the pancreas of the mouse. The effects of moderately elevated zinc (Zn) intake on copper (Cu) metabolism and on the pancreas were studied. Zn (80 mg/L) as acetate was added to the drinking water of male Swiss mice for up to 12 weeks, which increased the total daily Zn intake to about 5 times the adequate level. Total Cu and Zn in tissues was measured by atomic absorption spectrometry. 64Cu was used to measure some effects of the Zn suppletion on the Cu metabolism. Furthermore, ceruloplasmin and amylase in plasma and superoxide dismutase in erythrocytes were measured. The pancreas was examined by light microscopy. The Zn supplementation decreased the 64Cu absorption and increased the retention of 64Cu, but did not lead to a Cu deficiency. Microscopic examination of the pancreas revealed focal hypertrophy of acinar cells, occasionally accompanied by vacuolation of cytoplasm and/or the presence of degenerated cells. The results, obtained in mice given a moderately increased Zn intake, should be interpreted as a warning against the chronic use in man of high doses of Zn for non-medical purposes.


Configuration, in serial reconstruction, of individual axons projecting from area V2 to V4 in the macaque monkey. The detailed morphology of long extrinsically projecting axons in the neocortex has been difficult to investigate and is in fact poorly understood. Some data, based on extracellular injections of Phaseolus vulgaris leucoagglutinin (PHA-L), are available for individual axons projecting from area V1 to area V2 or MT. Like geniculocortical projections, axons projecting from area V1 to area MT are readily identifiable (they typically have a bistratified termination pattern and large terminal specializations and are of large caliber), but those projecting from area V1 to V2 are more variable. To provide a broader basis for interpreting constant and variable features of axon morphology, we used high-resolution serial section reconstruction to analyze small populations of PHA-L-labeled axons projecting from area V2 to V4. Reconstruction of 20 axons suggests that this system is variable in terms of overall configuration and laminar distribution. Most terminal arbors are located at the border between layers 3 and 4, but some remain entirely within layer 3 or 4, some target preferentially the superficial layers (1, 2, and 3A), and some have collaterals in layer 5 or, rarely, layer 6. Arbor size is fairly constant among the three visual cortical projections examined so far (typically about 200 microns in diameter). In area V4, however, axons frequently have three or four separate arbors, which branch divergently (in one instance, over 2.6 mm x 3.0 mm). These features may be correlated with aspects of the particular functional organization of area V4, such as coarse topography, large receptive field size, and modularity. Axonal variability may also denote differences, morphological or physiological, among neurons of origin in area V2.


On the engineering of rDNA proteins for purification by immobilized metal affinity chromatography: applications to alternating histidine-containing chimeric proteins from recombinant Escherichia coli. Recently we reported (D. B. Evans, W. G. Tarpley, and S. K. Sharma, 1991, Protein Expression Purif. 2, 205-213) the cloning, expression, and characterization of recombinant chimeric proteins with an N-terminal metal-binding peptide (mbp), His-Asp-His-Asp-His, and a renin cleavage site. Using these chimerics as examples, we describe here the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography (IMAC). In these chimerics, an alternate histidine-containing peptide was fused to the N-termini of HIV reverse transcriptase (HIV RT) and beta-galactosidase. These chimerics were retarded on immobilized nickel very strongly and could be completely eluted only by the use of 100 mM imidazole, whereas the wildtype HIV RT and Escherichia coli contaminating proteins were eluted between 10 and 35 mM imidazole. When the DNA coding for the mbp was removed, the resulting chimerics were recovered from the IMAC column at 35 mM imidazole. The strong and specific interaction between the chimeric protein and the immobilized metal ion was also abolished when the mbp was specifically cleaved by human renin. It is concluded from these studies that tailoring recombinant proteins with three or more alternate histidines should result in the isolation of such chimeric proteins from crude mixtures in a single step. Since IMAC is amendable to scale up, the tailored specificity engineered into the protein of interest via an mbp should allow one to achieve large-scale isolation of recombinant proteins from bacterial and nonbacterial hosts in a highly predictable manner.


Purification of recombinant antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein antigen using the expression system pH6EX3 followed by metal chelating affinity chromatography. A novel plasmid expression vector (pH6EX3) that directs the synthesis of a fusion protein with a histidine hexapeptide at its N-terminus and a foreign protein at its C-terminus was constructed. The fusion gene is controlled by a strong tac promoter, leading to high-level expression of recombinant protein in several bacterial strains; the protein is deposited mainly as an insoluble mass in inclusion bodies. The fusion protein can be purified from the insoluble cell fraction by one-step affinity chromatography based on the selective interaction between the histidine hexapeptide and a metal chelating matrix charged with Ni2+ ions. The principle of this new system was tested by expressing and purifying antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein autoantigen. With the use of column chromatography and pH gradient elution, about 25 micrograms recombinant protein/ml of bacterial culture was obtained.


Higher specific activity of the Escherichia coli glutamyl-tRNA synthetase purified to homogeneity by a six-hour procedure. The glutamyl-tRNA synthetase (EC 6.1.1.17) of Escherichia coli was purified to homogeneity from the overproducing strain DH5 alpha(pLQ7612) by a two-step procedure that takes only about 6 h and yields 10 mg of enzyme per gram of wet cells. The process consists of a two-phase polyethylene glycol-dextran partition, the top phase of which is diluted and directly applied to an anion-exchange FPLC MonoQ column. The purified enzyme has a specific activity about twice that of the same enzyme purified to homogeneity by the lengthy conventional procedure from either a normal strain or this overproducing strain. This difference is discussed in relation to the generation of microheterogeneity in proteins during their purification.


Resolution of microheterogeneity associated with recombinant HIV-1 heterodimeric reverse transcriptase. HIV-1 reverse transcriptase (RT) has been successfully expressed as a biologically active recombinant protein in Escherichia coli and purified to homogeneity. After partial purification, RT was obtained primarily in a heterodimeric form represented by two subunits of 66 and 51 kDa, but the preparation also included several forms distinguishable in size and charge by chromatography on ionic-exchange and gel-filtration columns. We have developed a purification method that yields a single heterodimeric form of RT. Our strategy involves the selection of RT molecules exhibiting uniformity in elution from QAE Sepharose anion-exchange columns and Superose 12 gel-filtration columns. In the former, RT is resolved into multiple peaks on the basis of enzymatic activity, one of which represents highly active and pure p66:p51 heterodimeric RT. This highly active RT fraction, after gel-filtration chromatography, yields a compositionally pure protein product free of observable microheterogeneity by 1D and 2D polyacrylamide gel electrophoresis under a variety of conditions. Furthermore, the RNAse H enzymatic activity associated with HIV-1 RT has been demonstrated to coelute with the purified polymerase activity during gel filtration at a size (120 kDa) consistent with its location on the heterodimeric protein molecule.


Comparative analysis of native and cysteine-deficient HIV-1 reverse transcriptase. To study the subunit structure and the active site of human immunodeficiency virus reverse transcriptase (RT), the enzyme was expressed in E. coli and purified to homogeneity in large quantities. The recombinant enzyme consists of two major polypeptides of 66,000 and 53,000 Da in equimolar amounts and a minor species of 51,000 Da. Amino acid sequence analysis of the recombinant proteins revealed that the amino termini of the two major subunits are identical to that of the virion-derived enzyme. The two cysteinyl residues at positions 38 and 280 in the RT amino acid sequence were replaced by alanine in an attempt to elucidate the role of the sulfhydryl groups in RT enzyme activities, heterodimer formation, and intrasubunit linkage. The results reported here show that the two cysteinyls are dispensable and their absence in the amino acid sequence of the reverse transcriptase does not affect DNA polymerase or ribonuclease H enzyme activities or the formation of heterodimer structures. Furthermore, inhibitors of polymerase activity such as 3-azidothymidine triphosphate, dideoxythymidine triphosphate, and tetrahydroimidazo[4,5,1-JK][1,4]benzodiazepens (1H)-one are equally effective on the mutant containing no cysteinyl residues and the wild-type enzyme.


Role of interferons in the therapy of melanoma. The increased incidence of disease, the relative unresponsiveness of advanced tumour to conventional therapies, and high socioeconomic costs make the malignant melanoma an aggressive cancer. During the last decade, several new biological agents have been developed, some of which have shown significant activity in the treatment of disease. However, the impact on the management of melanoma patients is still far from being conclusive. Among biological response modifiers (BRMs), interferons (IFNs) have generated a great deal of interest and have been extensively employed, although incorrectly. IFNs have been used without a specific rationale and at antiproliferative rather than biologically active doses; no extensive laboratory monitoring has been performed. In this paper data available in the current literature are reviewed and the efficacies of the different IFNs, used alone or in combination and in various treatment regimens, are compared in order to understand what is the place of IFNs in the management of patients with metastatic melanoma. Results are encouraging but still disappointing with the most effective treatment, with an overall response rate of 28.5% (10.5% complete responses). However, these results need confirmation. In conclusion, IFN is effective in the therapy of advanced melanoma, but improved response rates are necessary before it may be suitable for general, rather than investigative, use. Alternative biotherapeutical approaches and strategies are suggested.


