Phorbol ester induces down-regulation of CD4 molecule expression and resistance to in vitro infection by HIV1.

In vitro infectivity of the MT4 lymphoid cell line with human immunodeficiency virus (HIV) has been studied in correlation with the degree of expression of the CD4 molecule at the cell surface. To modulate this CD4 expression in vitro, pre-incubation with phorbol myristate acetate (PMA) was used. The lowest CD4 expression was obtained after 1 to 5 hours. Thereafter, a partial re-expression of OKT4 was observed, e.g., when the incubation time with PMA was extended to 20 hours. Reverse transcriptase (RT) activity decreased and was delayed proportionally to the length of incubation of cells with PMA. This observation was confirmed by the comparable variation of cytopathic effects and of p24 antigen release in culture supernatants. The decrease in HIV infectivity hence correlated with that of OKT4 expression when PMA treatment did not exceed a few hours. By contrast, after extended treatment, infectivity remained decreased although OKT4 expression reappeared.


[Treatment of prostatic pathology with local hyperthermia. Analysis of the results of a study on 60 patients].

The authors report the results of the hyperthermic treatment employed from January 1989 to April 1991 in 60 patients with BPH, PR and NP. Analysis of the results shows a determinant role for prostatitis (RC: 73.91%); the results for BPH are equally valid (RC: 56.25%). The authors used the same treatment for BPH with initially obstructive urinary symptomatology.


Immunohistochemistry with keratin monoclonal antibodies in canine tissues: urogenital tract, respiratory tract, (neuro-)endocrine tissues, choroid plexus and spinal cord.

Twelve oligo- or monospecific monoclonal antibodies (MoAbs) directed against human keratin types were used in an immunohistochemical study of the canine male and female urogenital tract, the respiratory tract, the adrenal gland, the (para-)thyroid gland, the choroid plexus and the spinal cord. The keratin MoAbs showed differences in staining patterns in the various epithelial tissues and the diverse epithelial cells. The kidney was characterized by a complex keratin staining pattern and the canine urothelium showed regional differences in keratin staining. Also in the female genital tract different keratin staining patterns were observed. Testicular and adrenal gland cells did not react with any of the keratin MoAbs. The keratin staining patterns in the various canine tissues showed, in addition to similarities, also distinct differences when compared to the staining patterns in corresponding tissues of other species, e.g. of man. These staining dissimilarities indicate that the reactivity patterns of the keratin MoAbs with restricted keratin immunoreactivity can not be always extrapolated from one species to another. Nevertheless, MoAbs directed against human keratin proteins can apparently be used to differentiate between various types of canine epithelia or epithelial compartments.


[The content of DNA, RNA and protein and the fresh weight:DNA, protein:DNA and RNA:DNA ratios in tissue of male calves, bulls, short-scrotum bulls and oxen during the growth period].

In male calves, in bulls, in short scrotum bulls and in oxen of different age the content of DNA, RNA and protein in various tissues (cerebrum, cerebellum, spinal cord, ventricles, lung, liver, cortex of the kidney, spleen, testicles, M. semimembranosus) was analysed. The fresh weight: DNA-, the protein: DNA- and the RNA: DNA-ratios were calculated. In the age of 78 weeks the short scrotum bulls had a body weight of 577 +/- 44, the bulls of 550 +/- 21 and the oxen of 462 +/- 41 kg. The testicles of the short scrotum bulls were smaller than that of the bulls. The differences in the content of DNA, RNA and protein per g tissue between male calves, bulls, short scrotum bulls and oxen of the same age were in part significant. The total content of DNA, of RNA and of protein for the 78 weeks old as well as the number of nuclei of the lung, the liver, the spleen and the testicles for the 6 and 78 weeks old animals were calculated. The biochemical parameters can be used for the characterization of the inhibition of growth of the different organs of calves and cattle by malnutrition and diseases.


Topical retinoic acid treatment of photoaged skin: its effects on hyaluronan distribution in epidermis and on hyaluronan and retinoic acid in suction blister fluid.

Topical treatment with retinoic acid (tretinoin, vitamin A acid) has been reported to partly reverse signs of photodamage. To determine whether the histochemical distribution of hyaluronan (hyaluronic acid, HYA) in the epidermis and dermis and the amounts of HYA and retinoic acid in suction blister fluid were influenced by such topical treatment, 14 subjects healthy apart from moderate photodamage were instructed to treat the lateral forearm with 0.01-0.05% retinoic acid cream for 6 months. In a study of the short-term effects, another six subjects applied 0.05% retinoic acid cream for 2 weeks. After 6 months the thickness of the vital epidermis had increased by 23%. The HYA staining was based on a specific immunohistochemical method in which hyaluronan-binding protein is used. Before treatment HYA was seen as a meshwork around the cells in the upper half of the stratum spinosum. After 6 months of treatment this meshwork had increased in thickness by 31% compared with pretreatment specimens. The HYA staining was already intense in the papillary dermis before treatment and no difference was observed after 6 months' treatment. The mean concentration of HYA in blister fluid had increased significantly (43%) after 2 weeks of treatment whereas after 6 months there was no significant difference in this respect between the treated and untreated arm. The increase in the thickness of the epidermal HYA meshwork after 6 months and the blister fluid HYA after 2 weeks may indicate that HYA is involved in the epidermal change induced by topical retinoic acid therapy.(ABSTRACT TRUNCATED AT 250 WORDS)


[The regulatory role of pulmonary surfactant in macrophage metabolism].

The surfactant was isolated from rat lung tissue and its effect on biosynthesis of DNA, RNA and protein as well as content of cyclic nucleotides and prostaglandins was studied in macrophages. When the surfactant was incubated simultaneously with lung macrophages within 6 hrs, an initial inhibition and subsequent activation of DNA, RNA and protein synthesis were observed. Stimulation of protein synthesis was found to follow the increase in the biosynthesis of DNA and RNA. At the same time, the surfactant induced elevation of cAMP in the macrophages within 1 hr of cultivation, while content of cGMP was decreased within 3 and 6 hrs. Content of prostaglandins E was increased in the cells and concentration of prostaglandin F2 alpha was decreased within 6 hrs. The findings suggest that the surfactant-mediated increase of DNA-induced protein synthesis was cAMP-, cGMP- and prostaglandin-dependent.


Galanin does not affect the growth hormone-releasing hormone-stimulated growth hormone secretion in patients with hyperthyroidism.

Patients with hyperthyroidism have reduced spontaneous and stimulated growth hormone (GH) secretion. The aim of our study was to evaluate the effects of galanin, a novel neuropeptide which stimulates GH secretion in man, on the GH response to GHRH in patients with hyperthyroidism. Eight untreated hyperthyroid patients with Graves' disease (6F, 2M, aged 25-50 years) and six healthy volunteers (3F, 3M, aged 27-76 years) underwent from -10 to 30 min in random order: (i) porcine galanin, iv, 500 micrograms in 100 ml saline; or (ii) saline, iv, 100 ml. A bolus of human GHRH(1-29)NH2, 100 micrograms, was injected iv at 0 min. Hyperthyroid patients showed blunted GH peaks after GHRH+saline (10.2 +/- 2.5 micrograms/l) compared to normal subjects (20.7 +/- 4.8 micrograms/l, p < 0.05). GH peaks after GHRH+galanin were also significantly lower in hyperthyroid subjects (12.5 +/- 3 micrograms/l) compared to normal subjects (43.8 +/- 6 micrograms/l, p < 0.05). That galanin is not able to reverse the blunted GH response to GHRH in hyperthyroidism suggests that hyperthyroxinemia may either increase the somatostatin release by the hypothalamus or directly affect the pituitary GH secretory capacity.


Defective stimulation of proliferation and collagen biosynthesis of human bone cells by serum from diabetic patients.

We studied the influence of fasting serum from nine insulin-dependent diabetic children and adolescents under insufficient metabolic control on normal human bone cells in vitro compared with serum from eight sex- and age-matched controls. Cell number 24 h after plating was significantly less under diabetic serum, indicating impaired cell attachment, spreading and initiation of cell proliferation. Cell number after five days was reduced by 1% diabetic serum, while higher serum concentrations had diverging effects on osteoblast proliferation. Collagen synthesis of human osteoblasts was significantly reduced by 8% diabetic serum compared to 8% control serum, while synthesis of non-collagenous proteins was not affected. Duration of diabetes (several weeks up to 12 years) had no influence on these parameters. The serum from one patient, which was studied a second time under excellent metabolic control three months later, however, had lost its inhibitory influence on collagen synthesis of osteoblasts. The pattern of the interstitial collagen types I, III and V was not altered by diabetic serum. These results indicate that defective regulation of proliferation and collagen synthesis of osteoblasts by components present in human diabetic serum may be an important factor in the development of diabetic osteopenia. The negative influence might be explained in part by reduced levels of IGF-I and elevated levels of IGF binding protein-1 in the diabetic sera.


Examination of antithyroid effects of smoking products in cultured thyroid follicles: only thiocyanate is a potent antithyroid agent.

We studied the antithyroid action of cigarette smoking products (nicotine, cotinine, and thiocyanate) in the physiological culture system of porcine thyroid follicles. Iodide uptake, iodine organification, de novo thyroid hormone formation, and iodide efflux were measured in the presence of 0-200 mumol/l nicotine, cotinine, or potassium thiocyanate. Nicotine and cotinine did not inhibit iodide transport or thyroid hormone formation. Thiocyanate concentrations equivalent to serum levels of smokers showed three independent antithyroid actions: (i) inhibition of iodide transport, (ii) inhibition of iodine organification, and (iii) increased iodide efflux. Inhibition of iodide transport by thiocyanate was competitive with iodide and independent of TSH concentration. Thiocyanate did not inhibit TSH mediated cAMP production or Na+K+ ATPase activity, a sodium pump for iodide transport. When 50 mumol/l thiocyanate was added 2 h after incubation with iodide or when 1 mumol/l thiocyanate was added from the beginning of incubation, iodine organification was inhibited without changing iodide transport. De novo thyroid hormone formation was clearly inhibited by 50 mumol/l thiocyanate. Thiocyanate increased iodide efflux although the degrees of iodide efflux by 10 mumol/l and 100 mumol/l thiocyanate did not differ significantly. In summary, thiocyanate, a product of smoking, has three independent antithyroid activities. The data of iodide transport kinetics suggest that thiocyanate can be an antithyroid agent particularly in iodine deficiency.


Studies on regulation of insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-4 production in human bone cells.

