Type VI collagen microfibrils: evidence for a structural association with hyaluronan. Type VI collagen, a widespread structural component of connective tissues, has been isolated in abundance from fetal bovine skin by a procedure involving bacterial collagenase digestion under nonreducing, nondenaturing conditions and gel filtration chromatography. Rotary shadowing electron microscopic analysis revealed that the collagen VI was predominantly in the form of extensive intact microfibrillar arrays. These microfibrils were seen in association with hyaluronan, which was identified by its ability to bind the G1 fragment of cartilage proteoglycan. Treatment with highly purified hyaluronidase largely disrupted the collagen VI microfibrils into component tetramers, double tetramers, and short microfibrillar sections. Subsequent incubation of disrupted collagen VI in the presence of hyaluronan facilitated a partial repolymerization of the microfibrils. In vitro binding studies have also demonstrated that type VI collagen binds hyaluronan with a relatively high affinity. These studies demonstrate that a specific structural relationship exists between type VI collagen and hyaluronan. This association is likely to be of primary importance in the growth and remodeling processes of connective tissues.


Retinoic acid receptor expression in human skin keratinocytes and dermal fibroblasts in vitro. Retinoic acid is essential for the normal differentiation of epithelia but its cellular function is obscure. The expression patterns of retinoic acid receptors (RARs) in skin cell types may give an insight into the role of retinoic acid in skin. We have compared the patterns of RAR expression in human keratinocytes and dermal fibroblasts in vitro, and studied the effects of retinoic acid on RAR expression. RAR-alpha and RAR-gamma were expressed in keratinocytes and fibroblasts: RAR-gamma was expressed at similar levels in both cell types but RAR-alpha was more abundant in fibroblasts. There were no differences in expression of either RAR-alpha or RAR-gamma between stratifying (high-calcium medium) and proliferating (low-calcium medium) keratinocytes and expression of these RARs was unaffected by retinoic acid. RAR-beta was undetectable in keratinocytes. In the majority of fibroblast cell lines, RAR-beta transcripts were either undetectable or expressed at a low level. Retinoic acid at low concentrations (10(-10) to 10(-9) M) rapidly induced the expression of RAR-beta. Cyclic adenosine monophosphate (cAMP) analogues inhibit RAR-beta induction in teratocarcinoma cells. However, dibutyryl-cAMP did not affect RAR-beta induction in fibroblasts. Forskolin, an adenylate cyclase activator, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) decreased constitutive RAR-beta mRNA levels but did not block induction of RAR-beta by retinoic acid. Since intracellular cAMP levels were only increased detectably in response to forskolin, the reduction in constitutive levels of RAR-beta mRNA may be mediated by mechanisms other than via cAMP.


High-performance liquid chromatographic determination of rufloxacin and its main active metabolite in biological fluids. A high-performance liquid chromatographic method is described for the determination of a fluoroquinolone, rufloxacin, and its N-desmethyl metabolite in plasma, urine and bile. Samples are chromatographed on a poly(styrene-divinylbenzene) column, the eluate being monitored with a fluorescence detector. The method was validated and a detection limit of 10 ng/ml for both rufloxacin and its metabolite in all the biological matrices considered was found. The method was successfully applied in pharmacokinetic studies.


Seasonality in human reproduction: an update. A seasonal distribution in human natural conception and birth rates has been consistently demonstrated by epidemiological studies in several geographical areas. Possible hypotheses abound to explain this seasonality. Though a seasonal effect on reproduction is well-documented and has been extensively investigated in mammals, information concerning the influence of seasonal variation and its mechanism on human and primate reproduction is scarce. Recent evidence suggests that deterioration in sperm quality during the host summer in sub-equatorial areas, may result in lower conception rates leading to a reduction in the birth rate in spring. In northern countries, however, in regions where a strong seasonal contrast in luminosity exists, activity of the anterior pituitary-ovarian axis and the conception rate are decreased during the dark winter months. In these areas, inversely, a peak in conception rate during summer leading to a maximum in birth rate in spring has been observed. It is believed that seasonality in the ovulation rate may cause this variability. However, changes in the quality of the ovum or in endometrial receptivity which may lead to a greater waste of ovulated eggs and peri-implantation conceptuses at specific times of the year, have also been suggested. These phenomena might have important implications for in-vitro fertilization and gamete intra-Fallopian transfer as well as infertility in general. The seasonal effects, which may influence primate and human fertility and reproduction and its possible mediators, are critically reviewed.