Reversible depigmentation of human melanoma cells by halistanol trisulphate, a novel marine sterol. The pigmented human melanoma cell line, MM418, became demelanized when treated continuously with a nontoxic level of halistanol trisulphate (HTS), a C29 steroidal detergent isolated from a marine sponge. Nontoxic levels of halistanol or of a range of anionic, cationic and neutral detergents had no such effect. Control MM418 cells varied greatly in size, appearance and pigmentation; HTS-treated cells were smaller than controls, had a uniform, generally bipolar appearance, and lacked pigment. HTS induced only minor changes in cell ultrastructure, with fewer mature melanosomes being found in treated cells. Suppression of melanin synthesis was apparent within 24 h of addition of HTS, as judged by inhibited incorporation of the false precursor, 5[125I]-2-thiouracil. Reversal of inhibition occurred within the same period after removal of HTS. Tyrosinase activity gradually decreased to 25% of the control value during a 19-day treatment with HTS, and expression of two carbohydrate-dependent tyrosinase epitopes, 5C12 and 2B7, was abolished. Expression of one other melanosomal protein and of vimentin was not affected. The results suggest that HTS inhibits maturation of tyrosinase to a form associated with melanin synthesis.


T cells and human autoimmune thyroid disease: emerging data show lack of need to invoke suppressor T cell problems. Human T cells recognize self and foreign antigens when such antigens are processed into small peptides and bound to molecules coded for by genes of the HLA region on chromosome 6. The part of the T-cell surface which is responsible for such recognition is a set of molecules coded for by a variety of genes and known as the T-cell-receptor complex. In animal models, T cells are able to transfer autoimmune thyroiditis and T cells have, therefore, long been implicated in the etiology of human autoimmune thyroid disease (AITD). Information gained from the study of intrathyroidal T cells and thyroid antigen-specific T-cell clones has shown that in patients with Graves' disease, mainly helper T-cell clones have been obtained, whereas in autoimmune (Hashimoto's) thyroiditis cytolytic T-cell clones may be predominant. Such thyroid antigen-specific T cells have now been shown to recognize one or other of the three major thyroid-specific antigens; thyroglobulin, thyroid peroxidase, or the TSH receptor and efforts are currently in progress to characterize the T-cell epitopes of these major thyroid autoantigens. Recent findings of restricted T-cell receptor V gene use amongst intrathyroidal T cells confirm the primary role of T cells in human thyroid autoimmune processes leading to AITD. However, the mechanisms whereby such autoreactive T cells escape deletion and anergy, and how they become activated, remain uncertain. There is compelling evidence that the thyroid cell itself, by expressing HLA molecules, and presenting antigen directly to the T cells, may initiate disease, perhaps after an external insult.


The effects of FK-506 and cyclosporin A on the proliferation of PHA-stimulated T cells in response to IL-2, IL-4 or IL-6. Stimulated by PHA, the T cells responded well to exogenous IL-2, IL-4 or IL-6, but the responses were inhibited by FK-506 or cyclosporin A (Cs A). In contrast, when stimulated by PMA, the T cells responded to IL-2 and IL-4, but not to IL-6 and the responses were not inhibited by FK-506 and Cs A. Kinetic studies showed that FK-506 and Cs A had no inhibitory effects on T cell proliferation in response to IL-2 and IL-4 after the resting T cells were pulsed with PHA alone for a certain time. However, the response of the PHA-pulsed T cells to IL-6 was still inhibited by FK-506 or Cs A, but the inhibitory effect gradually decreased as the time in which the PHA-pulsed T cells interacted with IL-6 was prolonged. In a control system, the proliferation of the T cells that were treated with FK-506 or Cs A for 3 h and washed 3 times was not inhibited when the T cells were stimulated with PHA in combination with either IL-2, IL-4 or IL-6. Our data suggest that FK-506 and Cs A interfere with the early steps of T cell proliferation after stimulation of PHA, but not PMA. It is likely that the two drugs inhibit the expression of lymphokine receptors, by interfering Ca(2+)-related signals and that IL-6 induces T cell proliferation in a different way than IL-2 and IL-4, which are FK-506- and Cs A-sensitive.


Effects of anti-CD11a, anti-CD11b and anti-CD18 on histamine release from human basophils primed with IL-3. This study was carried out to investigate the possible role of adhesion molecules in the histamine release from human basophils primed with recombinant IL-3 (rIL-3). Anti-IgE monoclonal antibody (0.1 microgram/ml) did not induce apparent histamine release by itself, however, remarkable histamine release was induced from rIL-3-primed human basophils triggered by anti-IgE. Bear-1 (anti-CD11b monoclonal antibody) inhibited the histamine release enhanced by rIL-3, however SPV-L7 (anti-CD11a monoclonal antibody) or CLB-LFA1/1 (anti-CD18 monoclonal antibody) did not inhibit the histamine release enhanced by rIL-3 at all. The present study suggests that alpha-chain of Mac-1 is involved in the priming effect of IL-3 to increase histamine release from human basophils.


Mast cell response to formaldehyde. 1. Modulation of mediator release. To examine the effects of the atmospheric pollutant formaldehyde on functionally distinct mast cells, peritoneal mast cells (PMC), intestinal mucosal mast cells (IMMC) and mouse bone-marrow-derived mast cells (BMMC) were incubated with various concentrations of formaldehyde. Pretreatment for 30 min with up to 100 micrograms/ml formaldehyde was not cytotoxic to mast cells. Formaldehyde (1-10 micrograms/ml) alone induced low levels of histamine release (< 10%) from IMMC and BMMC. Antigen-induced histamine release was significantly increased in both PMC pretreated with low concentrations of formaldehyde (5-20 micrograms/ml) and BMMC pretreated with 10 micrograms/ml formaldehyde but decreased in PMC pretreated with a higher concentration (100 micrograms/ml) of formaldehyde. By contrast, antigen-induced histamine release was decreased in IMMC pretreated with formaldehyde in a dose-dependent manner. Histamine release stimulated with A23187 was also increased in PMC pretreated with a low concentration (10 micrograms/ml) of formaldehyde but decreased in those pretreated with a higher concentration (100 micrograms/ml) of formaldehyde. Pretreatment with 10 micrograms/ml formaldehyde significantly enhanced beta-hexosaminidase release from PMC stimulated with antigen or A23187. Compared to sham-treated PMC, PMC pretreated with formaldehyde expressed a markedly depressed natural cytotoxicity for the tumor target WEHI-164 (an assay of tumor necrosis factor alpha activity). These results suggest that formaldehyde modifies various mast cell functions through alterations in cellular metabolism. Such effects may be important in respiratory and other diseases associated with formaldehyde exposure.


Mast cell response to formaldehyde. 2. Induction of stress-like proteins. Previously we established that immediately after pretreatment with low concentrations (5-10 micrograms/ml) of formaldehyde antigen- and ionophore-induced histamine secretion was enhanced from peritoneal mast cells (PMC) isolated from rats infected with Nippostrongylus brasiliensis. In contrast to immediately following pretreatment with low concentrations of formaldehyde, 3 h after a 30-min treatment with formaldehyde (10, 50 and 100 micrograms/ml) antigen-induced histamine secretion from PMC was significantly depressed, and 35S-methionine incorporation was also decreased. To further explore the effects of formaldehyde on mast cells, we investigated protein biosynthesis of PMC following formaldehyde treatment and compared this with the effects of hydrogen peroxide (H2O2) or heat treatment. One- and two-dimensional SDS-PAGE were used to assess the effects. Formaldehyde treatment induced the synthesis of 70- and 72-kD stress-like proteins in rat PMC. Pretreatment of PMC with 50 microM H2O2 and heat (45 degrees C) also induced proteins with the same molecular weight. Two-dimensional SDS-PAGE analysis established that formaldehyde-induced 70-kD proteins had the same pI values as 70-kD heat shock proteins previously observed in mammalian cells. These results suggest that formaldehyde, H2O2 and heat shock induce stress proteins in rat PMC. It will be important to establish whether or not these stress proteins are responsible for the functional alterations observed in the mast cells.


Differential increase in 12-HETE release and CD29/CD49f expression of platelets from normal donors and from patients with atopic dermatitis by Staphylococcus aureus. The generation of the arachidonic acid-derived inflammatory mediator 12-hydroeicosatetraenoic acid (HETE) and the expression of CD29 as well as CD49f from unstimulated and stimulated platelets has been studied in patients with atopic dermatitis (AD) as well as in healthy volunteers. Heat-killed clinical isolates of Staphylococcus aureus served as stimuli. Unstimulated platelets from patients with AD produced higher amounts of 12-HETE compared to platelets from normal donors. The absolute 12-HETE release from platelets of patients with AD was significantly higher compared to the control group after stimulation with heat-killed S. aureus, whereas the relative increase remained. The expression of CD29 and CD49f on unstimulated platelets of patients with AD was markedly enhanced compared to platelets from normal donors. Stimulation with S. aureus led to similar results as to the CD29 expression on normal and atopic platelets or to a markedly higher expression of CD49f on platelets from normal donors. As compared to platelets from normal donors the CD49f expression on atopic platelets was slightly enhanced by S. aureus. Our data emphasize that platelets may play an important role in the pathogenesis of AD by an increased preactivation and by an enhanced responsiveness to S. aureus which colonizes permanently the skin of patients with AD.