Previous studies have shown that the actions of IGF-II in bone are determined not only by its concentration, but also by the concentration of IGFBP-4 as well as other IGFBPs. In this study, we sought to determine by Western ligand blotting the effects of growth hormone, IGF-I and IGF-II on the production of IGFBP-3 and IGFBP-4 in TE89 human osteosarcoma cells and in untransformed normal human bone cells derived from rib. Human growth hormone at 10 micrograms/l decreased the amount of IGFBP-4 but had no effect on the IGFBP-3 level in the conditioned medium of low density cultures of TE89 cells and human bone cells derived from rib. Human growth hormone had no effect on IGFBP-3 or IGFBP-4 levels in the conditioned medium of high density human bone cell cultures. IGF-I and IGF-II, which increased human bone cell proliferation, decreased the level of IGFBP-4 (30% of control at 100 micrograms/l IGF-I and IGF-II) but increased the level of IGFBP-3 (3-10 fold at 100 micrograms/l IGF-I and IGF-II) after 48 h of treatment in the conditioned medium of both low and high density TE89 cell cultures. Similar changes in IGFBP-3 and IGFBP-4 levels were also seen in the conditioned medium of human bone cells derived from rib after treatment with IGF-I and IGF-II. Studies to determine the underlying molecular mechanisms by which IGF-II decreased the amount of IGFBP-4 in the conditioned medium revealed that IGF-II decreased the IGFBP-4 mRNA abundance and increased the IGFBP-3 mRNA abundance in human bone cells. Based on the above findings, we conclude that the production of both IGFBP-3 and IGFBP-4 is regulated in bone cells and that local and systemic agents may modulate the responsiveness of bone cells to IGFs by regulated secretion of IGFBP-3 and IGFBP-4.


[Platelet antigens].

Platelet specific antigens and platelet antigens shared with other peripheral blood cells are presented. The molecular characteristics of membrane glycoproteins which are platelet specific determinants is discussed.


Cytomegalovirus infection in children with blood diseases.

The study included 68 children aged from 1 to 16 years treated for acute leukemias and bone marrow aplasia. Cytomegalovirus antigen (CMV) was detected by immunofluorescence in urinary sediment cells and in cell cultures after their inoculation with CMV. Besides, the activity of IgG and IgM classes of antibodies against CMV was determined. Presence of one or more markers of CMV infection was demonstrated in 31 children, i.e., 45.5%. In eight children (11.7%) clinical manifestation of CMV infection were demonstrable with fever, hepatitis, pneumonia, rash. In all the children who completed the treatment with hyperimmune globulin, regression of clinical symptoms and signs of CMV infection with the elimination of virus antigen from urine was achieved.


Marital discord and early child development.

As part of a prospective study on the neuropsychiatric development of children born at varying degrees of organic and psychosocial risk, the effect of marital discord on the cognitive and social-emotional development of 315 first-born children into two parent households was investigated. Both the children and the quality of the parental relationship were evaluated when the children were 3 months and 2 years of age. The findings show that marital discord at 3 months does not affect child performance at 3 months, or at 24 months. Marital discord at 2 years does affect the emotional well-being of the 2 year olds, i.e. it is accompanied by a significant rise in psychopathological symptoms. When the effect of the stability of marital strife from the first assessment to the second was studied, a significant increase of behaviour problems and decrease in cognitive performance was found in the group of children whose parents' marital situation had deteriorated and failed in the meantime but surprisingly not in the group with chronic discordant relationships. Conversely, poor child performance and emotional adjustment improved with a positive change in the parental relationships. Analysis shows that negative marital change goes hand in hand with a decrease in parenting skills which affect children more adversely than their counterparts whose parents had a history of marriage and child-rearing problems. Further study of the course of marital relationships and child development at the next research waves (4.5 and 8 years) will show which results are of long-term versus short-term relevance.


NMDA-receptor blockers but not NBQX, an AMPA-receptor antagonist, inhibit spreading depression in the rat brain.

The effect of different glutamate-receptor antagonists on the induction of cortical spreading depression of Leao and of cortical anoxic membrane depolarization were investigated in the anaesthetized rat. Spreading depression (SD), elicited by mechanical stimulation of the cortical surface, was inhibited by the non-competitive N-methyl-D-aspartate (NMDA)-receptor blocker, (+-)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)-cyclo-hepten-5,10-imi ne maleate (dizocilpine or MK-801), (0.30 mumol kg-1 (0.10 mg kg-1)), and the competitive NMDA-receptor antagonists; cis-4-phosphonomethyl-2-piperidine carboxylate (CGS 19755), (3.36 mumol kg-1 (0.75 mg kg-1)), D-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 40116), (1.20 mumol kg-1 (0.25 mg kg-1)) and its carboxylester CGP 43487, (6.30 mumol kg-1 (1.50 mg kg-1)). The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepripriate (AMPA)-receptor blocker, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F) quinoxaline (NBQX), administered as an intravenous dose of 29.76 and 89.29 mumol kg-1 (10 & 30 mg kg-1), which is sufficient to block seizures and protect against ischaemic brain damage, did not inhibit spreading depression. None of the drugs utilized inhibited the anoxic membrane depolarization. The data demonstrate that NMDA-receptor activation is essential for the initiation and propagation of spreading depression, while activation of AMPA-receptors is not obligatory. The observed initiation and propagation of SD, during AMPA-receptor blockade, suggest that activation of voltage-operated ion channels may contribute to release the magnesium block of the NMDA-receptor operated channel and to the initiation of SD.


Effects of nerve growth factor and acetyl-L-carnitine arginyl amide on the human neuronal line HCN-1A.

The HCN-1A clonal cell line, derived from the cortical tissue of a patient with unilateral megencephaly, was shown to differentiate into a mature neuronal-like state in the presence of the nerve growth factor, dibutyryl cyclic adenosine, 3',5'-monophosphate and either 1-isobutyl-3-methylxanthine or forskolin. Differentiation was assessed by measuring the percentage of cells that displayed branched, varicose processes that stained for synaptophysin. Treatment of cultures with a cocktail containing forskolin increased immunocytochemical staining for gamma aminobutyric (GABA), neurofilament protein and the nerve growth factor receptor species p75NGFR. Treatment with acetyl-L-carnitine alone had some effects on the cell morphology while acetyl-L-carnitine arginyl amide and nerve growth factor together increased the GABA content. Positive staining levels for the neurotransmitters gamma aminobutyric acid, glutamate, somatostatin, cholecystokinin and vasoactive intestinal polypeptide were measured quantitatively for HCN-1A under basal conditions.


Microcephalic cerebrum with hypomyelination in the pygmy mouse (pg).

To determine whether somatomedin has a direct action on cerebral development instead of an indirect action of a growth hormone, we examined the central nervous system of the pygmy mouse (pg), a mutant with normal somatomedin activity. Our findings are: (A) the weights of the pg/pg cerebrum and cerebellum weighted were significantly less than those of the normal controls (pg/+), 14 and 15% less, respectively; (B) the total DNA content was reduced by 17% in the cerebrum and cerebellum of the pg/pg mouse; (C) the total RNA content was reduced in the cerebrum and cerebellum, proportional to the reduction in DNA; (D) CNPase activity was reduced selectively in the cerebrum of the pg/pg mouse by 25%; and (E) the pg/pg mice exhibited a strikingly reduced level of activity with an indistinct diurnal periodicity. Therefore, the present findings suggest that the action of somatomedin on the proliferation and maturation of glial cells might be a necessary precondition to myelin formation.


Determination of genome size of Haemophilus influenzae Sb: analysis of DNA restriction fragments.

Genomic DNA size was measured in clinical isolates of Haemophilus influenzae by Pulsed-Field Gel Electrophoresis of DNA restriction fragments. Because of the high (64%) A+T content of H. influenzae DNA, restriction enzymes that recognize sequences with at least four GC base pairs were expected to be rare cutters. Five enzymes that produced fragments greater than 200 kb in size were used to digest intact chromosomes and fragments resolved by TAFE and/or FIGE: ApaI (GGGCCC), EagI (CGGCCG), NotI (GCGGCCGC), RsrI (CGGA/TCCG), and SmaI (CCCGGG). All five had recognition sequences with at least six GC base pairs. The genomic DNA size of H. influenzae serotype b, estimated with ApaI, EagI, NotI, RsrII, and SmaI, is 1,950 kb.


Staphylococcal lipase affects phagocytosis of Staphylococcus aureus by human granulocytes and monocytes.

The rate of immunological and non-immunological phagocytosis of staphylococci by lipase pre-treated human granulocytes and monocytes was compared. It was found that the effect of this enzyme on two types of cells is opposite. Lipase decreases phagocytosis by granulocytes and increases by monocytes. The revealed differences between phagocytosing cells studied prompted us to investigate the influence of lipase on Fc receptors on these cells (rosette EA test). The different susceptibility of Fc receptors on non-activated phagocytes to lipase was found. This could be at least partially responsible for the difference observed between phagocytic activity of granulocytes (decreased) and monocytes (increased) pretreated with staphylococcal lipase. Inactivated enzyme showed a similar effect as active enzyme in the case of granulocytes. However, inactivated enzyme had no effect on rosette formation by lipase pretreated monocytes, indicating an enzymatic effect.


Transformation of Thiobacillus versutus with plasmid DNA.

The representative of the facultatively chemolithotrophic thiobacilli, Thiobacillus versutus has been successfully transformed for the first time with plasmid DNA. The plasmid used for the transformation study was pKK2, a derivative of the broad host range pSa plasmid conferring Km resistance being effectively expressed in T. versutus. Different methods inducing an artificial state of competence were tested. Transformants were obtained at the efficiency of about 10(3) per micrograms of DNA. pKK2 appeared to be compatible with T. versutus indigenous plasmids, but for stable maintenance it required constant selective pressure.


A rapid agar-diffusion test for quantifying the toxic effects of copper on microorganisms.

The rapid agar diffusion test for quantifying the toxic effects of copper on microorganisms was elaborated. The assay was run in standard Petri dishes containing Czapek Solution Agar (Difco). Immediately after inoculation with the organism tested 3.2 mm diameter wells were punched in the agar. Wells were filled (20 microliters) with distilled water (control) and a range of solutions containing 128 to 4096 ppm Cu2+ (as CuSO4). After incubation (2-5 days, 26 degrees C) the diameters of Cu-inhibition zones were measured. Cu-sensitivity indices (ICu's) were calculated according to the formula: ICu = a2/(3.2-b), where: a--slope of the linear regression equation: log [Cu] (x)--inhibition zone diameter (y), b--intercept of the same equation. Additionally for characterization of Cu-sensitivity of the organism tested the predicted highest non-inhibitory Cu concentration (10c) was calculated, where: c = (3.2-b)/a. Both ICu's and adjusted average diameters of Cu-inhibition zones characterized well the Cu-sensitivity in organisms tested, as indicated by the statistical analyses. ICu's correlated very well (negative correlation) with 50% effective doses (Ed50's) of Cu2+ for mycelial growth of Cylindrocarpon destructans. The applicability of the elaborated assay in ecotoxicological studies was discussed.


Effect of hyperthermia on the antitumor actions of interferons.