Clinical data which influence semen parameters in infertile men. Spermiograms, Sims-Huhner postcoital tests, in-vitro sperm penetration tests and hormone stimulation tests, including gonadotrophin-releasing hormone, thyrotrophin-releasing hormone and the adrenocorticotrophic hormone (ACTH) test, were obtained in 225 infertile men. The present paper deals with the influence which anamnestic factors have on the spermiogram and on hormone levels. Adrenocortical function decreased with age. Blue-collar workers had poorer sperm parameters than white-collar workers. Smokers exhibited hormone alterations without significant changes in the spermiogram. Regular alcohol consumption was associated with elevated dehydroepiandrosterone-sulphate and oestradiol concentrations and poor postcoital test results. Emotional stress had a negative influence on sperm parameters. Environmental influences warrant additional attention in the evaluation of men with abnormal spermiograms.


Cytomegalovirus DNA detection in sera from patients with active cytomegalovirus infections. Using a specific and sensitive polymerase chain reaction method, we detected reliably the presence of human cytomegalovirus (HCMV) DNA directly in serum samples collected at an early stage of HCMV infection, even before immunoglobulin M (IgM) antibodies were measurable. HCMV DNA was detected in serum from all patients with active HCMV infection; in 91% of these patients, HCMV DNA was found in the acute-phase serum. In 13 of 44 patients, HCMV DNA was found in serum before HCMV-specific IgM. For four kidney transplant recipients, the occurrence of HCMV DNA in serum, virus isolation from urine and leukocytes, and HCMV IgG and IgM serology were determined. We found a correlation between HCMV DNA in serum and positive virus isolation from leukocytes. In three of five congenitally infected infants, HCMV DNA and HCMV IgM were detected in the same sample. Two other infants were HCMV DNA positive, although no HCMV IgM antibodies were measurable. HCMV was found in urine from these infants either by virus isolation or with the polymerase chain reaction. Serum from one of the 22 healthy HCMV-seropositive blood donors was HCMV polymerase chain reaction positive.


Detection of salivary antibodies in cats infected with feline immunodeficiency virus. The saliva of cats infected with feline immunodeficiency virus was examined for total immunoglobulin content and antiviral antibodies. Seropositive cats showed an increase in salivary immunoglobulin G levels, which was only partly attributable to the enhanced prevalence of oral inflammatory lesions, compared with the levels in seronegative cats. Immunoglobulin G, but not immunoglobulin M, levels in serum were also increased. Salivary antibodies were determined by indirect immunofluorescence and Western blot (immunoblot) analysis. All but 1 of the 16 seropositive cats examined were positive, while all 16 control cats were negative. The presence of oral lesions was not a prerequisite for antibody detection in saliva. It was concluded that salivary antibody might be usefully exploited for diagnostic and epidemiologic purposes.


Modified MacConkey medium which allows simple and reliable identification of Providencia stuartii. This work describes a modified MacConkey medium (MCP medium) enabling the simple identification of Providencia stuartii, an emerging nosocomial pathogen. A total of 813 strains, belonging to the families Enterobacteriaceae and Pseudomonadaceae, were tested on MCP medium; all P. stuartii strains were phosphatase positive, as were 97.5% of Morganella morganii strains, in contrast with all other tested organisms. A simple discriminating test, such as the ornithine or citrate test, allowed identification of strains of these species. We have also compared the reliabilities of P. stuartii identification by commercial kits (API 20E system) by using a standard MacConkey or MCP medium. Sixteen and three-tenths percent of P. stuartii strains were misidentified by using the former procedure, while with the latter all strains were correctly identified. Finally, the MCP medium was used over a 6-month period in our routine clinical laboratory. Of a total of 1,278 seeded urine samples from elderly patients, we isolated 103 P. stuartii strains which were all correctly identified by coupling MCP medium and the API 20E system. Seventeen and one-half percent of these strains were misidentified when the API 20E system was used in combination with standard MacConkey medium.