Pharmacological studies on the role of protein kinase C in signal transduction of human basophils. Recent investigations have demonstrated that activation of basophils involves the activation of protein kinase C (PKC). In the present study the effects of different nonselective and selective PKC inhibitors on IgE-mediated histamine release from human basophils were investigated. While potent but nonselective inhibitors such as staurosporine exerted a dose-dependent inhibition of Fc epsilon-receptor-mediated histamine release, staurosporine derivatives with high selectivity for PKC potentiated the IgE-mediated response. The results provide evidence that the histamine release-inhibiting activity of protein kinase inhibitors is inversely correlated with their specificity for PKC. This may confirm the hypothesis that PKC exerts a negative modulatory role during the process of stimulus secretion-coupling following receptor aggregation in basophils. Moreover, investigations with phorbol esters and diacylglycerol derivatives as potent PKC activators show that direct cellular PKC activation and antigen-stimulated mediator release are not closely correlated.


Epitope analysis of aztreonam by antiaztreonam monoclonal antibodies and possible consequences in beta-lactams hypersensitivity. Three cell lines producing monoclonal antibodies, Az-1 (IgG1), Az-2 (IgG1) and Az-3 (IgM) against aztreonam were established. The epitopes and the cross-reactions of the antibodies with various beta-lactams, which were conjugated with human serum albumin (HSA), were examined by enzyme-linked immunosorbent assay (ELISA) and ELISA inhibition test. In ELISA, Az-1 and Az-2 reacted only with aztreonam and ceftazidime, which have the same acyl side chain. Furthermore, Az-2 showed a strong cross-reaction with carumonam. In the ELISA inhibition test, Az-1 and Az-2 were inhibited from binding to aztreonam-HSA by aztreonam, ceftazidime, aztreonam hydrolysate, aztreonam-epsilon-amino-n-caproic acid (EACA) and ceftazidime-EACA. Az-2 was also inhibited with carumonam. From the above results, it seems that Az-1 can recognize only the degraded structure of monobactam nucleus, and Az-2 can recognize the degraded nucleus moiety and the acyl side chain. On the other hand, Az-3 displayed broad cross-reaction to various beta-lactams in ELISA. Furthermore, the MAb showed no inhibitory reaction with various beta-lactams except aztreonam- and ceftazidime-EACA conjugates in the ELISA inhibition test, suggesting that Az-3 recognize a new antigenic determinant (NAD), which is formed by the conjugation of beta-lactam and carrier protein. The above results indicate that antibodies can recognize at least three epitopes of the degraded product(s) of aztreonam nucleus, acyl side chain and NAD in aztreonam-protein conjugate.


Estradiol augments while tamoxifen inhibits rat mast cell secretion. Mast cells have been studied extensively for their involvement in allergic reactions, where they secrete numerous powerful mediators in response to immunoglobulin E and specific antigens. However, they are also triggered by neuropeptides, they have been found in close contact with neurons, and they are activated in diseases such as angioedema, interstitial cystitis and irritable bowel disease, the prevalence of which is much higher in women. When tested on purified rat peritoneal mast cells, 17 beta-estradiol augmented secretion of histamine and serotonin, starting at 1 microM and in a dose-dependent manner, whether stimulated by the mast cell secretagogue compound 48/80 or the neuropeptide substance P. However, 17 beta-estradiol did not augment mast cell secretion stimulated by immunoglobulin E and specific antiserum indicating that immunologic stimulation is under different regulation. Testosterone inhibited secretion induced by compound 48/80. Tamoxifen, an estrogen receptor antagonist used in the treatment of breast cancer, inhibited serotonin and histamine release from purified rat peritoneal mast cells triggered by compound 48/80 or substance P. Tamoxifen also inhibited the increase in intracellular free Ca2+ originating from an influx of extracellular Ca2+ in response to compound 48/80. Moreover, tamoxifen antagonized the synergistic effect of phorbol myristate and the cation ionophore A23187 on mast cell secretion, suggesting that tamoxifen's inhibition may be due to regulation of protein kinase C activity. Tamoxifen may, therefore, have a beneficial effect in other neuroimmunoendocrine disorders both through estrogen receptor blockade and inhibition of mast cell secretion.


[Genetic monitoring of human population exposed to chemical and radiation hazards]. It is impossible to protect human heredity from the ecological consequences of environmental pollution if there is no permanent control of hereditary variability in human populations, i.e. genetic monitoring. One of the urgent problems in genetic monitoring planning is the magnitude of representative samples necessary for establishing the significant mutation effect of environmental factors. The elevation of the mutation frequency can be determined either by the dynamics of the frequencies of hereditary pathology or by comparison of the frequencies in populations that differ in harmful factor exposures. Comparison of the effects evaluated by cytogenetic, epidemiological (registration of congenital malformation frequencies) and molecular-genetic methods in various population groups will make it possible to solve the problem of safety in the investigated regions.


[Prenatal diagnosis as a method of prevention of congenital and hereditary diseases]. The program of the prevention of congenital and hereditary diseases with the aid of prenatal diagnosis includes a complex of different methods: ultrasonography, invasive procedures made at different times of pregnancy, obstetrical monitoring, immunochemical blood tests, fetal cytogenetic analysis, pathological, anatomical, and syndromological studies in abortuses. Emphasis is laid on the use of the data on ultrasound screening of the pregnant and screening of the mother's blood for some factors that form a group of women at a greater genetic risk, who require prenatal diagnosis. The efficacy of the preventive measures can be enhanced with combined use of instrumental, obstetrical and laboratory research methods. The establishment of the correct and early diagnosis may, on the one hand, remove the tension and concern in the family; on the other hand, it may prevent bearing a sick child and provide the married couple with a based genetic counselling about progeny.


[A program of prevention of hereditary lysosomal diseases in the USSR]. The organization of genetic counselling for the families of patients with lysosomal storage diseases (LSD) was based on the interaction of the genetic counselling units of this country with a laboratory of inherited metabolic diseases of the National Research Center of Medical Genetics, USSR AMS. All the patients from 705 families at risk were examined using biochemical techniques and methods of somatic cell genetics. In total the loci differentiation was performed for 309 patients with mucopolysaccharidoses, glycoproteinoses, mucolipidoses, sphingolipidoses and other LSD. 53 families at risk (of 277) were prenatally diagnosed. 66 fetuses were diagnosed for mucopolysaccharidoses, type I, II, III, A and B, VI, Tay-Sachs disease, Sandhoff's disease, GM1-gangliosidosis, metachromatic leukodystrophy, mannosidosis, and multiple sulfatidosis. In total 18 affected fetuses were diagnosed and aborted. All the prenatal diagnoses were verified. The prevalence of mucopolysaccharidoses in two Central Asian republics was evaluated as 1:15,000. An Uneven ethnic distribution of different mucopolysaccharides in the USSR has also been shown.


[Computerized analysis in the study of multiple congenital defects connected with chromosome abnormalities: phenotype-karyotypic relations and genetic markers]. Computerized comparisons of phenotypes observed in different kinds of chromosomal imbalance and presented in the form of sparse matrices of traits were made to study the specificity of the indicated phenotypes, the possibility of differential diagnosis of the clinically similar forms, the presence of genetic markers, and the correspondence of the compared phenotypes to syndrome criteria. Stable enough, though variable trait associations characteristic of definite forms of imbalance of chromosomes 4, 5 and 9 were revealed, which were especially manifest when the respective trait frequency profiles were compared. Phenotypic distinction of 9p- and 11q-segmental monosomies was demonstrated and respective "phenotypic nuclei" were isolated. It has been shown that reliability of identification increases when the case to be analyzed is compared with a large enough number of primary descriptions. Analysis of 35 cases of 4p-segmental monosomies allowed the conclusion that Wolf-Hirschhorn syndrome is associated with deletion within 4 (p14-pter) region.


[Hereditary pathology load of the population and regional medical-genetic counseling]. Analysis of hereditary disease load of the populations with different genetic structure has demonstrated the necessity of a regional approach to the organization of the medico-genetic aid to the community. It has been shown that in Central Asia, the requirement of the medico-genetic aid is almost 2 times higher than in the Russian Federation. Bearing in mind the age-associated reproduction data, those differences are three times as higher. To make the work of the medico-genetic consultations more efficient, it is necessary to increase the portion of prospective counselling at the expense of an active approach to identifying patients in the populations.


[Gene manifestations in negative man-made environment: a selective genetically determined sensitivity to asbestos]. Genetic polymorphism of the systems of haptoglobin, transferrin, alpha 1-antitrypsin and complement C3 was investigated in patients with asbestosis and in healthy individuals who contacted with asbestos for a long time. The significant differences were discovered between the groups under comparison in the distribution of the phenotypic and genic frequencies in these loci. Simultaneous studies of the levels of these proteins depending on the phenotype demonstrated differentiation of the patients and healthy subjects according to the mean values and dispersions. The data obtained provide evidence in favour of occupational selection as regards the genetic factors investigated.


[Medical Genetics Center of the Russian AMS: retrospective, status quo, perspectives]. The paper is concerned with the brief history of the Institute of Medical Genetics, USSR AMS. Recently it has been reorganized to the USR AMS Research Center of Medical Genetics which consists of two Institutes: the Institute of Human Genetics and the Institute of Clinical Genetics. The list of the departments and laboratories of both Institutes, their research programs and projects are summarized. The research programs are under discussion.