Hyperthermia treatment has been shown to enhance the in vitro antiproliferative effects of IFN-alpha, IFN-beta, and IFN-gamma, with IFN-gamma being more strongly enhanced than IFN-alpha. The comparative effects of hyperthermia on the in vivo antitumor activities of IFN-alpha and IFN-gamma were evaluated in the murine system using both subcutaneous and intraperitoneal B16 melanoma tumor model systems. Heat-induced whole body hyperthermia, resulting in a 2 degree C rise in body temperature, was administered by incubating the mice for 8 hours in a dry incubator at 37.1 degrees C. Whole body hyperthermia was found to enhance the antitumor activity of IFN-alpha by approximately 1.0 fold and 1.2 fold for the subcutaneous and intraperitoneal tumor models, respectively. This represented an additive effect of hyperthermia and IFN-alpha. Hyperthermia was found to enhance the antitumor activity of IFN-gamma by approximately 2.9 fold and 2.2 fold for the subcutaneous and intraperitoneal tumor models, respectively. This represented a synergistic effect of hyperthermia and IFN-gamma. The results of this in vivo study confirm and extend the in vitro observation that hyperthermia more strongly enhances the antitumor action of IFN-gamma than IFN-alpha. These results may have clinical importance because they suggest that hyperthermia may be used in combination with IFN-gamma to provide a synergistically enhanced antitumor action.


Effect of Penicillium expansum culture conditions on patulin production.

Penicillium expansum strains grown on Capek-Dox liquid medium excreted patulin to the medium. Its amount increased until day 12, then smaller amounts of the toxin were observed. Maximum patulin excretion took place at 25 degrees C, pH 6, although at 5 degrees C, pH 3 the presence of this mycotoxin was also observed. The highest amount of patulin produced was observed in medium containing fructose.


Effect of preservatives on patulin production by Penicillium expansum.

Penicillium expansum has been grown on Capek-Dox medium using glucose and fructose as carbon source. Preservatives used in fruit processing and introduced in the medium were sorbic acid, formic acid, benzoic acid, SO2 and saccharose. Sulphur dioxide had a most inhibitory effect on mycelium growth and patulin production, formic acid concentration of 0.025% increased the amount of patulin by about 30% as compared to the culture with no preservatives. However its higher concentrations inhibited synthesis of this mycotoxin. Sorbic acid concentration of 0.1% stimulated the fungus strains examined in patulin synthesis but its highest amounts were detected using 0.0125% benzoic acid increased patulin secretion from 8 to 50% as compared to the control, depending on the strain examined. Saccharose concentration up to 50% clearly decreased patulin content in the medium until its total disappearance.


Localization of basic fibroblast growth factor (bFGF) in a metastatic cell line (AT-3) established from the Dunning prostatic carcinoma of rat: application of a specific monoclonal antibody.

Localization of basic fibroblast growth factor (bFGF) in a metastatic cell line, AT-3, established from the Dunning prostatic carcinoma of rat was determined by two immunological techniques using a specific monoclonal antibody against bFGF. Concentration of bFGF in cell extract was measured by sandwich radioimmunoassay (RIA) with heparin-Sepharose and 125I-labeled monoclonal antibody. bFGF concentration in the extract of AT-3 cells increased with increasing concentration of NaCl in extraction buffer. Localization of bFGF in AT-3 cells was determined by counting radioactivity of 125I-labeled monoclonal antibody binding to AT-3 cells before or after increasing permeability of the cells. The binding increased significantly by this treatment, indicating that bFGF within the cells was detected.


Use of a reconstituted basement membrane to study the invasiveness of tumor cells.

We have used an extract of basement membranes which can be reconstituted into a biologically active gel matrix composed predominantly of collagen IV, laminin, nidogen, and heparin sulfate proteoglycan, in order to study the mechanisms involved in tumor cell invasion. When layered onto a porous filter in a Boyden chamber, the gel forms a barrier to the passage of normal cells. Malignant cells are able to cross this layer when the conditioned medium of NIH 3T3 cells is used as a chemoattractant to stimulate cell migration. A variety of human tumor cells have thus been studied in this system and we find a high correlation between their invasiveness in vitro and their malignant behavior as exhibited in vivo. We have used this in vitro invasion assay to test for factors which might inhibit tumor cell invasion. Collagenase IV is produced by malignant cells and is thought to be required for invasion. Indeed, inhibitors of this enzyme have demonstrated reduced tumor cell invasiveness. One site of five amino acids, on the B1 chain, which has been shown to promote cell adhesion, migration and binding to laminin receptor, was found to inhibit the invasion of tumor cells. In addition, factors which elevated cAMP levels were also able to suppress the invasiveness of tumor cells. These data suggest that the assay system described herein can be successfully utilized to study the invasive activity of tumor cells and those factors that may inhibit the spread of malignant cells.


Nucleolar organizer regions in prostate cancer.

A silver colloid technique for the staining of nucleolar organizer regions (NORs) was applied to paraffin sections of 52 clinical prostate cancers, 5 incidental carcinomas of the prostate, 12 benign prostatic hypertrophy (BPH) specimens and 7 normal prostates. The mean numbers of silver-stained NORs (AgNORs) in these lesions were 3.12 +/- 0.52 in clinical cancer, 2.65 +/- 0.64 in incidental cancer, 1.66 +/- 0.16 in BPH, and 1.76 +/- 0.22 in normal prostate. There was a statistically significant difference in agNORs numbers between cancer and benign prostatic tissues (p < 0.001). However, no significant difference was observed in AgNORs numbers between incidental and clinical carcinoma of the prostate. In clinical cancer, only poorly differentiated adenocarcinoma showed a statistically larger number of AgNORs than the well or moderately differentiated group (p < 0.02). Correlation between AgNORs numbers and clinical stage was not obvious. There was no relationship between the number of AgNORs and serum values of tumor markers such as PAP, PSA and gamma-Sm. Moreover, the AgNORs numbers did not show a relation to decreasing rates of serum marker levels during successful anti-androgen therapy. If the patients with prostate cancer were divided into two groups by 2.9 of AgNORs number, the group with the smaller number of AgNORs (n = 14) was found to have a tendency towards a longer disease-stabilizing period than the larger group (n = 17).


Computed tomographic evaluation of bone metastases in prostatic cancer patients.

This report provides new morphological insights, based on diagnostic methods, into metastases to the bone in prostate cancer patients. Lumbar CT examination is available to evaluate whether metastases are truly present or not, especially in aged patients with positive bone scans. The evaluation of response is clearer on CT. Furthermore, CT is useful not only in estimating the presence of metastases, CT also provides definite details on the extent of the metastatic condition.


Radiation treatment of prostate bone metastases and the biological considerations.

This contribution on the biology and management of bone metastases from prostatic cancer is divided into three parts. The first details a study conducted at Stanford University on the prevention of bone metastases in the lumbar spine, in patients in whom the lumbar spine has been irradiated coincidental to the radiation treatment of the paraaortic lymph nodes. The incidence of metastases was significantly reduced in 71 patients in whom the apparently normal lumbar spine was irradiated, as compared to the incidence of metastases in 65 patients who received no lumbar irradiation. The implications of these observations on developing strategies for early, or preemptive, irradiation for bone metastases are discussed. In the second part, the optimum radiation dose and fractionation scheme for the palliation of overt bone metastases is addressed. Drawing largely from the work of Arcangeli et al., a total dose of 40-50 Gy*, fractionated at 2 Gy per day, seems to be the regimen of choice for enduring pain relief for most patients with prostatic metastases to bone. Finally, the recent utilization of strontium-89 in the palliation of advanced bone metastases is addressed. *The Gy is the current international unit of radiation. 1Gy = 100 Rad; 1cGy (centigray) = 1 Rad.


Clinical course of bone metastasis from prostatic cancer following endocrine therapy: examination with bone x-ray.

X-ray findings of bone metastatic lesions from 81 cases of stage D2 prostatic cancer were examined before and following endocrine therapy. Untreated lesions were classified into five types; osteoblastic (15%), mixed, but mainly osteoblastic (31%), mixed, but mainly osteolytic (17%), osteolytic (10%), and undetermined with a positive bone scan (27%). Patients with two mixed types had a tendency of widely speeded areas of metastasis and elevated serum prostatic acid phosphatase. Temporal enlargement of sclerotic lesion immediately after the start of therapy did not indicate disease progression. In many cases, changes from osteolytic to osteoblastic patterns were noticed in the same lesion regardless of the effects of endocrine therapy. Remodeling to the sclerotic pattern appeared as curative changes. From these findings, it was concluded that the natural course of bone lesions showed a tendency to change from the osteolytic to osteoblastic type and relapse was often accompanied by an increase of the osteolytic type lesion. Evaluation of therapeutic effects based on remodeling, changes in number and areas of lesions, and the appearance of new lesion correlated well with prognosis.


Palliative radiotherapy of bone metastasis.

We analyzed the effects of radiotherapy on 226 sites of metastatic bone tumors from 1981 to 1984, and on 157 sites of bone metastases of prostate cancer from 1970 to 1988. The radiation effect on pain relief was recognized in about 90% of cases within the dose of 20Gy to 30Gy, and there were not many differences in these effects according to the original tumors or histological types. In prostate cancer, the sites which needed re-irradiation were not recognized within 6 months after irradiation, and only 12 out of 80 sites (15%) that could be observed after more than 6 months needed re-irradiation due to recurring pain. As more than 60% of the patients with prostate cancer who needed irradiation to control bone metastases died within a year, to get pain relief by irradiation immediately and safely was thought to be very useful from the viewpoint of useful life. One patient was irradiated on 16 sites and 2 of these 16 sites received 4 treatments of irradiation and the shortest interval was 10 months and the longest one was 18 months.


Comparative study of prostatic carcinoma bone metastasis among Japanese in Japan and Japanese Americans and whites in Hawaii.

Metastatic prostate carcinomas in autopsy cases from three populations 49 cases of indigenous Japanese, 29 cases of Japanese Americans and 14 from whites in Hawaii) were compared in terms of their clinicopathological, immunohistochemical (tenascin and ras p21) and lectin binding (Helix Pomatia antigen, HPA) properties. Only the clinicopathological features were analyzed in the cases of whites in Hawaii. The results indicate that poorly differentiated carcinoma is less common, whereas distant metastasis is more frequent, in indigenous Japanese. Some of the Japanese-American cases with poorly differentiated carcinomas did not show any distant metastases. HPA and ras p21 expression are more common, but tenascin is less common in indigenous Japanese. HPA expression is more common in cases with metastasis, especially with metastasis to the bone and other organs, than nonmetastatic cases. Prostatic cancer cases in indigenous Japanese were more aggressive biologically than those in Japanese Americans, but no phenotypic differences were seen relevant to the presence or absence of bone metastases.


[Value of PSA in the staging of prostatic cancer].