Evaluation of a live attenuated, cold-adapted parainfluenza virus type 3 vaccine in children. Cold passage 18 (CP18) parainfluenza virus type 3 (PIV-3) vaccine was evaluated in a double-blind, randomized, placebo-controlled study of 95 infants and young children. None of 19 seropositive older children 41 to 124 months old became infected when 10(6) 50% tissue culture infective doses (TCID50) of vaccine virus was administered intranasally. Two of nine and seven of twenty-four young seropositive children given 10(5) or 10(6) TCID50 of CP18 PIV-3, respectively, became infected. Each of four seronegative young children became infected, as indicated by virus shedding and antibody response, when given 10(6) TCID50 of CP18 PIV-3 intranasally. Illness was not observed in seropositive children. Two of the four seronegative children developed a mild illness characterized by rhinorrhea and wheezing on auscultation; none had fever. In one case, vaccine virus spread from a vaccine to a sibling control but did not cause illness. The vaccine is attenuated relative to wild-type PIV-3, but additional attenuation will be required to achieve a satisfactory PIV-3 vaccine.


Use of aminotransferase, hepatitis C antibody, and hepatitis C polymerase chain reaction RNA assays to establish the diagnosis of hepatitis C virus infection in a diagnostic virology laboratory. Clinical and therapeutic decisions for hepatitis C virus (HCV) infection depend on factors that include documentation of past infection as well as identification of those who might benefit from antiviral chemotherapy with systemic interferon. To evaluate the ability of a diagnostic laboratory to accurately identify such patients, we compared results obtained with serum transaminase assays, two HCV antibody assays (enzyme immunoassay [EIA] and immunoblot), and a polymerase chain reaction (PCR)-based assay for HCV RNA using a group of consecutively submitted samples within our university-based diagnostic virology laboratory and sera from a population of random blood donors. One hundred percent of specimens with R values of greater than 3.0 in the HCV EIA were positive in the confirmatory immunoblot. However, 25% of specimens with EIA R values of between 1.0 and 3.0 were not confirmed by either recombinant immunoblot assay (RIBA) or RNA PCR assay (false-positive specimens). A significant correlation (P less than 0.01) between increasing reactivity in the RIBA and positivity in the RNA PCR assay was found. The incidence of HCV viremia, as determined by the RNA PCR assay, was 73% for confirmed seropositive specimens, 33% for seropositive specimens with indeterminate RIBA results, 12% for seronegative specimens obtained from infected patients, and 2.0% for seronegative specimens obtained from uninfected blood donors. In contrast, serum transaminase testing did not correlate with the RNA PCR assay for HCV. Use of the EIA and immunoblot assay followed by RNA PCR testing will identify most patients who are viremic with HCV.


Detection of group C rotavirus antigens and antibodies in animals and humans by enzyme-linked immunosorbent assays. Enzyme-linked immunosorbent assays (ELISAs) were developed to detect group (gp) C rotavirus antigens and antibodies. Both assays were confirmed to be specific for gp C rotavirus by using serogroup A, B, and C rotaviruses; hyperimmune antisera to these serogroups of rotaviruses; and paired serum specimens from animals infected with gp C rotaviruses. The ELISA for antigen detection reacted not only with porcine gp C rotaviruses but also with human and bovine gp C rotaviruses. Following experimental challenge of gnotobiotic pigs with porcine gp C rotavirus, the virus was found by ELISA in all diarrheic feces. A high prevalence of antibodies to gp C rotaviruses was detected in sera from adult pigs (93 to 97%) and cattle (47 to 56%) in the United States and Japan. However, no antibody to gp C rotavirus was detected in the sera (n = 20) of adult horses in the United States. In human sera from Hokkaido, Japan, 3% of children and 13% of adults possessed antibody to gp C rotaviruses. These results suggest that the ELISA that we developed may be useful for surveying gp C rotavirus infections in animals and humans. On the basis of serology, gp C rotavirus infections are common in pigs and cattle in the United States and Japan, but they occur at lower levels in humans from the Hokkaido area of Japan.