[Genetic aspects of the action of immunosuppressive agents]. The data were obtained formerly that mice of certain strains essentially differ in the sensitivity to the immunodepressive and antiproliferative action of the alkylating agents (so-called opposite strains: DBA/2--highly sensitive, BALB/c--resistant). It is shown in the present work that with the use of the other non-alkylating immunodepressive agents (cytarabine, cyclosporin A, dexamethasone) that differ in the action mode, DBA/2 mice retain a high sensitivity whereas BALB/c mice a low sensitivity to all the immunodepressants. The sensitivity to the immunodepressive action in vivo directly correlates with that of the immunocompetent cells in vitro. Potential mechanisms determining the same type sensitivity to diverse immunodepressant in mice belonging to the above-indicated genotypes are under discussion.


[Use of the calculation of the micronuclei in human cells in the detection of cytogenetic injuries by environmental factors]. The review is concerned with an analysis of the data reported in this country and abroad on the use of micronuclei calculation for revealing cytogenetic injuries by environmental factors. It has been shown that the micronucleus test possesses a rather high sensitivity, provides information and affords an objective judgement about the action of mutagenic environmental factors on man.


[Assessment of total mutagenic activity of harmful factors of the industrial environment at the metallurgy plants of South Ural]. Harmful factors of the industrial environment of the steel plants are represented by sulfur- and arsenic-containing compounds, nitrogen and carbon oxides, aerosols of heavy and rare metals, fluorine compounds whose concentrations exceed the maximum allowable ones in the air of the working place. The results of the cytogenetic examination of metallurgists attest to the pronounced total mutagenic activity of unfavourable factors of the industrial environment, among which the key role is played by arsenic, fluorine and heavy metal compounds.


[Arthrology: processes of differentiation and integration]. Modern arthrology, the science of disease of and damages to the joints, is a multidisciplinary section of medicine (rheumatology, orthopedics, pediatrics, allergology, oncology, and so forth). About 4% of the population of the earth suffer from different diseases of the joints. It is noted that the prevalence of joint pathology is non-uniform in different countries and continents. In the XX century, orthopedic arthrology, surgical methods of treatment are developing fairly intensively. The following issues have become a matter of growing concern: the effect of ionizing radiation, including low doses, on the articular cartilage, the role played by stress, disruption of the ecological balance in the genesis of arthrological disease, connection between dysplasia of the joints and subsequent dystrophic processes in them (the sequence dysplasia-dysplastic arthrosis), disturbances of the load of the joints as a pathogenetic mechanism of dystrophic and necrotic processes. In arthrology, special attention should be paid to aseptic necrosis of the jointed bones and to protruding coxarthrosis. The topicality of joint endoprosthetics is stressed as is the necessity of the setting up of the arthrological centres where endoprosthetics and restorative surgery might be up to standard. Unfortunately, the manufacture of joint endoprostheses in this country lags behind the requirements. Lately the problem of infantile and juvenile arthritides has become very urgent, posing the problem of the development of arthrological techniques for diagnosis and surgical treatment of the joints. The Academy of Medical Sciences of the Russian Federation should strengthen its coordinating and integrating roles in further development of arthrology.


[Deforming gonarthrosis (questions of pathogenesis)]. Based on the biochemical, morphological, radionuclide, electron microscopy and arthroscopic data it has been established that there are significant changes in collagen proteins of the joint cartilage in the first stage of gonarthrosis deformans when degenerative and dystrophic alterations can only be seen in the joint cartilage. The determination of type II collagen which is commonly called procollagen brings something new in the understanding of the mechanism of degenerative dystrophic alterations in the joint cartilage. A considerable increase of a local proteolytic activity has been ascertained, which should be considered as the result of deep redox processes in the cartilage tissue. Gonarthrosis-associated microcirculatory disorders in the epiphyses with the development of vascular stasis, thrombosis, mural and total erythrocyte microaggregation cells, and perivascular hemorrhages have been confirmed. For the first time two forms of gonarthrosis (acute and latent) have been defined arthroscopically. The pathogenetic treatment methods have been elaborated in terms of the stages of the morphological changes identified.


[Immunomodulation in dystrophic diseases of the joints]. Immunological examinations of 357 patients with mixed arthritis, coxarthrosis and aseptic necrosis of the femoral head have revealed substantial immunopathological deviations requiring immunomodulation and influencing surgical outcomes, with inhibition or dysfunction of T and B cellular reactions, inversion of the regulatory index, remarkable autoimmune component, and bacterial sensitization in a considerable part of the examined. In addition to the conventional immunomodulating agents (levamisole, thymalin, T-activin, sodium nucleinate), it is recommended that hemodes and polyglucin may be used, provided they are chosen individually in vitro. The use of ELISA made it possible to reveal a direct strong correlation between the level of antibodies to glycolipid cartilaginous antigen and arteparon as well as a decrease of the number of antibodies in therapeutic administration of the drug. This suggests the immunological mechanisms of the action of arteparon, namely according to the principle of hapten inhibition.


[A systemic approach to the surgical treatment of degenerative-dystrophic diseases of the hip joint]. Based on the experience gained with the surgical treatment of over 900 patients with different forms of degenerative dystrophic diseases of the hip joint the current principles of the choice of different types of surgical interventions were defined. As far as the patients with aseptic necrosis of the head of the femur are concerned, the main problem in this case lies in the early diagnosis of osteonecrosis. The surgical treatment is specified by the leading factor of the pathogenesis at every stage of the development of the process (vascular disorders, changes in the biomechanism of the joint). The planning of operations in patients with dysplastic coxarthrosis rests on the consideration of 8 basic factors that influence the disease by means of factorial analysis and determination of the prognostic estimations of the treatment efficacy.


[Surgical treatment of rheumatoid lesions of the joints]. Thirty-four years of experience gained with the treatment of patients suffering from rheumatoid arthritis made it possible to develop a complex of orthopedic treatment methods (conservative and surgical) based on the clinical, x-ray and morphological classification. These methods allow arresting inflammation in the joint, preventing destruction of the joint and formation of concordant and disconcordant deformities, correcting deformities, recovering function, the patients activity, returning ability to active life, to social useful work, and minimizing the patients' disability.


[Rheum-orthopedics in the system of complex treatment of rheumatoid arthritis]. The author presents an original conceptual scheme of the complex medical rehabilitation of patients with RA on the basis of the conservative and surgical treatment of RA (3,000 cases). Successful results were attained in 70% of cases (follow-up of 5 years).


[Thyroliberin: new physiological effects and prospects of clinical use]. In addition to the well-known activities of TRH (stimulation of respiration, antidepressant action, increase of motility, antishock action, therapeutic effects against ataxia, and so forth), some new properties were established. TRH was found (1) to stimulate the contractility of lymph vessels in ultra-low doses and to be useful in the treatment of acute pancreatitis; (2) to normalize cerebral circulation in rats and neonates after asphyxia; (3) to improve the electrophysiological parameters of the retina and pigmented epithelium in rats and man, to exert a long-term positive influence on the health status of patients suffering from senile retinal macular dystrophy; (4) to potentiate the analgesic effect of low doses of morphine in a definite range of doses (analog PR-546). It is of paramount importance that most of the indicated activities of TRH may hold promise for clinical application.


[Last-generation synthetic opioid analgesics as alternative to opiates in oncologic surgery]. The present-day problem is the development of effective methods of general anesthesia and postoperative anesthesia on the basis of nonopiate central analgesics possessing no hazardous side effects of opiates. A study was made of synthetic analgesics of the last generation as agents for intra- and postoperative anesthesia as compared with conventional opiates. It has been established that synthetic analgesics belonging to the class of opiate agonists-antagonists, namely moradol and norphine, compare favourably enough with fentanyl. They are superior to fentanyl in antistressor properties, provide for a more powerful and longer analgesic effect and can replace conventional opiates in all the stages of surgical treatment of the patients. This is particularly important for the oncological clinic where opiates are to be preserved as reserve for the treatment of chronic painful syndrome.


[Blood platelet as a test system in the evaluation of neurohormonal imbalance in pregnant women with diabetes mellitus]. A study was made of platelet alpha 2- and beta 2-adrenoreceptors stimulated by adrenaline and alupent as well as of hemostatic system parameters the levels of ACTH, cortisol, glucagon and C-peptide in the blood of women with type I diabetes mellitus during pregnancy, surgical delivery, and the postoperative period. It is shown that the changes in the sensitivity of both subtypes of platelet adrenoreceptors are closely related to the activation of the stress-realizing systems and may serve as a test for estimating the intensity of neurohumoral imbalance both during pregnancy and surgical intervention. The sensitivity of platelet adrenoreceptors to agonists may be examined by a simple retention test allowing rapid information to be derived, which is of paramount importance under clinical conditions.