The clinical value of Serum Prostate Specific Antigen (PSA) in the staging of prostatic carcinoma was evaluated in 62 patients who underwent radical retropubic prostatectomy. Preoperative levels of PSA were compared with the final pathological stage obtained from all surgical specimens examined for capsular penetration, seminal vesical invasion and lymph node involvement. PSA level was closely correlated with the volume and the stage of the prostatic carcinoma. 93% of the patients with PSA < or = 10 ng/ml had tumor confined to the gland. All patients with PSA > 20 ng/ml had extraprostatic tumor extension (stage C or D). Patients with histologically proved prostatic carcinoma, PSA > 20 ng/ml and negative bone scan can be assumed to have extraprostatic disease and/or lymphatic involvement. Patients with PSA (drawn in the requested conditions) < or = 10 ng/ml can be considered to have organ confined disease, and can be spared a bone scintigraphy. Our study indicate an increasing role of PSA in the clinical staging of patients with prostatic carcinoma.


The concentrations of GABA, 5-HIAA and HVA in the cerebrospinal fluid of children with infantile spasms and the effects of ACTH treatment.

Children with infantile spasms (IS) are generally treated with ACTH although little is known of the biochemical basis of the symptoms and the mechanism of this therapy. We have measured the concentrations of gamma-aminobutyric acid (GABA), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in the CSF of IS children, followed the effect of ACTH treatment on these parameters and correlated CSF GABA values with the cause of IS, cranial CT findings and antiepileptic treatment. While significant differences in GABA concentrations were found between the children with IS and those with febrile seizures or nonconvulsive symptoms, these could be accounted for by age, not the disease present. The CSF GABA level was highest in the IS children with normal CT, cryptogenic cause and no antiepileptic treatment, and lowest in those with abnormal CT, symptomatic cause and antiepileptic treatment. The basal level of CSF 5-HIAA in the IS children was higher than that in the nonconvulsive children, but HVA levels did not differ. ACTH therapy did not change the CSF levels of GABA, 5-HIAA and HVA significantly.


Neuroleptic malignant syndrome-like state in a patient with Down syndrome and basal ganglia calcification.

We report a case of rhabdomyolysis in a 13-year-old Down syndrome patient with progressive quadriplegia, choreoathetosis and dystonia. Cranial CT demonstrated bilateral basal ganglia calcification. He experienced the sudden onset of high fever, cloudiness of consciousness, muscle rigidity and severe opisthotonus. The diagnosis was made on the basis of the marked increases in serum creatine kinase and myoglobin. There was remarkable elevation of 5-hydroxyindoleacetic acid, homovanillic acid and methoxy-hydroxyphenyl glycol in the cerebrospinal fluid during hyperpyrexia. This case exhibited almost all the diagnostic criteria of the neuroleptic malignant syndrome. It was suggested that abnormalities of monoamines in the central nervous system may be related to the pathologic etiology of this state and rhabdomyolysis.


Significant factors in the disinfection and sterilization of flexible endoscopes.

Many nosocomial infection outbreaks have been linked to improper disinfection of the flexible endoscopes used in hospitals and clinics. The objective of this study was to evaluate the efficacy of scope disinfection with glutaraldehyde and hydrogen peroxide in manual and mechanical protocols. Bacillus subtilis and Pseudomonas cepacia were the test organisms. Each channel in two different endoscopes was seeded and evaluated separately. Residual chemical germicide levels in the channels and in the work environment were also measured. Parametric analyses were carried out on log transformations of number of colony-forming units recovered. Repeated measures analysis demonstrated that both the type of disinfectant and the method of washing were significant factors for disinfection. Hydrogen peroxide proved to be more efficacious than glutaraldehyde for killing or removing B. subtilis in a 10-minute contact period. Automatic disinfection was more efficacious than manual disinfection for killing or removing B. subtilis in a 10-minute contact period. The channel being disinfected also proved to be a significant factor, with carbon dioxide and elevator channels the most difficult to disinfect consistently.


[Classical conditioning of anaphylaxis in sensitized guinea pigs].

An attempt of classical conditioning of anaphylaxis by odor was carried out, using actively sensitized guinea pigs with an inhalation of ovalbumin (OA). One month after sensitization, animals were divided into the conditioned group; group C, and the unconditioned group; group U, consisted of 6 animals, respectively. Dimethylsulfied (DMS: sulfur odor), was inhaled in group C as a conditioned stimulus with OA which is an unconditioned stimulus, while only OA was inhaled in group U. Four days after these procedures, saline was inhaled in group C and DMS was solely inhaled in group U in order to equalize the total inhaled dose of OA and DMS in both groups. These sessions were repeated once a week for seven weeks. After final sessions, all animals were inhaled DMS, saline and OA separately, and blood samples were drawn after each inhalation to measure plasma histamine levels. After an inhalation of DMS only, plasma histamine levels of group C and U were 47.5 +/- 9.7 and 25.7 +/- 1.2 ng/ml, respectively. In group C, plasma histamine levels were 32.9 +/- 4.7 at the inhalation of saline and 59.0 +/- 9.2 ng/ml at OA inhalation. Plasma histamine level after an inhalation of DMS only was significantly higher in group C than that in group U (p < 0.05). These results suggest that the conditional stimulus (DMS inhalation) may induce histamine release in the absence of any antigenic stimulus and support the evidence for mast cell-neuron interaction.


Heterophile antigens in serous cystadenocarcinoma of the human ovary.

As the aberrance of carbohydrate moieties, the appearance of heterophile antigens in human ovarian cancer tissue was studied using various heterophile antigens and antibodies together with antisera raised in rabbits by immunization with extracts of human ovarian cancer tissues. In the immunodiffusion test, rabbit antisera reacted with a heat-stable, perchloric-acid extractable antigen that was different from CEA and CA125 and possessed Hanganutziu-Deicher (HD)-type specificity in all 4 specimens of serous cystadenocarcinoma of the ovary. Furthermore, a pathologic serum with strong HD-antibody complex formed a precipitin line with one of them. In the cancer tissue, we also immunochemically demonstrated the presence of rat-erythrocyte antigens. However, other types of heterophile antigens, such as Paul-Bunnell and Forssman, were not detected. These results suggest that HD-type antigens appear in serous cystadenocarcinoma of the human ovary, one in broad distribution and some in specific cases.


Rapid axonal transport velocity is reduced in experimental ethylene oxide neuropathy.

Chronic exposure of rats to ethylene oxide (EO) causes distal axonal neuropathy of lumbosacral primary sensory neurons. To study the pathogenesis of this neuropathy, we measured rapid axonal transport in peripheral nerves. Rats were exposed for 6 h to 500 ppm EO in a chamber three times a wk for 15 wk. Rapid axonal transport and quantitative histological alterations of peripheral nerves were studied. After [35S]methionine injection into the dorsal root ganglion, the velocity of rapid anterograde axonal transport of radioisotope-labeled protein was measured. The velocity in the rats exposed to EO was 33% less than that in control rats exposed to filtered room air. However, histological differences were slight. Morphometric studies showed that in EO-exposed rats, only the distal portions of the sural nerve had significantly greater incidental degeneration of myelinated fibers than did controls. There were significantly fewer large myelinated fibers only in the distals peroneal nerve. Therefore, a decrease in the velocity of anterograde axonal transport, related to these slight histological abnormalities of the peripheral nerve, may play a causative role in the development of the distal axonal neuropathy owing to chronic EO exposure.


[Intralesional therapy with natural interferon-beta in refractory squamous epithelial cancers of the ENT area].

It has been the aim of the present investigation to study the effect of intratumorally applied human fibroblast interferon (nIFN-beta; Fiblaferon 5 for the first two weeks, and Fiblaferon 3 three times a week) in a phase-II clinical trial of thirteen patients with advanced head and neck squamous cell carcinomas. All of the patients had failed established therapeutic modalities before and could not be treated by conventional procedures. nIFN-beta was injected intratumorally and its effect on tumour size was assessed by an independent, second observer as well as via CT and MR imaging. All assessments were done prior to treatment, 8 weeks after beginning treatment and at 16 weeks. Three female and ten male patients with primary tumours of the hypopharynx (n = 5), the larynx (n = 4), the oropharynx (n = 1), the glandula submandibularis (n = 1), the oral cavity (n = 1) and the oesophagus (n = 1) have undergone outpatient treatment three times a week. Tumour size showed no change in six patients while progressive disease occurred in seven cases after eight weeks of treatment. Radiological findings did not change in the nine patients continuing treatment while five showed progressive disease. There were no serious local or systemic side effects due to the intratumoral nIFN-beta treatment. The survival time was 9.73 months after the onset of nIFN-beta treatment.


Rifamycins as inhibitors of retroviral reverse transcriptase from M-MuLV, RAV-2, and HIV-1.

29 Rifamycins were tested for inhibition of Reverse Transcriptase (RT) as potential anti HIV drugs. Two purified commercial enzymes from M-MuLV and RAV-2 were used. Anti-RT activity was also measured on a crude lysate of HIV-1. The results show that some derivatives have interesting levels of activity on isolated M-MuLV and RAV-2 RTs, while they are less active on the RT in the crude HIV-1 lysate. The active derivatives include oximes and hydrazones, alkylaminoderivatives, open ansa-chain derivatives and derivatives carrying a modified nucleoside.


Conformational analysis of receptor selective tachykinin analogs: senktide and septide.

The conformational behavior in solution of two receptor selective tachykinin agonists, senktide (succinyl-D-F-MeF-G-L-M-NH2) and septide (pQ-F-F-P-L-M-NH2) is described. Two dimensional cross relaxation NMR spectroscopy is used together with coupling constant data to obtain interproton distance constraints. These results are used in conjunction with semi-empirical energy computations to indicate favorable conformations. Senktide is found to have a high degree of conformational order which is attributed to rotational restriction associated with the N-methylation of phenylalanine. The lowest energy conformation in accord with the experimental interproton distances contains a beta-turn. Interproton distances indicate that septide exists as a random coil or in an extended chain conformation. Energy computations suggest that septide is primarily an extended chain with internal reorientation restricted by the proline residue. These results may be related to the selectivity of these peptides for different receptors, in that the analogs, with conformations more stable than tachykinins, are more receptor selective.


An algebraic representation of RNA secondary structures.

This paper develops mathematical methods for describing and analyzing RNA secondary structures. It was motivated by the need to develop rigorous yet efficient methods to treat transitions from one secondary structure to another, which we propose here may occur as motions of loops within RNAs having appropriate sequences. In this approach a molecular sequence is described as a vector of the appropriate length. The concept of symmetries between nucleic acid sequences is developed, and the 48 possible different types of symmetries are described. Each secondary structure possible for a particular nucleotide sequence determines a symmetric, signed permutation matrix. The collection of all possible secondary structures is comprised of all matrices of this type whose left multiplication with the sequence vector leaves that vector unchanged. A transition between two secondary structures is given by the product of the two corresponding structure matrices. This formalism provides an efficient method for describing nucleic acid sequences that allows questions relating to secondary structures and transitions to be addressed using the powerful methods of abstract algebra. In particular, it facilitates the determination of possible secondary structures, including those containing pseudoknots. Although this paper concentrates on RNA structure, this formalism also can be applied to DNA.