Effect of dietary acid detergent fiber on responses to high environmental temperature. Twenty-seven multiparous Holstein cows averaging 120.7 DIM, 9 per diet, were assigned at random to 20, 17, or 14% ADF diets of corn, corn silage, and soybean meal, following a covariant period when all cows received the 14% ADF diet. An 18-d period (period 1) of cool weather preceded the onset of high environmental temperature from June 1988 to the end of July 1988 (period 2). Mean daily maximum air temperatures were 35.2, 36.8, and 34.7 degrees C for the covariant period and for periods 1 and 2, respectively. Minimum temperatures were 14.5, 16.0, and 21.5 degrees C. Milk production during both periods was higher, and the decrease in milk production associated with the onset of hot weather was lower, in cows fed the 14% ADF diet. Intake was not affected by diet during period 1 but was lower in cows fed the 17 and 14% ADF diets in period 2 relative to period 1. At any given environmental temperature, DMI was higher in cows fed lower ADF diets. Although DMI declined more rapidly with increasing daily minimum temperature with lower ADF diets, milk production was less sensitive to daily minimum temperature in cows fed the 14% ADF diet.


Energy balance and lactation response in Holstein cows supplemented with cottonseed with or without calcium soap. Holstein cows (n = 58, 21 primiparous), fed corn and wilted grass silages (63:37, DM basis) for free choice consumption, were assigned to control concentrate or supplemented concentrate during wk 1 to 16 postpartum with linted whole cottonseed (15% of projected DMI) alone or with Megalac (.54 kg/d). Our objective was to examine the effects of fatty acids on energy and N balances, total tract digestibility, and milk fatty acids in wk 7 and 16 and to assess total lactation responses. During balance measurements, fatty acids constituted 4.1, 6.8, and 8.6% of DM in control, oilseed, and oilseed plus protected fatty acid diets. Fat additions reduced fiber digestion (attributed to oilseed) and, to some degree, DMI and milk yield, but enhanced fat test without affecting protein percentage. Supplementary fat increased the proportion of C18:0 in milk at the expense of short-chain fatty acids. Supplemental oilseed with or without protected fatty acids reduced total heat production by 6% and reduced heat in excess of maintenance by 8%. Best estimates of NEL in linted whole cottonseed and of fat in Megalac were 1.81 and 5.69 Mcal/kg of DM. In total lactation, primiparous cows yielded more milk and FCM when fed oilseed plus Megalac and less of each when fed oilseed alone than controls. In pluriparous cows, milk yield was reduced by 2.7 kg/d relative to other treatments when oilseed plus Megalac was fed; FCM yield increased about 2 kg/d only when oilseed was supplemented alone. Overall, data suggest that basal ration fat and oilseed supplementation were too high or that supplementation should have been delayed until feed intake was higher.


Dynamic model prediction of the value of reduced solubility of alfalfa silage protein for lactating dairy cows. A net carbohydrate and protein system was used to develop model diets for lactating dairy cattle with various protein solubilities in the alfalfa silage component of the diet. The objective was to determine the level to which alfalfa silage could be used to replace supplemental protein sources as the silage protein solubility decreased and to estimate the value of silage treatments needed to reduce protein solubility. Four cow groups were considered: early lactation multiparous cows, primiparous cows, midlactation cows, and late lactation cows. Diets were balanced for metabolizable protein, metabolizable energy, and ammonia and peptides for rumen bacteria; limits on DMI and effective NDF were enforced. Lower protein solubility was predicted to increase the yield of bacteria per unit of alfalfa silage DM and the yield of metabolizable protein per unit of alfalfa silage CP. Because of reduced protein supplements, diet costs were decreased. The savings per unit of silage in these rations increased as alfalfa silage protein solubility decreased. For example, with a reduction in solubility from 61 to 51% of CP, the savings ranged from $2.96 to $3.26/tonne of silage across the four cow groups. The value of acid treatment of silage needed to effect these reductions exhibited diminishing returns as application rate increased and appeared to be most cost effective when used on high quality alfalfa fed to high producing cows with application rates less than 2 kg/tonne. Management practices that reduce silage temperatures were predicted to save $.50 to $1.50/tonne of silage when the diets were balanced to account for protein degradability.