Epilepsy in the first 10 years of life: findings of the child health and education study. To identify children with afebrile seizures in a national cohort, classify the seizures, and document progress in the first 10 years of life. Population based birth cohort study. The child health and education study, which includes 16,004 neonatal survivors (98.5% of infants born in the United Kingdom during one week of April 1970). 14,676 children for whom relevant information was available. Responses to parental and general practitioner questionnaires and hospital records at 5 and 10 years after birth. 84 children (42 boys, 42 girls) had had one or more afebrile seizure (incidence 5.7/1000). 63 children (31 boys, 32 girls) had epilepsy (incidence 4.3/1000). 49 of 55 children had a second seizure within a year of the first. The commonest seizure types were tonic-clonic (42) and complex partial (25). A greater proportion of children with complex partial seizures had recurrences. Children who had infantile spasms or a mixed seizure disorder had a poor outcome. All six children who died had symptomatic seizures in the first year, but seizures were not the direct cause of death. The results of this study are probably representative of seizure patterns in the general population. Outcome after seizures is determined more by the underlying disease than by the seizures themselves.


Cytotoxic drug and cytotoxic drug/G-CSF mobilization of peripheral blood stem cells and their use for autografting. We analysed 99 courses of leukapheresis after the use of cytotoxic drugs or cytotoxic drugs plus G-CSF (cytotoxic/G-CSF) to mobilize peripheral blood stem cells (PBSC) in 68 patients with hematologic or solid malignancies. Mean yields of granulocyte-macrophage progenitor cells (CFU-GM) with cytotoxic/G-CSF mobilization were significantly higher than those with cytotoxic mobilization (18.6 vs 8.40 x 10(4)/kg). The optimal timing of collection was different between these two mobilizations; the mean number of days to a peak level of circulating CFU-GM after cytotoxic/G-CSF mobilization was less than that after cytotoxic mobilization (24.2 vs 27.7 days). The leukocyte level on the day of peak CFU-GM was significantly higher in cytotoxic/G-CSF mobilization than that in cytotoxic mobilization (mean 12.8 vs 2.7 x 10(9)/l), whereas the platelet level was not different (mean 132 vs 125 x 10(9)/l). Increasing patient age was not a major adverse factor for PBSC collection. Synchronous recovery of both leukocytes and platelets was critical for achieving a high CFU-GM yield in these two mobilizations. Following PBSC autotransplantation, the rate of trilineage hematologic reconstitution showed a significant correlation with the infused dose of CFU-GM, whether they were collected with cytotoxic or cytotoxic/G-CSF mobilization. These results suggest that G-CSF can expand the PBSC pool and that CFU-GM yield after cytotoxic/G-CSF mobilization may predict trilineage hemopoietic reconstitution after ABSCT, as well as cytotoxic mobilization.


Increased yield of myeloid progenitor cells in bone marrow harvested for autologous transplantation by pretreatment with recombinant human granulocyte-colony stimulating factor. Pretreatment with haemopoietic cytokines prior to marrow harvest may result in improved quality of bone marrow harvested for autologous bone marrow transplantation (BMT). Such improvements may reduce the risk for graft failure and decrease time to engraftment. Patients undergoing autologous BMT received recombinant human G-CSF (rhG-CSF) immediately prior to marrow harvest. rhG-CSF was administered as daily subcutaneous injections for 5 days at 5 micrograms/kg body weight. Comparison of bone marrow samples before and after rhG-CSF treatment showed an increased bone marrow cellularity and a ninefold increase in the number of marrow leucocytes per volume aspirated. The mean marrow myeloid:erythroid ratio increased from 2.6 to 4.0. The mean numbers of immature (CD38 positive) and proliferating (CD71 positive) myeloid cells increased significantly from 41.6 to 50.8% and from 17.0 to 34.8%, respectively. Other subsets studied, including CD34 positive stem cells, were unchanged. The relative numbers of day 7 and 14 granulocyte-macrophage colony-forming units (day 7/14 GM-CFU) were unchanged. Long-term marrow cultures revealed that the numbers of 'long-term culture initiating cells' were unchanged after rhG-CSF treatment in spite of the ninefold increase in cellularity. To date, five of the patients have been transplanted with autologous marrow harvested after rhG-CSF treatment. Time to trilineage engraftment was unchanged compared with historical controls. We conclude that pretreatment with rhG-CSF prior to marrow harvest may improve the graft by increasing the total number of myeloid lineage restricted progenitor cells, resulting in stable but not accelerated myeloid engraftment of autologous marrow.


Characterization of chemotherapy mobilized peripheral blood progenitor cells for use in autologous stem cell transplantation. Twenty patients were treated with chemotherapy to mobilize progenitors into the blood. Peripheral blood stem cells were quantitated in peripheral blood or leukapheresis products using colony assays and flow cytometric measurement of CD34+ cells. In four patients where complete sets of serial samples were obtained, the appearance of CD34+ cells preceded the increase in CFU-GM by 24-48 h. Peak levels of CD34+ cells ranged from 0.6-5% and coincided with the peak increase in CFU-GM. Mobilized CD34+ cells contained subsets expressing CD33, CD13, CD45RA, CD38, HLA-DR, CD61 and CD41. Subsets of CD34+ cells expressing CD33, CD13, or CD45RA represent committed myeloid progenitors. In contrast to bone marrow CD34+ cells, few mobilized CD34+ cells expressed CD71, CD7, CD19 or CD10. Prompt engraftment of granulocytes greater than 500 x 10(6)/l at a median of 13 days and platelets greater than 50 x 10(9)/l at a median of 15 days was observed in patients reconstituted with mobilized cells. These data indicate that CD34+ cells mobilized during recovery from chemotherapy are predominantly myeloid in phenotype and contain few actively proliferating cells or cells with lymphoid phenotypes.


Expression of phosphatidylinositol anchored membrane proteins in paroxysmal nocturnal haemoglobinuria after bone marrow transplantation. A 20-year-old male with severe bone marrow failure associated with paroxysmal nocturnal haemoglobinuria (PNH) underwent an allogeneic bone marrow transplantation (BMT). Flow cytometric analysis of phosphatidylinositol (PI) anchored membrane proteins prior to BMT showed a markedly reduced expression of monocyte CD14 and neutrophil CD16 molecules. On day +17 after BMT expression of both antigens reached normal values and remained stable throughout a follow-up period of 10 months, thus confirming the eradication of the PNH clone. To date, this is the first case in which normal expression of PI-anchored proteins after BMT is reported.


Collection of peripheral blood stem cells mobilized by high-dose Ara-C plus VP-16 or aclarubicin followed by recombinant human granulocyte-colony stimulating factor. We developed an effective method for harvesting large numbers of peripheral blood stem cells (PBSC) for use in autotransplantation. Twenty patients with hematological malignancies were treated with high doses of Ara-C (12 g/m2) and VP-16/aclarubicin followed by administration of rhG-CSF (50 micrograms/m2). The optimal time for starting PBSC collection was determined by monitoring the CD34-positive stem cells in blood using immunomagnetic beads. PBSC were collected with a CS-3000 blood cell separator. A total blood volume between 7000 and 9000 ml was processed in each apheresis. Under these conditions, a total of 64 apheresis procedures was performed in the 20 patients. The mean numbers of mononuclear cells and of CFU-GM harvested per apheresis were 4.1 x 10(8)/kg and 110 x 10(4)/kg, respectively. A number of CFU-GM sufficient for engraftment (> 30 x 10(4)/kg) could be harvested by a single apheresis in 15 of the 20 patients. So far, 11 patients have been transplanted with PBSC and obtained rapid hematopoietic recovery. The median time to recover neutrophils more than 0.5 x 10(9)/l was 10 days, and that for platelets 50 x 10(9)/l was 11 days. This method for harvesting large numbers of PBSC allows safer autotransplantation in patients with chemoradiosensitive tumors, and is applicable to older patients.


[Non-surgical treatment of benign prostatic hypertrophy. Current status of the problem]. Benign prostatic hypertrophy is a highly prevalent disease, with a significant morbidity. Classical surgical therapy (open or transurethral adenomectomy) is very effective, but not without risks and complications. Better understanding of the pathogenesis of the disease have led to the development of several alternative treatments. Hormonal treatments are briefly reviewed, especially the preliminary results obtained with the 5-alpha-reductase inhibitors.


The effects of calcitonin gene-related peptide on formation of intra-articular oedema by inflammatory mediators. 1. The temporal and quantitative effects of inflammatory mediators on plasma extravasation in the rat knee were investigated by use of a perfusion technique. 2. Intra-articular perfusion of substance P (SP), bradykinin or histamine over a 5 min test period produced rapid-onset and prolonged plasma extravasation in a dose-dependent fashion. The rank order of potency was bradykinin greater than SP greater than histamine. 3. Calcitonin gene-related peptide (CGRP) did not induce plasma extravasation but enhanced substance P-induced plasma extravasation in a dose-dependent fashion. A 5 min co-perfusion of the two agents produced short-term enhancement lasting 10 min while continuous co-perfusion produced enhancement for the duration of the perfusion. 4. A 5 min perfusion of CGRP enhanced plasma extravasation when co-perfused with bradykinin but not histamine. However, when CGRP and histamine were continuously co-perfused over a 20 min test period, an enhanced response was apparent. 5. The results indicate that intra-articular perfusion of CGRP enhances synovial plasma extravasation induced by agents that increase vascular permeability, but suggest that the response is not uniform and is critically dependent on the duration of perfusion within the joint.