FTIR study of specific binding interactions between DNA minor groove binding ligands and polynucleotides.

The use of FTIR spectroscopy is made to study the interactions between polynucleotides and two series of minor groove binding compounds. The latter were developed and described previously as part of an ongoing program of rational design of modified ligands based on naturally occurring pyrrole amidine antibiotic netropsin, and varying the structure of bisbenzimidazole chromosomal stain Hoechst 33258. Characteristic IR absorptions due to the vibrations of thymidine and cytosine keto groups in polynucleotides containing AT and GC base pairs respectively are used to monitor their interaction with the added ligands. Although the two thiazole based lexitropsins based on netropsin structure differ in the relative orientation of nitrogen and sulfur atoms with respect to the concave edge of the molecules, they interact exclusively with the thymidine C2 = O carbonyl groups in the minor groove of the alternating AT polymer as evidenced by specific changes in the IR spectra. In the second series of compounds based on Hoechst 33258, the structure obtained by replacing the two benzimidazoles in the parent compound by a combination of pyridoimidazole and benzoxazole, exhibits changes in the carbonyl frequency region of poly dG.poly dC which is attributed to the ligand interaction at the minor groove of GC base pairs. In contrast, Hoechst 33258 itself interacts only with poly dA.poly dT. Weak or no interaction exists between the ligands and any of the polynucleotides at the levels of the phosphate groups or the deoxyribose units.


Mapping of two new human B-cell epitopes on HIV-1 gp120.

To map epitopes on gp120 defined by human antibodies and to examine the neutralizing activity of these antibodies. Serum from HIV-1-antibody-positive individuals was used to screen a random fragment expression library representing gp120 from the HIVIIIB clone BH10. The library was based on the pUEX1 expression vector. Serum was tested for in vitro neutralizing activity using H9 cells and the HIVIIIB isolate. Four different epitopes defined by human antibodies were mapped on gp120. Two of these have not previously been reported and are located within amino acids (aa) 90-100 in the C1 region and aa 355-365 in the semi-conserved region between V3 and V4. The other two are located within aa 140-145 and aa 286-309. These epitopes are situated in regions that have been shown to demarcate human epitopes. Three serum samples with neutralization titres > or = 1024 were identified. None of the purified antibody fractions defining the mapped epitopes on gp120 had any neutralizing capacity against HIVIIIB. This study is the first demonstration of the applicability of random fragment expression libraries for the direct screening of human serum in order to map epitopes on gp120. Two new epitopes and two previously identified epitopes were mapped in this way. However, none of the linear epitopes was defined by antibody fractions neutralizing HIVIIIB, and it was not possible to map epitopes defined by neutralizing antibodies in the serum samples capable of neutralizing HIVIIIB infection of H9 cells. Thus, it appears that the neutralizing activity of serum in this study was not due to anti-gp120 antibodies defining linear epitopes.


The safety and pharmacokinetics of a reverse transcriptase inhibitor, 3TC, in patients with HIV infection: a phase I study.

To determine the safety and pharmacokinetics of the nucleoside analogue, 3TC. A Phase I, open-label, single-centre study. Twenty asymptomatic, HIV-infected male patients with CD4 lymphocyte counts < 500 x 10(6)/l who had not received previous antiretroviral therapy completed the study. Each patient received a single intravenous dose followed by a single oral dose of 3TC. Four patients were dosed at each of five dose levels (0.25, 1.0, 2.0, 4.0 and 8.0 mg/kg). The most commonly reported adverse event was headache, which was generally reported to be mild. The mean bioavailability of 3TC was 82% following oral administration. The majority of the dose (approximately 70%) was excreted unchanged in the urine. Overall, 3TC was well tolerated following dosing, and there were no significant changes in the safety parameters measured. Phase I/II clinical trials with 3TC are ongoing to evaluate its safety, pharmacokinetics and preliminary activity.


Adriamycin, bleomycin and vincristine chemotherapy with recombinant granulocyte-macrophage colony-stimulating factor in the treatment of AIDS-related Kaposi's sarcoma.

To determine the maximum tolerated dose of granulocyte-macrophage colony-stimulating factor (GM-CSF) that would reduce the severity and duration of neutropenia from combination cytotoxic chemotherapy in the treatment of AIDS-related Kaposi's sarcoma (KS). Phase I, dose escalation. Outpatient clinic of a university hospital. HIV-seropositive patients with advanced KS. Combination chemotherapy consisting of adriamycin, bleomycin, and vincristine (ABV), with escalating doses of recombinant human GM-CSF (rhGM-CSF). Patients were treated for a median of six cycles (range, between two and seven cycles) of biweekly chemotherapy with GM-CSF administered in divided daily subcutaneous doses on days 2-12. Serum cytokine levels of interleukin (IL)-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha were measured before, during, and after therapy to correlate with response to therapy. A GM-CSF dose of 250 micrograms/m2 was well tolerated, whereas the next dose escalation, of 500 micrograms/m2, was associated with dose-limiting toxicities, including grade 3 fever, fatigue, and diarrhea. GM-CSF produced predictable cyclic increases in granulocytes, allowing for delivery of full-dose chemotherapy on schedule. All patients were HIV-p24-antigen-negative at study entry; no activation of p24 antigenemia was observed after repeat testing. Consistent changes in cytokine levels were not observed. Responses included one complete and three partial responses, and two patients with stable disease parameters. We conclude that GM-CSF can be administered safely to patients with AIDS-related KS receiving myelosuppressive chemotherapy, resulting in granulocytic response, without up-regulation of HIV p24 antigen levels in serum.


AIDS-associated mucocutaneous Kaposi's sarcoma treated with bleomycin.

To evaluate efficacy and tolerance of bleomycin in AIDS-associated mucocutaneous Kaposi's sarcoma. A previous study showed that bleomycin was effective and well tolerated in this setting. A non-comparative, open, prospective study. Seventy patients were treated with 5 mg per day intramuscular bleomycin on 3 consecutive days every 2 weeks. Two patients achieved a complete response and 50 a partial response (overall response rate, 74%). Median time to treatment response was 4 weeks (range, 2-12 weeks) and median time to relapse 10 weeks (range, 2-36 weeks). Dose-limiting toxicity consisted of cutaneous adverse reactions. Bleomycin is active against AIDS-associated mucocutaneous Kaposi's sarcoma; relapse occurs after discontinuation of therapy.


Isolation of a gramicidin S hyperproducing strain of Bacillus brevis by use of a fluorescence activated cell sorting system.

A gramicidin S (GS) hyperproducing mutant of Bacillus brevis was isolated by using a protein-staining fluorescence dye (fluorescein isothiocyanate, FITC), and a fluorescence-activated cell sorting system (FACS). By flow cytometry (FCM) analysis after staining with FITC, higher producing cells of the wild-type had higher fluorescence signals than cells with low productivity or cells from a GS non-producing mutant. Staining with FITC did not affect the viability of cells under the conditions chosen for FCM analysis. This enabled us to recover viable cells after sorting. After wild-type cells were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, mutants with higher fluorescence than the parental strain were obtained by cell sorting. Among them, strain 18 was chosen as a GS hyperproducer; it produced 590 micrograms GS/ml compared to 350 micrograms/ml by the wild-type strain. This method has the advantage of being able to screen large numbers of cells in a short time. Furthermore, use of the fluorescence dye technique will expand the use of FACS to the improvement of other cultures that produce metabolites that do not have a specific fluorescence or strong enough fluorescence for normal cell sorting.


Rapid assessment of bacterial viability by flow cytometry.

The ability of a flow cytometer to rapidly assess microbial viability was investigated using three vital stains: rhodamine 123 (Rh123); 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] and fluorescein diacetate (FDA). Rh123 was found to clearly differentiate viable from non-viable bacteria. The methodology for staining bacteria with this dye was optimised. Rh123 was shown to stain and discriminate several different species of viable bacteria although this was not universal. Viable cells of Bacillus subtilis were found to stain better with FDA than with Rh123. The results demonstrate the ability of flow cytometry to rapidly detect and estimate the viability of bacterial populations.


Antibody epitopes sensitive to the state of human immunodeficiency virus type 1 gp41 oligomerization map to a putative alpha-helical region.

Two antibodies, affinity-purified from human immunodeficiency virus-positive human plasma with synthetic peptides in the region gp41(566-596), were found to recognize oligomeric gp41 more strongly than the monomeric form in an immunoblot assay. In contrast, a murine anti-gp160 monoclonal antibody, which maps within this sequence to gp41(581-596), recognized only monomeric gp41 after disruption of the oligomer with sodium dodecyl sulfate. This monoclonal anti-gp160 antibody did not recognize chemically crosslinked oligomeric gp41 that had been treated with similar conditions used to disrupt the gp41 oligomer. These results indicate that this epitope is inaccessible to binding by this antibody when gp41 is oligomeric. Cyanogen bromide cleavage of gp41 resulted in a 17-kD fragment Thr-541-Met-631. A significant proportion of this fragment was oligomeric when derived from chemically crosslinked gp41. The region Ala-566-Gln-596, within the cyanogen bromide fragment, contains the oligomerization-sensitive epitopes as well as two lysine residues available for crosslinkage. This region is relatively conserved and has the propensity to form an amphipathic alpha-helix.


Early changes of urinary amylase isoenzymes in diabetes mellitus.

The altered excretion of isoenzymes of amylase in urine was used as an early indicator of the loss of electric charges in the glomerular basement membrane, in 202 juvenile-onset insulin-dependent diabetic patients, compared with the pattern of excretion in 51 normal subjects matched for age and sex. Diabetics showed an increased excretion of salivary amylase. The salivary to pancreatic amylase ratio in urine (S/P ratio) was always below 1 in control subjects, but was elevated in 33.2% of diabetics, although microalbuminuria was present in only 26.2% of diabetic patients. The concentrations of other proteins in urine were within the reference ranges in nearly all patients, indicating that the kidney was not seriously affected. The increased salivary amylase excretion was not due to changes in the plasma concentration of any of the isoamylases, but to a real increase in excretion, as its fractional excretion in relation to creatinine clearance was clearly increased (1.0 +/- 0.7 vs. 1.52 +/- 1.99, p < 0.05), and the ratio of their clearances was also increased (0.35 +/- 0.18 vs. 0.49 +/- 0.61, p > 0.05). Moreover, the prevalence of altered S/P ratios was higher than the prevalence of microalbuminuria (36.6% vs. 18.8% of patients in the first decade of evolution of insulin-dependent diabetes mellitus). Altered S/P ratios were most prevalent in the first decade, whereas microalbuminuria was most prevalent in the second decade of the disease.(ABSTRACT TRUNCATED AT 250 WORDS)


Alpha 1-microglobulin (protein HC): features of a promising indicator of proximal tubular dysfunction.

alpha 1-Microglobulin (protein HC) is a stable urinary indicator protein which reflects acute and chronic dysfunctions of the proximal renal tubule. High urinary concentrations were found to be indicative of tubular drug toxicity, interstitial nephritis or chronic renal failure. The protein is synthesized by liver cells and readily associates with serum immunoglobulin A. Only the free form is filtered through the glomerulus and is reabsorbed by proximal tubular cells. The exact physiological function of this member of the new superfamily of lipocalins is still unknown, but there are indications that alpha 1-microglobulin (protein HC) may serve as an excretion vehicle for fluorescent, charge-heterogeneous substances of unknown nature. Additionally, it seems to be associated with the humoral and cellular immune response.


Masking the cryptodeterminant on the 54-kilodalton mouse sperm surface antigen.

Previously, we reported that the epitope of a 54-kDa sperm surface sialoglycoprotein on the flagellum is masked by sialic acid residues. The epitope is referred to as a hidden determinant or cryptodeterminant. This paper reports the manner in which the epitope is masked as evaluated qualitatively and quantitatively by means of SDS-PAGE/immunoblots (Western blot) and ELISA. Immunoblotting with four specific monoclonal antibodies to the 54-kDa sialoglycoprotein--T21 (IgM), MC71 (IgG1), MC81 (IgM), and MC91 (IgM)--demonstrated that not only IgM but also IgG antibody MC71 and the Fab fragment MC71 are masked. Quantitative evaluation with ELISA to compare the antibody titration curves of the masked and unmasked antigens on sialidase-treated and untreated sperm, respectively, indicated that sialidase caused the antibody-binding ability of the epitopes to increase to a different level for each antibody. There were 32-256-, 8-16-, 16-, and 2-4-fold increases in binding to T21, MC71, MC81, and MC91 antibodies, respectively. These results suggest that the antigen-masking through the cryptodeterminant does not depend upon the subtype or the molecular mass of the antibody, but upon the biochemical nature of the epitope region that is closely related to the sialic acid. The mechanism and physiological roles of the antigen-masking are discussed.


Distribution of beta 1 integrin subunit in rat seminiferous epithelium.

We have studied the presence and distribution of beta 1 integrins in the seminiferous epithelium of prepubertal and adult rats. Our immunofluorescence data show that in the adult the antibody recognizes specific areas localized around the heads of elongating and maturing spermatids and above spermatogonia at stages I-VII. The following were found to be negative: a) areas adjacent to spermatogonia at stages IX-XIV and adjacent to spermatocytes and to round spermatids; b) spermiated spermatozoa. In the prepubertal rat, positive tubules are first apparent around Day 17 of age. Immunofluorescence and immunoprecipitation studies show that Sertoli cell monolayers from 3-wk-old rats express beta integrins in vitro.


Polyclonal antibodies against the rat NK1 receptor: characterization and localization in the spinal cord.

We have developed antibodies against the NK1 receptor and have investigated its cellular distribution. Rabbit polyclonal antibodies were generated against peptide (19-32) of the rat brain NK1 receptor. They were very specific to the NK1 site as shown by ELISA against various epitopes of NK1, NK2 and NK3 receptors and by immunoblotting of proteins from bacteria transfected with rat brain NK1 receptor cDNA and from rat cortex. Determining how immunostained NK1 receptors are distributed in the rat spinal cord made it possible to identify the cellular structures on which NK1 receptors are located and where they form synapses with SP terminals. In the superficial layers of the dorsal horn, the NK1 receptors appeared mainly of dendritic nature and were, like SP, abundant. In the deep layers of the dorsal horn and in the ventral horn, they were associated mostly with cell bodies.


Chronic NO synthase inhibition fails to protect hippocampal neurones against NMDA toxicity.

Since nitric oxide (NO) is supposed to mediate excitotoxicity in various brain structures, the effects of two NO synthase inhibitors were studied on rat hippocampal lesions induced by the focal injection of N-methyl-D-aspartate (NMDA). Although both drugs (NG-nitro-L-arginine methyl ester: L-NAME and L-NG-nitroarginine: L-NOARG) were given twice daily for 4 days before NMDA injection, at doses which are known to profoundly inhibit NO synthase activity, no significant decrease of NMDA-induced damage could be observed. These results do not confirm the current hypothesis of a NO involvement in NMDA toxicity at least on hippocampal neurons, in vivo.


Accelerated expression of cholecystokinin gene in the brain of rats rendered tolerant to morphine.

Results from behavioural studies have shown that central cholecystokinin octapeptide (CCK-8) is a powerful antagonist against opioid effects and that an increased release of central CCK-8 plays an important role in the mechanisms of tolerance to morphine analgesia. No information is available concerning the rate of biosynthesis of CCK-8 in response to chronic morphine administration. Blot hybridization experiments made in the present study revealed a marked increase in preproCCK mRNA in the brain of rats receiving chronic morphine treatment for 1, 3 and 6 days, showing an increment of 52% (p < 0.05), 62% (p < 0.05) and 97% (p < 0.01) respectively. The results suggest that an acceleration of the expression of the CCK gene during long-term morphine treatment might constitute one of the mechanisms for morphine tolerance.


U-50,488H into A10 reduces haloperidol-induced elevations of accumbens dopamine.

Injections of kappa (k) opioid agonists into the A10 ventral tegmental area (VTA) induce behavioral inhibitions and decreased interest in incentive stimuli, behavioral changes indicative of decreased mesolimbic dopamine (DA) transmission. In seeming contrast, three separate laboratories have recently reported that intra-VTA injections of k agonists do not affect nucleus accumbens septi (NAS) basal levels of extracellular DA. In the present experiment, we investigated whether intra-VTA injections of a k agonist would decrease pharmacologically-stimulated increased levels of extracellular NAS DA. It was found that intra-VTA injections of the k agonist U-50,488H significantly attenuated haloperidol-induced elevations of DA, as measured by in vivo microdialysis.


The trophic effect of beta-amyloid 25-35 peptide is not mediated by NK1 or bombesin receptors.

beta-Amyloid peptide and spantide have previously been described to have trophic effects on hippocampal neurones in vitro. We report here that bombesin and [Pro9]-substance P also show a neurotrophic effect on cultured hippocampal neurones. The neurotrophic effect of spantide or a beta-amyloid fragment containing amino acids 25 to 35 was not blocked by addition of the NK1 receptor agonist, substance P or the nonpeptide NK1 antagonist, RP 67580. For the bombesin-related peptides, the antagonist [Tyr4-DPhe12]-bombesin also provokes a trophic response, but the agonist alytesin and the antagonist [Leu13-psi-(CH2NH) Leu14]-bombesin have no effect on neurite growth. These results suggest that the observed trophic responses are unlikely to be mediated by a classical NK1 or bombesin receptor.


Modulatory role of substance P on gonadotropin and prolactin secretion in the rabbit.

Substance P (SP) is present in large quantities in the brainstem and hypophysiotropic areas of the brain, but its roles in gonadotropin and prolactin secretion are controversial. The aim of this study was to measure luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin (PRL) release from the pituitary after either intracerebroventricular (ICV) injection or infusion of SP or its C- and N-terminal fragments in intact (INT) and ovariectomized (OVX) conscious rabbits. A single injection of SP into the 3rd cerebral ventricle (3CVT) in INT and OVX rabbits augmented plasma LH concentrations, especially when SP was applied during the initial phase of an LH peak. Injection of SP during the declining phase of LH release was not effective. Injection of SP into the 3CVT was followed by increased plasma PRL concentrations in OVX but not in INT rabbits. Both SP 1-11 and SP 1-7 failed to alter LH, FSH, and PRL secretion when the peptides were slowly infused into the 3CVT, although ICV infusion of SP 6-11 did cause a delayed increase in LH release. The results support a stimulatory role of SP on LH and prolactin release. The results further indicate that although the stimulatory effect of SP on LH is ovarian steroid-independent, in the absence of ovarian steroids, SP is stimulatory only during the rising phase of an LH pulse. A dual role of SP-ergic transmission in modulating LH secretion is discussed.


[Palliative interventions].

The German Gastric Cancer TNM Study included 1335 patients, of whom 657 (49.2%) underwent palliative surgery. The rate of resection was 50.6%. Postoperative complications occurred twice as often after palliative resectional surgery than after non-resectional procedures. In contrast, operative mortality rates did not differ. The median time of survival was 3 months after non-resectional procedures and 11 months after palliative resections. A considerable restriction of the quality of life occurred 2-3 months before death in both patient groups. Therefore, patients with non-resectional surgery hardly profit from the operation.


[Conservative, palliative therapy of malignant pleural mesothelioma].

No effective conventional therapy for malignant pleural mesothelioma has yet been described. Radiotherapy does not increase median survival, but there is a palliative analgesic benefit. Chemotherapy with a different regimen in 182 patients led to a median survival time of 12 months. In comparison to an untreated historical group of 142 patients, there is an advantage in survival of 5 months. Additional surgical treatment has not prolonged the life expectancy. The percentage of long-time survivors was 5.5%.


[Conservative therapy of malignant pleural effusion].

The nonsurgical treatment of malignant pleural effusion is palliative in principle. This aim must be achieved with a minimum of side effects and risks to the patients. Before treatment, a clear histologic diagnosis must be made. Thoracoscopy offers a fast and safe result with a more than 90% reliability. Systemic cytotoxic treatment and local procedures are available. Using pleurodesis, talkum is clearly superior to tetracycline in achieving a very low recurrence rate. The median survival in 287 patients was 9 months.


[Pelvic multivisceral resection from the viewpoint of gynecology].

The pelvic exenteration procedure must be considered a multimodal therapeutic concept from a humanistic-medical point of view. Careful patient selection, radical resection of all tumor-infiltrated organs, complete interdisciplinary organ reconstruction, and optimal perioperative management including full psychosomatic rehabilitation are mandatory. This concept should only be realized in medical centers with highly sophisticated infrastructures.


Activation of Ca(2+)-dependent currents in cultured rat dorsal root ganglion neurones by a sperm factor and cyclic ADP-ribose.