Phosphodiesterase inhibition in ventricular cardiomyocytes from guinea-pig hearts. 1. The present study compared the cyclic nucleotide phosphodiesterase (PDE) activities in cardiomyocytes and ventricular cardiac tissue from guinea-pigs. The aim of the study was to determine whether PDE activities in ventricular tissue accurately reflect the isoenzymes present in cardiomyocytes. 2. In homogenates of cardiomyocytes and multicellular ventricular tissue, four distinct soluble PDE activities could be separated by DEAE-sepharose chromatography. 3. In multicellular cardiac tissue as well as in cardiomyocyte preparations, adenosine 3':5'-cyclic monophosphate (cyclic AMP) PDE isoenzymes I-IV were comparable in terms of substrate affinities, and inhibition or stimulation by guanosine 3':5'-cyclic monophosphate (cyclic GMP). However, in cardiomyocytes the Vmax values of PDE I-IV were lower by a factor of about 2 to 7 and the basal activities were lower by a factor of about 3 to 5 as compared to multicellular cardiac tissue. 4. To investigate whether the PDE I-IV activities were similarly inhibited by PDE inhibitors in both preparations, we studied the effects of 3-isobutyl-1-methylxanthine (IBMX), UD-CG 212 Cl (2-(4-hydroxy-phenyl)-5-(5-methyl-3-oxo-4, 5-dihydro-2H-6-pyridazinyl)benzimidazole HCl) and rolipram. UD-CG 212 Cl was a selective PDE III inhibitor in cardiomyocytes (IC50 0.3 mumol l-1) and in ventricular tissue (IC50 value 0.1 mumol l-1). Rolipram selectively inhibited PDE IV in cardiomyocytes (IC50 1.4 mumol ml-1) and in ventricular tissue (IC50 1.1 mumol l-1) whereas IBMX was a nonselective PDE inhibitor in both preparations.5. It is concluded that the PDE isoenzymes I-IV from multicellular ventricular tissue can be used as a representative system for investigating PDE inhibiting properties of PDE inhibitors in the myocardium since comparable PDE isoenzymes I-IV exist in guinea-pig ventricular cardiomyocytes and multicellular ventricular tissue.


Differential modulation of extracellular levels of 5-hydroxytryptamine in the rat frontal cortex by (R)- and (S)-zacopride. 1. The ability of various anxiolytic and potential anxiolytic agents to modify 5-hydroxytryptamine (5-HT) release in the frontal cortex of the rat was assessed by the microdialysis technique. 2. The benzodiazepine receptor agonist, diazepam (2.5 mg kg-1, i.p.), the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT, 0.32 mg kg-1, s.c.) and the 5-HT1A receptor partial agonist buspirone (4.0 mg kg-1, i.p.) maximally reduced extracellular levels of 5-HT in the rat frontal cortex by approximately 50-60%, 70-80% and 30-40%, respectively. 3. (R)-zacopride (1.0-100 micrograms kg-1, i.p.) dose-dependently reduced extracellular levels of 5-HT in the rat frontal cortex (approximately 80% maximal reduction) whereas the other 5-HT3 receptor antagonists ondansetron (10 micrograms kg-1, i.p.) and (S)-zacopride (10-100 micrograms kg-1, i.p.) were ineffective. 4. In contrast to (S)-zacopride (100 nM; administered via the microdialysis probe), (R)-zacopride (1.0-100 nM; administered via the microdialysis probe) induced a concentration-dependent reduction in extracellular levels of 5-HT in the rat frontal cortex (approximately 70% maximal reduction). 5. In contrast to ondansetron (100 micrograms kg-1, i.p.), (S)-zacopride (10-100 micrograms kg-1, i.p.) dose-dependently reversed the (R)-zacopride (10 micrograms kg-1, i.p.) induced reduction in extracellular levels of 5-HT in the rat frontal cortex. The highest dose of (S)-zacopride (100 micrograms kg-1, i.p.) completely prevented the (R)-zacopride response.In addition, (S)-zacopride (100 nM; administered via the microdialysis probe) attenuated the inhibitory action of (R)-zacopride (10 nM; administered via the microdialysis probe) on extracellular levels of 5-HT in the rat frontal cortex.6. In conclusion, the present study provides further evidence of the ability of diazepam, 8-OH-DPAT and buspirone to reduce the activity of the central 5-hydroxytryptaminergic system in vivo. Furthermore,the results indicate that the ability of (R)-zacopride to reduce the in vivo release of 5-HT in the rat frontal cortex does not correlate with its 5-HT3 receptor antagonism. However, the differential affinity of (R)- and (S)-zacopride for a (S)-zacopride-insensitive (R)-zacopride site in rat cerebral cortex mirrors the relative activity of the two zacopride stereoisomers to modify the in vivo release of 5-HT in the frontal cortex of the rat and their ability to release suppressed behaviour in animal models of anxiety.


Tachykinin receptors in the guinea-pig renal pelvis: activation by exogenous and endogenous tachykinins. 1. The contractile response to substance P, neurokinin A, selective agonists for the NK1, NK2 and NK3 tachykinin receptors and the activity of receptor-selective antagonists has been investigated in circular muscle strips of the guinea-pig isolated renal pelvis in the presence of indomethacin (3 microM). 2. Neurokinin A was the most potent agonist tested, being about 32 times more potent than substance P. The action of both substance P and neurokinin A was enhanced by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). The selective NK2 receptor agonist [beta Ala8] neurokinin A (4-10), was slightly less potent and effective than neurokinin A itself. The selective NK1 receptor agonist [Sar9] substance P sulphone was effective at low (nM) concentrations but its maximal effect did not exceed 30% of maximal response to substance P or neurokinin A. The NK3-selective agonist [MePhe7] neurokinin B was effective only at high (microM) concentrations. 3. The pseudopeptide derivative of neurokinin A(4-10), MDL 28,564, displayed a clear-cut agonist character, although it was less potent than neurokinin A. 4. The responses to roughly equieffective (25-35% of maximal response) concentrations of [beta Ala8] neurokinin A (4-10), MDL 28,564 and [MePhe7] neurokinin B were antagonized to a similar extent by MEN 10,376 (3 microM), a selective NK2 tachykinin receptor antagonist, while the response to [Sar9] substance P sulphone was unchanged. 5. The response to [Sar9] substance P sulphone was inhibited by the NK1 receptor-selective antagonist, GR 82,334 (3 microM) while the response to [beta Ala8] neurokinin A (4-10) was unchanged. 6. The selective NK2 receptor antagonists MEN 10,376, L 659,877 and R 396 antagonized competitively the response to [PAla8] neurokinin A (4-10) with the following rank order of potency (pA2 values in parentheses): MEN 10,376 (7.41)>L 659,877 (7.15)>R 396 (6.43). MEN 10,376 and L 659,877 also competitively antagonized the response to neurokinin A, although with lower potency as compared to the selective NK2 receptor agonist.7. MEN 10,376, L 659,877 and R 396 reduced in a concentration-dependent manner the contractile response produced by electrical field stimulation (1 Hz, 100 V, 0.25 ms pulse width, trains of 10 s). The rank order of potency of NK2 receptor antagonists in blocking the response to electrical stimulation (MEN 10,376> L 659,877> R 396) closely mimicked their potency in antagonizing exogenous tachykinins.8. The inhibitory effect of MEN 10,376 toward responses produced by electrical field stimulation was significantly reduced when tested in the presence of peptidase inhibitors, which increased significantly the response to nerve stimulation.9. GR 82,334 (3 pM) did not significantly affect the response to nerve stimulation in untreated preparations and slightly reduced it in the presence of peptidase inhibitors.10. We conclude that both NK, and NK2 receptors mediate the contractile effect of tachykinins in the circular muscle of the guinea-pig renal pelvis and that the response ascribable to NK2 receptor stimulation is larger than that ascribed to NK, receptor stimulation. The NK2 receptor in the guinea-pig renal pelvis belongs to the same subtype previously identified in the rabbit pulmonary artery. NK2 receptors play a dominant role in the physiological response determined by the release of endogenous tachykinins and a contribution of NKI receptors becomes evident after inhibition of peptide degradation.


Participation of protein kinase C in endothelin-1-induced contraction in rat aorta: studies with a new tool, calphostin C. 1. Calphostin C at 10(-6) M was shown to be selective and highly effective in inhibiting contractile responses of rat aortae to 12-o-tetradecanoylphorbol-13-acetate, while it had no effect on contractile responses to elevated KCl. 2. In the rat aorta, endothelin-1 (ET-1) developed a sustained tonic contraction dose-dependently in both normal Ca(2+)-containing Krebs and Ca(2+)-free Krebs containing 1 mM EGTA. Calphostin C (10(-6) M), a selective protein kinase C inhibitor, antagonized the maximal tensions for cumulative addition of 10(-8) M ET-1 by 13.2% in Ca(2+)-containing medium and 25.8% in Ca(2+)-free Krebs containing 1 mM EGTA. 3. In both Ca(2+)-containing medium and Ca(2+)-free Krebs containing 1 mM EGTA, precontraction with 10(-8) M ET-1 had no effects on the contractile response to subsequently added 10(-6) M 12-o-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C. 4. In Ca(2+)-free Krebs containing 1 mM EGTA, precontraction with 10(-6) M TPA potentiated the contractile response to subsequently added 10(-8) M ET-1, whereas this potentiation was abolished by pretreatment with 10(-6) M calphostin C. The mechanism of the TPA-induced potentiating effect remains to be determined. 5. These results suggest that the participation of protein kinase C in the 10(-8) M ET-1-induced contraction may be 13.2% and 25.8% in the presence and absence of extracellular Ca2+, respectively, and that mechanisms other than protein kinase C may be predominantly responsible for ET-1-induced tonic contraction.