The effects of intracellular application of two novel Ca2+ releasing agents have been studied in cultured rat dorsal root ganglion (DRG) neurones by monitoring Ca(2+)-dependent currents as a physiological index of raised free cytosolic Ca2+ ([Ca2+]i). A protein based sperm factor (SF) extracted from mammalian sperm, has been found to trigger Ca2+ oscillations and to sensitize unfertilized mammalian eggs to calcium induced calcium release (CICR). In this study intracellular application of SF activated Ca(2+)-dependent currents in approximately two-thirds of DRG neurones. The SF induced activity was abolished by heat treatment, attenuated by increasing the intracellular Ca2+ buffering capacity of the cells and persisted when extracellular Ca2+ was replaced by Ba2+. In addition, activity could be triggered or potentiated by loading the cells with Ca2+ by activating a series of voltage-gated Ca2+ currents. Ca(2+)-activated inward current activity was also generated by intracellular application of cyclic ADP-ribose (cADPR), a metabolite of NAD+, which causes Ca2+ release in sea urchin eggs. This activity could also be enhanced by loading the cells with Ca2+. The cADPR induced activity, but not the SF induced activity, was abolished by depleting the caffeine sensitive Ca2+ store. Ruthenium red markedly attenuated SF induced activity but had little action on cADPR induced activity or caffeine induced activity. Our results indicate that both SF and cADPR release intracellular Ca2+ pools in DRG neurones and that they appear to act on subtly distinct stores or distinct intracellular Ca2+ release mechanisms, possibly by modulating CICR.


Colony-stimulating factor (CSF)-dependent growth of two leukemia cell lines.

The progressive accumulation of leukemic cells in acute myeloblastic leukemia (AML) results from the self-renewal capacity of leukemic blast progenitors. The growth of leukemic blast progenitors is supported by growth factors and colony-stimulating factors (CSFs) have been shown to stimulate this phenomenon in vitro. After repeated subculture of leukemic cells obtained from a patient with AML M4 in the presence of recombinant G-CSF, a cell line dependent on G-CSF was established. This cell line, designated OCI/AML1a, does not respond to GM-CSF, interleukin-3 (IL-3), IL-1 or stem cell factor as well as G-CSF. The stimulatory effect of G-CSF on OCI/AML1a cells is almost completely blocked by monoclonal anti-G-CSF antibody. With G-CSF added in the culture, the OCI/AML1a cell line has been growing exponentially for over 5 years now. Another cell line, with growth dependent on IL-3, has also been established from a patient with chronic lymphocytic leukemia in the acute phase. This cell line TMD2 does not respond to G-CSF, GM-CSF, IL-1, or stem cell factor and anti-IL-3-antibody blocks the stimulatory effect of IL-3 on these cells. Receptors for IL-3 have been found on the surface of TMD2 cells. Although the TMD2 cell line is not derived from AML, the novel character of IL-3-dependency provides useful information for the study of the role of growth factor(s) in leukemic cell proliferation. These two CSF-dependent cell lines are expected to be excellent models for the investigation of the precise mechanism by which G-CSF and IL-3 stimulate the growth of leukemic cells.


The prognostic significance of the CD34 antigen in acute myeloid leukaemia.

The prognostic value of CD34 expression on leukaemic blast cells was assessed in 38 patients with acute myeloid leukaemia. Nineteen patients had more than 10% CD34 positive blast cells. Median survival for the CD34 positive patients was 125 days and for the CD34 negative patients the median survival has not yet been reached at day 575 (p = 0.06). Of those patients who received intensive chemotherapy, CD34 positive patients (n = 13) had a median survival of 150 days while for CD34 negative patients (n = 14) the median survival has not yet been reached (p = 0.01). Adjustment for age and pre-existing myelodysplastic syndrome did not affect the correlation of CD34 positivity with survival (p = 0.02). Over the period of observation (median 10 months, range 2-19 months) the relative risk of death was 5 times greater for the CD34 positive patients. This study suggests that CD34 expression is an adverse prognostic marker, independent of age and pre-existing myelodysplasia.


The clinical significance of cytokines and soluble forms of membrane-derived activation antigens in the serum of patients with Hodgkin's disease.

In a search for specific serum markers with prognostic impact in Hodgkin's Disease (HD), we evaluated the clinical significance of several cytokines (IL-1 beta, IL-2, IL-3, IL-6, G-CSF, GM-CSF, TNF-alpha) and soluble forms of membrane-derived antigens (sCD4, sCD8, sCD23, sCD25, sCD30) in the serum of patients with untreated HD. Elevations of three groups of serum factors were observed: Firstly, elevations of the hematopoietic cytokines GM-CSF (detected in 39%), IL-6 (57%) and IL-3 (13%), which occurred simultaneously in the majority of the cases; secondly, simultaneous elevations of the inflammatory cytokines TNF-alpha and IL-1 beta (detected in 7%); and finally, elevations of membrane-derived activation antigens sCD8, sCD25, and sCD30. While the cytokine levels did not correlate with other obvious parameters, the membrane-derived activation antigens sCD8, sCD25 and sCD30 were associated with a poor prognosis. Only sCD30 correlated with disease activity and holds promise for the follow-up of patients in remission. Further investigations of these parameters at the cellular level might help to elucidate the enigmatic biology of HD.


Automated multiple peptide synthesis.

A fully automated instrument for multiple simultaneous peptide synthesis was constructed to provide large numbers of peptides for immunological research. The synthesis is performed in a flow-through mode with the conventional solid supports contained in 48 individual reaction columns. The instrument is based on a commercial autosampler equipped with a motor-driven syringe for accurate delivery of reagents and a robot arm carrying a dispenser needle. Dedicated software was developed to compile overlapping peptides from a given protein sequence and to control all functions of the robot. In situ activation by BOP was chosen as the optimized chemistry protocol. The peptides are cleaved from the resin in the reactors used for synthesis, thus minimizing handling. Performance of the instrument was demonstrated by synthesis of overlapping 14-mer peptides derived from the sequence of HIV reversed transcriptase. A second mode of operation allows the synthesis to be carried out on the surface of polyethylene pins. Peptides derived from the sequence of human TNF were synthesized using this method and used to characterize antibodies raised against the intact protein.


The current position of allogeneic and autologous BMT in multiple myeloma.

The projected outcome of allogeneic BMT for myeloma based on the EBMT data is as follows: for patients transplanted after first line therapy there is a 30% risk of transplant related death, a 55% chance of being alive at 5 years and a 35% chance of being alive in complete remission at 5 years. If BMT is performed later in the course of the disease, the risk of transplant related death is increased and the chance of long term relapse-free survival correspondingly reduced. Conversely, ABMT has a low mortality risk, but there is currently no evidence that ABMT using unpurged marrow can produce long term cure as no series has shown any evidence of a plateau in remission duration. The early results of maintenance interferon are encouraging but longer follow-up is needed to determine whether the proportion of patients in continuing remission at 5 years will approach that seen after allogeneic BMT. Early results of peripheral blood stem cell transplantation are also encouraging but longer follow-up is required. It remains extremely important when comparing results of ABMT with chemotherapy alone to compare similar patient groups, bearing in mind that patients who have autologous transplantation in first response are by definition responders with good performance status and without significant renal failure. In order to address this problem in a prospective manner, a randomised study has been planned by the EORTC in collaboration with the EBMT. In this study, patients with adequate renal function will be randomised at diagnosis to chemotherapy followed by either autologous BMT or continuing chemotherapy.(ABSTRACT TRUNCATED AT 250 WORDS)


Down regulation of stem cell colony formation by purified CD8 lymphocytes and CD8 conditioned medium: potential importance for bone marrow transplantation in leukemia.

A methodology for selection of the CD8 cell subset from the peripheral blood and bone marrow mononuclear cells was developed using anti-T8 (CD8) antibody and magnetic microspheres coated with anti-mouse IgG. Following optimization of antibody:cell binding ratio and microsphere:cell ratios, CD8(+)-cells in the peripheral blood and bone marrow were effectively removed, with an overall final recovery of 34.9% +/- 8.6%, and 56% +/- 8.5% respectively with complete recovery of stem cells and very little contamination with effector cells. CD8(+)-depleted cell preparations demonstrated a 3-4 fold increase in CFU-C and CFU-E colony formation over non-depleted preparations when stimulated with G-CSF, GM-CSF or IL-3 and erythropoietin. The largest increase in colony formation was evident when IL-3 was used to stimulate colony formation. Purified autologous CD8+ T-cells or culture supernatant from in vitro cultures of purified autologous CD8+ T-cells added back to CD8 depleted preparations, induced 20%-90% suppression of CFU-C and CFU-E colony formation in a dose dependent manner. Colony formation by CD34+ cells, purified by anti CD34 antibodies and magnetic microspheres, were also inhibited by either pure CD8(+)-cell populations or CD8-culture supernatant. Preliminary fractionation studies indicate that the inhibitory factor is a protein of > 50 kd. In contrast, when purified autologous CD4+ cells were added to purified CD34+ stem cells, an increase (< 50%) in colony formation was observed. These results, taken together, suggest that CD8+ T-cells are negative effectors of normal hematopoiesis whereas CD4+ T-cells function as positive effectors.


Multidrug resistance in acute leukemia: a conserved physiologic function.

Native resistance to conventional chemotherapy remains an important cause of treatment failure in the adult acute leukemias. Delineation of cellular mechanisms of drug resistance therefore represents a prerequisite to the development of more effective treatment strategies. The multidrug resistance (MDR) phenotype represents one such mechanism of resistance with direct clinical relevance. This phenotype occurs normally in certain mammalian tissues, and is detectable in tumor cell lines selected for resistance to naturally occurring antineoplastics. The mdr1 gene or its glycoprotein product, P-glycoprotein, is detected with high frequency in secondary acute myeloid leukemia (AML) and poor-risk subsets of acute lymphoblastic leukemia. In prospective studies in AML, MDR overexpression is an independent determinant of response to treatment and overall survival with conventional-dose induction regimens. Investigations of mdr1 regulation in normal hematopoietic elements has shown a pattern which corresponds to its regulation in acute leukemia, explaining the linkage of mdr1 to specific cellular phenotypes. Therapeutic trials are now in progress to test the ability of various MDR-reversal agents to restore chemotherapy sensitivity in high-risk acute leukemias.


Patient-controlled analgesia for older adults.

Use of patient-controlled analgesia (PCA) was compared with nurse-administered intermittent intramuscular (IM) injections of morphine in older adults during their postoperative recovery. Data analyses indicated that the PCA and IM groups did not differ in pain intensity, pain distress, and satisfaction. The PCA group had significantly less sleep disturbance from pain than the IM group. Neither group was considered to have acceptable pain management.


The postoperative pain experience: a description based on the McGill Pain Questionnaire.

In surgical nursing practice, postoperative pain is of particular concern because of its documented effect on recovery and behaviors associated with recovery. Yet, little is known about the nature of this pain, other than its intensity. In this study, a description of the nature of the postoperative pain experience was generated from analysis of patient responses to the McGill Pain Questionnaire in four nursing intervention studies. The combined sample included 246 adult cholecystectomy patients. Patient descriptions of pain on the third postoperative day suggest a circumscribed sensory experience of moderate intensity. Sensory descriptors selected reflect two levels of noxious stimulation, one at the deeper somatic level (e.g., throbbing, stabbing, cramping, pulling, and burning) and one at a more superficial level (e.g., pricking, sharp, pinching, itching, sore, and tender). Descriptors of deeper pain tended to reflect greater intensity. The overall experience was characterized by 16 descriptors, selected by more than 30% of the sample and reflecting sensory, affective, and evaluative dimensions of the pain experience. Comparison of the findings from this combined sample drawn from hospitals in the northeastern United States were remarkably similar to those reported by Taenzer in Canada. The descriptors selected support, extend, and validate the kind of "sensation information" needed in preoperative instruction used in nursing practice and for abdominal surgery and can be useful in the assessment and management of postoperative pain.


Slow potentials activated by afferent and direct stimulation in spinal interneurons of laminae III-V.

Stimulation of hairy skin afferent fibers and/or direct depolarizing current injection initiates persistent changes in membrane potential (slow potentials), lasting up to 1 min or longer, in some deep dorsal horn neurons. Intracellular staining with horseradish peroxidase shows that these cells are interneurons, suggesting that slow potentials modulate moment-to-moment impulse transmission in local spinal circuits.


Substance P stimulates IL-1 production by astrocytes via intracellular calcium.

There is increasing evidence that local substance P (SP) exacerbates peripheral inflammations, partly by stimulating production of inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha). SP may play similar roles in certain central nervous system inflammations. Multiple sclerosis plaques, for example, form around veins which are innervated by unmyelinated SP-containing fibers, and astrocytes in multiple sclerosis plaques stain for SP. We tested whether SP could stimulate IL-1 and TNF alpha production by cultured astrocytes and whether calcium was the second messenger in this process. We found that both SP and the calcium ionophore A23187 raised intracellular calcium ([Ca2+]i) and stimulated IL-1 production in astrocytes. SP also nonsignificantly increased TNF alpha production by astrocytes. Treatment with dibromo BAPTA/AM, an intracellular calcium buffer, blocked SP-induced IL-1 production. These findings indicate that SP induces IL-1 production by astrocytes and uses calcium as a second messenger. Our results indicate local SP may play a role in multiple sclerosis and certain other central nervous system inflammations.


Evidence that [3H]glutamate binding sites are masked by biologically relevant endogenous factor on cell membranes of frog spinal cord.

We identified the possible endogenous factor effective to modulate the binding of [3H]-labeled excitatory amino acid agonists and antagonists in the 100,000 x g supernatant of Triton X-100 (0.01%)-treated cell membranes from frog spinal cords. The factor inhibited the binding of [3H]glutamate to Triton X-100-treated cell membranes, to which the binding capacity of [3H]glutamate increased much more than that to intact cell membranes. The binding capacities of [3H]AMPA (an AMPA type agonist) and [3H]CPP (an NMDA type antagonist) to cell membranes remained low by Triton treatment, but they were enhanced significantly by the addition of the factor. The effect of the factor on the [3H]kainate binding was hardly observable. The factor may provide key information on receptor structures and the classification of receptor types concerning excitatory amino acids in the mammalian central nervous system.


Numerous SP-positive pyramidal neurons in cat neocortex are glutamate-positive.

An immunocytochemical technique that allows visualization of two antigens in the same neuron was used to verify the possibility that some neocortical pyramidal neurons contain both glutamate (Glu) and substance P (SP) immunoreactivity. The results show that a large fraction of SP-positive pyramidal neurons are also Glu-positive, and indicate that in a small population of cortical neurons a fast excitatory synaptic transmitter and a slow peptidic modulator coexist.


Impact of a galanin antagonist on exogenous galanin and natural patterns of fat ingestion.

The peptide galanin (GAL) has a potent stimulatory effect on fat ingestion after administration into the hypothalamic paraventricular nucleus (PVN). This study examined a newly synthesized GAL antagonist, M40, in two separate experiments involving: (1) PVN injections of M40 alone in freely feeding animals, to investigate the importance of endogenous GAL receptor activity in determining natural patterns of fat ingestion, and (2) PVN injections of M40 in combination with exogenous GAL, to determine whether endogenous GAL receptors mediate this peptide-induced response. The results demonstrate that PVN injection of M40 by itself dose-dependently (2-108 pmol) reduces spontaneous ingestion of the fat diet. This phenomenon is robust, behaviorally specific and opposite to that induced by GAL itself. Moreover, the stimulatory effect of PVN-injected GAL on fat ingestion can be blocked by prior PVN administration of M40 at relatively low doses (2-6 pmol), indicating that M40 is a potent antagonist of GAL receptors in the hypothalamus. Together, these results provide the first evidence for the existence of endogenous GAL receptors in mediating the action of exogenous GAL in the hypothalamus. They also constitute a crucial step in demonstrating a physiological function of these PVN GAL receptors in controlling natural patterns of fat ingestion.


Axonal flow blockade induces alpha-CGRP mRNA expression in rat motoneurons.

Axonal flow of motoneurons was blocked in the sciatic nerve by the application of vinblastine at a low concentration (100 microM) not considered likely to cause axonal degeneration. The number of motoneurons expressing alpha-calcitonin gene-related peptide (CGRP) mRNA was significantly increased by vinblastine application, while the expression of beta-CGRP mRNA was not influenced. These findings suggest that the synthesis of these two peptides is differentially regulated and that alpha-CGRP mRNA is possibly down-regulated by unknown factor(s) which normally undergo retrograde transport from the periphery.


Decrease of enkephalins in cerebellum during Wobbler mouse motoneuron disease.

The Wobbler mouse possesses an inherited motoneuron disease, which expresses itself primarily at cervical spinal levels and in cranial motor nuclei. Cell degeneration is sporatic and negligible in other motor regions of the brain (e.g., cerebellum, corpus striatum). However, enkephalin concentrations are consistently lower in the Wobbler cerebellum throughout the motoneuron disease, whereas substance P concentrations are significantly higher late in the disease compared with the normal phenotype littermates. The data imply that early changes in enkephalin (also shown for leucine enkephalin in the spinal cord and brainstem) may be important to the etiology of the Wobbler disorder. Like the late increase of substance P, this may reflect a yet-to-be described response to parent cell degeneration in the raphe nuclei. TRH remained unchanged in Wobbler cerebellum and corpus striatum, wherein the other peptides studied herein also maintained similar concentrations to the normal phenotype littermates.


Differential projections to the superior collicular layers from the perihypoglossal nuclei in the cat.

The primary objective of the present study is to demonstrate the presence of a projection to the superficial layers of the superior colliculus (SC) from the perihypoglossal nuclei, specifically from the nucleus intercalatus (INT) in the cat. Iontophoretic application of WGA-HRP into the perihypoglossal complex produced orthogradely labeled terminals in the SC contralaterally forming two bands: one is in the superficial gray layer, and the other in the intermediate gray layer. The superficial band was evenly distributed in the upper portion of the superficial gray layers (layers II1-2) and the deeper band existed in the intermediate gray layer (layer IV) being arranged in a discontinuous manner. Injections of the tracer into the superficial layers of the SC yielded retrogradely labeled cells only in the rostral part of the contralateral INT; by contrast, the injection confined to the deep layers produced labeling of cells exclusively in the nucleus prepositus hypoglossi (PH). Thus, the INT and the PH each project separately to the functionally different superficial and intermediate layers of the SC, respectively. On the basis of the present anatomical findings, it is suggested that the perihypoglossal nuclei as a whole contribute not only to the oculomotor but also to the visuosensory regulatory function in the SC.


Intrathecal oxymetazoline does not produce neurotoxicity in the spinal cord of the rat.

To determine if intrathecal (i.t.) oxymetazoline (OXY) induces histological evidence of spinal neurotoxicity, male, Sprague-Dawley rats (300-450 g; implanted with an i.t. catheter) were treated with i.t. saline or 100 nmol OXY twice daily for 3 days, or 200 or 300 nmol OXY once daily for 3 days. Spantide (D-Arg1, D-Try7,9, Leu11-substance P; 0.067 nmol = 0.1 microgram, 0.167 nmol = 0.25 microgram or 0.334 nmol = 0.5 microgram) or capsaicin (0.164 mumol = 50 micrograms), given as a single i.t. injection, were used as positive controls. Animals were killed 12 h after the last injection of saline or OXY, and 72 h after spantide or capsaicin. Spinal cord sections (L1 and adjacent segments) were examined by light microscopy for changes in gross morphology, substance P-like immunoreactivity (SP-IR) and calcitonin gene related peptide-like immunoreactivity (CGRP-IR). All doses of i.t. OXY produced antinociception (tail-flick ED50 = 53.7 nmol, paw pressure withdrawal ED50 = 93.3 nmol). Rectal temperature decreased by 1.5-2.4 degrees C up to 12 h after 100 nmol of i.t. OXY. There were no signs of inflammation or necrosis, and no detectable loss or damage to either spinal afferents or motor neurons as judged by SP-IR and CGRP-IR structures in spinal cords of OXY-treated animals (all doses) as compared to i.t. saline controls. Spantide (0.1 microgram) had no antinociceptive or neurotoxic effect; 0.25 microgram induced irreversible loss of the TF reflex and transient hind limb paralysis; 0.5 microgram induced irreversible loss of TF and PP responses, permanent hind limb paralysis, bladder and bowel dysfunction. The spinal cords from these animals showed signs of extensive necrosis, cavitation, and haemorrhage in the ventral horn accompanied by a loss of CGRP-IR motor neurons. Capsaicin-treated rats exhibited a permanent loss of the TF but not the PP response and a marked reduction of SP-IR spinal afferents in the dorsal horn. It is concluded that i.t. OXY produces antinociception in the rat with no detectable spinal neurotoxicity as assessed by parameters which are sensitive to the neurotoxins, spantide and capsaicin.


[Inhibition by pentoxifylline of procoagulant activity produced by endotoxin-active monocytes].

When appropriately stimulated, monocytes are able to initiate blood coagulation through the membrane expression of tissue factor. This procoagulant activity is thought to play a role in activating coagulation in response to inflammatory stimuli in vivo. We found that pentoxifylline, a methylxanthine derivative already reported to regulate some monocyte functions, inhibits the procoagulant activity developed by monocytes in vitro in response to endotoxin. This effect was accompanied by an early increase in intracellular levels of cyclic AMP and was mimicked by compounds that induce an increase in cyclic AMP levels. These results suggest that the suppressive effect of pentoxifylline occurs at least in part via an increase in intracellular cyclic AMP levels.


X-inactivation in girls with Rett syndrome.

Cytogenetic studies have been carried out on a series of nine girls with Rett syndrome, six of their mothers and nine normal female controls. No abnormality of the X-chromosome has been observed in any subject. X-inactivation studies using various methods of detecting the timing of individual band replication were performed. The overall pattern seen was essentially the same in all subjects, but in the patients with Rett syndrome there may be an alteration in the timing of the X-inactivation process in the region Xp11.3 or 4-->Xp21.