The actions of two sensory neuropeptides, substance P and calcitonin gene-related peptide, on the canine hepatic arterial and portal vascular beds. 1. The two peptides, calcitonin gene-related peptide (CGRP) and substance P (SP) were administered individually as bolus injections into the separately perfused hepatic arterial and portal vascular beds of the anaesthetized dog to assess their actions and relative molar potencies at these sites. 2. CGRP caused an immediate dose-related increase in hepatic arterial flow when injected close-arterially, reflecting a fall in resistance. This vasodilator effect was slightly increased by the prior administration of the selective beta 2-adrenoceptor antagonist, ICI 118,551. 3. On a molar basis, CGRP was more potent as an hepatic arterial vasodilator than the non-selective beta-adrenoceptor agonist, isoprenaline (Iso). 4. Intra-portal injection of CGRP also evoked hepatic arterial vasodilatation unaccompanied by other cardiovascular changes. 5. CGRP in doses up to 10 nmol had no effect on portal vascular resistance when administered intra-portally. 6. SP evoked a rapid, dose-related increase in hepatic arterial flow when injected intra-arterially. The molar ED50 for this hepatic vasodilatation was 40.2 fmol, significantly less than the ED50 for either CGRP or Iso. SP was the most potent hepatic arterial vasodilator yet examined. The vasodilator effect of SP was slightly potentiated by prior beta 2-adrenoceptor blockade. 7. SP caused hepatic arterial vasodilatation when administered by intra-portal injection; its absolute and relative potency was much reduced. 8. SP when injected intra-portally caused a graded increase in hepatic portal inflow resistance. The molar potency for this portal vasoconstriction was significantly greater than that for noradrenaline (NA); however, the maximum increase in portal resistance was significantly less to SP than to NA.9. In view of the location of the peptides CGRP and SP within the afferent innervation of the liver, it is proposed that they play an important function in controlling the hepatic microvasculature in response to sensory stimuli, particularly those arising from changes in portal blood composition secondary to change in metabolic activity within the gastrointestinal tract (GIT).10. Since the peptides are released from the GIT into the hepatic portal inflow, they may modify hepatic arterial blood flow, the extent of which is related to events within the GIT.


Effects of the bradykinin antagonist, HOE 140, in experimental acute pancreatitis. 1. The novel bradykinin antagonist, HOE 140, completely blocked the fall in rabbit blood pressure caused, not only by i.v. bradykinin, but also by i.v. kallikrein. This shows that both the effects of exogenously administered bradykinin and those of endogenously released kinins are antagonized by HOE 140. 2. Acute pancreatitis was induced in rats by i.v. infusion of the cholecystokinin analogue, caerulein. This treatment resulted in massive oedema of the pancreas, increased activities of amylase and lipase in serum and a characteristic, biphasic fall in blood pressure. 3. HOE 140 prevented the caerulein-induced pancreatic oedema and the second phase of hypotension whereas NPC 349, a widely used, but short-acting, bradykinin antagonist did not show a significant inhibition. HOE 140, in contrast to its inhibitory effects on caerulein-induced pancreatic oedema and hypotension, significantly augmented the increases in amylase and lipase activities in serum. 4. It is concluded that in this model of acute pancreatitis, the release of kinins induces pancreatic oedema and hypotension. Prevention by HOE 140 of the kinin-induced oedema allows the pancreatic enzymes to leave the tissue without hindrance and thus will diminish subsequent pathological events. It is suggested that the results obtained with the highly potent and long-acting bradykinin antagonist, HOE 140, provide a pharmacological basis for a clinical trial in acute pancreatitis.


Nitric oxide synthase responsible for L-arginine-induced relaxation of rat aortic rings in vitro may be an inducible type. 1. Characteristics of L-arginine-induced non-endothelial nitric oxide (NO) formation in rat isolated thoracic aorta were investigated. 2. Relaxation to L-arginine in arterial rings devoid of endothelium developed about 2 h after the first challenge with L-arginine and reached a maximum after a further 4 h of incubation. 3. After the arteries had relaxed in response to L-arginine, guanosine 3':5'-cyclic monophosphate (cyclic GMP) production was stimulated. In fresh arteries that had not yet relaxed in response to L-arginine, L-arginine failed to elevate cyclic GMP levels. 4. Glucocorticoids, actinomycin D and polymyxin B prevented the development of L-arginine-induced relaxation and L-arginine-stimulated increase in cyclic GMP formation. 5. Once L-arginine-induced relaxation developed, these agents no longer suppressed the relaxation and increase in cyclic GMP formation to L-arginine. 6. From these results, it is suggested that in the isolated thoracic aorta of the rat, endotoxin in the medium triggers induction of a non-endothelial NO synthase during prolonged incubation, which accelerates production of NO from added L-arginine to cause relaxation of the arteries via cyclic GMP. Glucocorticoids and protein synthesis inhibitors may prevent induction of NO synthase. It is suggested that the NO synthase mediating the production of muscle-derived NO from L-arginine is an inducible type.


The actions of capsaicin applied topically to the skin of the rat on C-fibre afferents, antidromic vasodilatation and substance P levels. 1. Single applications of solutions of capsaicin were made to the intact skin of anaesthetized rats and the effects on cutaneous blood flow and the firing of C-nociceptor afferents determined. Blood flow was measured by laser-Doppler flowmetry. C-fibre activity was recorded from filaments dissected from the saphenous nerve. 2. Following the application of a capsaicin solution (concentration > or = 1 mM) to rat saphenous skin, low frequency firing occurred in C-polymodal nociceptors that sometimes continued for > 10 min. At the some time, large increases in skin blood flow occurred exceeding 300% in some instances. 3. After the initial excitation, some C-polymodal nociceptors lost their sensitivity to pressure whilst their sensitivity to heat was lost or enhanced depending on the vehicle used. 4. Sensitivity of C-polymodal nociceptors to heat recovered in < 1 day following a single application of 33 mM capsaicin. Thresholds to mechanical pressure, however, were still significantly elevated by 123% on day 1, but had recovered on day 2. 5. Vasodilatation in response to saphenous nerve stimulation ('antidromic vasodilatation') was significantly reduced by 35%, 2 days after a single application of 33 mM capsaicin, but was normal at 4 days. 6. Following a single application of 33 mM capsaicin, skin substance P levels fell to only half the normal value at day 1 and remained at this level throughout the 4 day period examined. 7. It is suggested that the ability of relatively low concentrations of capsaicin to desensitize C-fibre nociceptors may underlie the analgesic action of topical capsaicin in man.


Control of cyclic AMP levels in primary cultures of human tracheal smooth muscle cells. 1. [3H]-adenosine 3':5'-cyclic monophosphate ([3H]-cyclic AMP) responses were studied in primary cultures of human tracheal smooth muscle cells derived from explants of human trachealis muscle and in short term cultures of acutely dissociated trachealis cells. 2. Isoprenaline induced concentration-dependent [3H]-cyclic AMP formation with an EC50 of 0.2 microM. The response to 10 microM isoprenaline reached a maximum after 5-10 min stimulation and remained stable for periods of up to 1 h. After 10 min stimulation, 1 microM isoprenaline produced a 9.5 fold increase over basal [3H]-cyclic AMP levels. The response to isoprenaline was inhibited by ICI 118551 (10 nM), (apparent KA 1.9 x 10(9) M-1) indicating the probable involvement of a beta 2-adrenoceptor in this response in human cultured tracheal smooth muscle cells. However, with 50 nM ICI 118551 there was a reduction in the maximum response to isoprenaline. Prostaglandin E2 also produced concentration-dependent [3H]-cyclic AMP formation (EC50 0.7 microM, response to 1 microM PGE2 6.4 fold over basal). 3. Forskolin (1 nM - 100 microM) induced concentration-dependent [3H]-cyclic AMP formation in these cells. A 1.6 fold (over basal) response was also observed following stimulation with NaF (10 mM). 4. The nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (0.1 mM) and the type IV, cyclic AMP selective, phosphodiesterase inhibitor rolipram (0.1 mM) both elevated basal [3H]-cyclic AMP levels by 1.8 and 1.5 fold respectively. IBMX (1-100 microM) and low concentrations of rolipram (< 10 microM), also potentiated the response to 1 microM isoprenaline. Inhibitors of the type III phosphodiesterase isoenzyme (SK&F 94120 and SK&F 94836) were without effect upon basal or isoprenaline-stimulated cyclic AMP responses in these cells.5. Carbachol (1 nM-I 00 microM) produced concentration-dependent inhibition of the [3H]-cyclic AMP response to 1 microM isoprenaline in human cultured tracheal smooth muscle cells (IC50 0.24 JM). Carbachol(1 JM) inhibited the [3H]-cyclic AMP response to 1 JM isoprenaline by 60%. This effect of carbachol was itself inhibited by atropine (50 nM) (KA 2.3 x 109 M-') indicating the involvement of a muscarinic receptor.6. These results show that primary cultures of human tracheal smooth muscle cells demonstrate cyclic AMP responses to direct receptor stimulation, adenylyl cyclase activation and inhibition with nonselective and type IV-selective cyclic AMP phosphodiesterase isoenzyme inhibitors, and that the cyclic AMP response to isoprenaline can be inhibited by muscarinic receptor stimulation.


Tachykinin receptors in rabbit airways--characterization by functional, autoradiographic and binding studies. 1. In many species, both NK1 and NK2 tachykinin receptors appear to be important in mediating the contraction of airway smooth muscle. We have examined the distribution and characterization of receptors for tachykinins in rabbit airways using functional length tension studies, autoradiography and radioligand binding studies. 2. Contractile responses to tachykinins were elicited in four different areas of the respiratory tree--trachea, and three progressively more distal areas of the right bronchus. The NK2 receptor-preferring agonists, neurokinin A (NKA), neuropeptide gamma (NP gamma) and the NK2-selective [Lys5 MeLeu9, Nle10]-NKA(4-10) [NKA (4-10) analogue] produced similar contraction in all four areas. Substance P (SP) and the NK1-selective [Sar9,Met(O2)11]-SP (Sar-SP) exhibited a marked location-dependence in the magnitude of contraction, producing minimal contraction in the trachea and more proximal bronchi with contractions becoming progressively larger in the more distal airways. Senktide (which is selective for the NK3 receptor) produced negligible contraction in all areas. 3. The NK2-selective antagonist, MDL29,913, was a weak antagonist of NKA and NKA(4-10) analogue. At a concentration of 2 microM, it produced a small but significant shift in the response curve to NKA and a greater shift (8 fold) in the curve to NKA(4-10) analogue, but it had no effect on responses to Sar-SP. The non peptide NK1 receptor antagonist, CP-96,345, was also unexpectedly weak in this preparation. The pD2 value for Sar-SP was decreased 27 fold by CP-96,345 at a concentration of 1 microM, without alteration in the maximum response.4. Autoradiographic binding sites to ['251I]-NKA were sparse over smooth muscle in proximal airway preparations and markedly increased in density in the more distal airways. There was negligible binding over vascular smooth muscle and epithelium.5. Radioligand binding studies revealed binding to ['251I]-NKA which was 82% specific. The order of potency for inhibition of ['251I]-NKA binding was SP> = Sar-SP> NKA = NPy>CP-96,345> NKA(4-10) analogue >NKB>>>MEN 10207 (the NK2 subtype selective antagonist) >MDL 29,913> senktide. This profile indicates binding predominantly to NK, receptors.6. These results suggest that there are at least two types of tachykinin receptors in rabbit airways, a population of NK, receptors, the density of which is greatest in the periphery and, in addition, NK2 receptors which are uniformly distributed throughout the airways. These receptors have unusual characteristics in that the NK, antagonist, CP-96,345 and the NK2 antagonist, MDL 29,913 respectively exhibited only weak potency.


Thimerosal blocks stimulated but not basal release of endothelium-derived relaxing factor (EDRF) in dog isolated coronary artery. 1. The effect of an acetly-coA lysolecithin acyltransferase inhibitor, thimerosal, on the release of endothelium-derived relaxing factor (EDRF) was examined in the greyhound isolated coronary artery. 2. Thimerosal (1-10 microM) relaxed fully, ring segments of coronary artery which were contracted with the thromboxane A2-mimetic, U46619 (30 nM). The response was endothelium-dependent, slow in both onset and time to reach maximum. The maximum relaxation to the highest concentration of thimerosal (10 microM) was maintained for 10-20 min before the tissue slowly regained active force (1-2 h) to the same or higher level as that prior to the addition of thimerosal. At this time the endothelium-dependent relaxation responses to acetylcholine (ACh), substance P (SP), bradykinin (BK) and the calcium ionophores, ionomycin and A23187 were abolished. The endothelium-dependent contractions to the nitric oxide synthase inhibitors, NG-nitro-L-arginine (L-NNA; 10-100 microM) and NG-monomethyl-L-arginine (L-NMMA: 10-100 microM), however, were unaffected. 3. Thimerosal (10 microM) did not affect the relaxation curve to sodium nitroprusside (SNP) nor the contraction curve to the thromboxane A2-mimetic, U46619. 4. Both the relaxation response to thimerosal and the selective block of the relaxation responses to stimulated EDRF release were unaffected by either indomethacin (10 microM) or superoxide dismutase (150 u ml-1). 5. L-NNA (100 microM) significantly blocked the relaxation curves to thimerosal and A23187 but not that to SNP.6. Abolition of stimulated EDRF-mediated responses with thimerosal was unlikely to result from maximal and maintained stimulation of EDRF release even when active U46619-induced force had returned to pre-thimerosal levels, since the relaxation curves to glyceryl trinitrate (GTN) and SNP were markedly attenuated in the presence of SNP and GTN respectively when active force was restored with endothelin-1 (ET-1).7. Melittin (1 microM), ionomycin (1 microM) and A23187 (1 microM) each had selective effects on stimulated but not basal EDRF responses, similar to those of thimerosal.8. We propose that stimulated but not 'basal' release of EDRF is dependent on the release of arachidonic acid or one of its non-cyclo-oxygenase metabolites, possibly by Ca2'-dependent activation of phospholipase A2.


Effects of nitric oxide synthase inhibitors, L-NG-nitroarginine and L-NG-nitroarginine methyl ester, on responses to vasodilators of the guinea-pig coronary vasculature. 1. The effects of L-NG-nitroarginine (L-NOARG) and L-NG-nitroarginine methyl ester (L-NAME) on vasodilatation induced by ATP, substance P, 5-hydroxytryptamine (5-HT), bradykinin and sodium nitroprusside (SNP) were examined in the guinea-pig coronary bed, by use of a Langendorff technique. The effects of these inhibitors of nitric oxide synthesis were assessed on their ability to inhibit both the amplitude and the area of the vasodilator response. 2. The vasodilator responses evoked by low doses of 5-HT (5 x 10(-10)-10(-8) mol) were almost abolished by L-NAME and L-NOARG (both at 10(-5), 3 x 10(-5) and 10(-4) M), although L-NOARG (3 x 10(-5) M) was significantly less potent than L-NAME (3 x 10(-5) M) as an inhibitor of vasodilator responses to 5-HT (5 x 10(-8) mol). 3. The vasodilator responses evoked by substance P (5 x 10(-12)-5 x 10(-9) mol) were reduced in the presence of L-NAME and L-NOARG (both at 10(-5) and 3 x 10(-5) M). The response to substance P was almost abolished by L-NAME and L-NOARG (both at 10(-4) M). 4. The amplitude of the vasodilator responses to ATP (5 x 10(-11) and 5 x 10(-9)-5 x 10(-7) mol) was little affected by either L-NAME or L-NOARG (both at 10(-5), 3 x 10(-5) and 10(-4) M).7. It is concluded that in the guinea-pig coronary vasculature, the vasodilatation evoked by substance P and low doses of 5-HT is mediated almost exclusively via nitric oxide, whereas the vasodilatations evoked by ATP and bradykinin appear to involve other mechanisms in addition to the release of nitric oxide. L-NAME was a more effective agent than L-NOARG in inhibiting the vasodilator actions of 5-HT and ATP in this preparation.


Can serum prostate-specific antigen replace bone scintigraphy in the follow-up of metastatic prostatic cancer? Bone scintigraphy is the most sensitive imaging technique for the initial detection of bone metastases and is widely used in the staging of prostatic cancer. This study was performed to assess whether the development of further bone metastases can be detected by serial measurements of the serum glycoprotein prostate-specific antigen (PSA) as an alternative to follow-up scintigraphy. The bone scintigrams and PSA levels of 101 patients with metastatic prostate cancer entered into two therapeutic trials have been reviewed. Serial results of both investigations were available in 59 cases. In three cases new bone deposits were observed without a corresponding rise in PSA. In two other cases the scintigrams were considered to be suspicious of progression with no change in PSA levels; however, further follow-up indicated that these changes were not due to metastases. In 13 cases PSA levels were rising in advance of new deposits on the scintigrams. In the remaining 41 cases the PSA levels and scintigraphic findings paralleled each other. We conclude that serial estimation of PSA levels is a simpler marker for disease progression than bone scintigraphy in metastatic prostatic cancer, but that neither technique in isolation gives complete accuracy.


Is urethral pressure profilometry useful in the preoperative assessment of benign prostatic hyperplasia? The principal aim of this study was to provide an objective assessment of the potential role of urethral pressure profilometry as a technique for assessing prostatic size by direct comparison with endoscopic assessment of prostatic length and computed tomographic measurement of prostatic volume and length. There was a poor correlation between pre-operative prostatic length and amount resected in the operating theatre. The results obtained with urethral pressure profilometry correlated poorly with those obtained using the other techniques, and cannot therefore be relied upon in routine clinical practice. Although there was good correlation between the length of the prostate and prostatic size, assessed by pre-operative computed tomography, this correlated poorly with the amount of tissue resected at operation. Further studies need to be conducted to compare objectively the completeness of prostatic resection with the outcome following prostatectomy.