Effects of varying forage and concentrate carbohydrates on nutrient digestibilities and milk production by dairy cows. Five Holstein cows with ruminal cannulas were used in a 5 x 5 Latin square design to determine the effect of replacing forage NDF with soyhull NDF and varying concentrations of nonstructural carbohydrates on nutrient digestion and milk production. Diets in which NDF percentage from forage (corn silage: alfalfa hay, 1:1) was 80 (control), 70, or 60 were formulated by substituting soyhulls for forage; total forage was 43.2, 36.7, and 31.1% of the diets, respectively, but total NDF was 31%. Nonstructural carbohydrates were formulated to be 47 (control), 35, or 25% by substituting soyhulls, roasted soybeans, and Ca soaps for concentrate. Ruminal acetate: propionate ratio decreased linearly when diets lower in forage NDF were fed, but it increased quadratically when dietary nonstructural carbohydrates were reduced. Apparent digestibility of OM increased quadratically, but NDF digestibility and lactation performance were unaffected when diets lower in forage NDF were fed. Digestibility of NDF increased linearly when nonstructural carbohydrates were reduced, perhaps because of greater digestibility of soyhull NDF and smaller negative associative effects. Fat from soybeans and Ca soaps was increased as nonstructural carbohydrates decreased. Added fat probably increased fatty acid digestibility and decreased milk protein percentage. Greater FCM production without correspondingly greater feed intake or BW loss increased feed efficiency as nonstructural carbohydrates decreased. In dairy rations containing soyhulls, 60% of dietary NDF from forage should maintain lactation performance, and decreasing nonstructural carbohydrates to 25 to 35% of feed DM, coupled with adding dietary fat, may decrease negative associative effects and improve efficiency of milk production.


Development of buffer systems for pH control and evaluation of pH effects on fiber digestion in vitro. An in vitro buffering system capable of pH control between pH 5.8 and 6.8 was developed to examine the effect of media pH on disappearance of NDF at various times of fermentation and to assess initially the effect of media pH on kinetics of NDF digestion. The pH conditions selected for evaluation of these buffer systems were 5.8, 6.2, and 6.8. Use of McIlvaine's solution with sodium bicarbonate was not successful because of rapid drifting of pH downward during fermentation. To evaluate the effectiveness of citric or phosphoric acids as components of phosphate-bicarbonate buffer systems, alfalfa silage and a mixture of alfalfa silage and corn grain (1:1 mixture, dry basis) were fermented for 0, 12, 24, 48, and 72 h. The pH of each flask was measured at 0, 4, 12, 24, 48, and 72 h postinoculation, and pH was readjusted with bicarbonate solution when necessary. Drifting of media pH downward was more noticeable when phosphoric acid was used to adjust the buffer pH than with citric acid. Citric acid had no adverse effects on NDF digestion compared with phosphoric acid when used to adjust a phosphate-bicarbonate buffer system. Alfalfa hay, bromegrass hay, and corn silage were incubated for 0, 12, 24, 48, 72, or 96 h at pH 5.8 or 6.8 using the phosphate-bicarbonate buffer system adjusted with citric acid. Estimation of kinetics of NDF digestion indicated that a decrease in media pH from 6.8 to 5.8 resulted in a marked reduction in NDF digestion; the largest apparent difference was extended digestion lag time.


Quantitative relationships between cyclic adenosine-3',5'-monophosphate and lipolysis in adipose tissue during the peripartum period. The objective was to study the control of lipolysis and to determine kinetic relationships between subcutaneous adipose tissue cyclic AMP concentrations and rates of lipolysis in primiparous dairy cows in late pregnancy and early lactation. Adipose tissue was biopsied from primiparous cows at -30, -15, -5, 5, 15, 30, and 60 d around parturition. Tissue was incubated with the following treatments: basal, no additions; isoproterenol at 10(-5) M; adenosine deaminase at 1 x 10(6) munits/ml; combined isoproteranol and adenosine deaminase; isoproteranol, adenosine deaminase, and 1 mM theophylline. Cyclic AMP was highest at .25 h and remained elevated for 2 h. Response of cyclic AMP at .25 h was 5-, 9-, 27-, and 38-fold for the four stimulatory treatments, respectively. Glycerol release at 2 h increased 3-, 2.3-, 2.7-, and 3-fold, respectively. Lipolysis was related logarithmically to cyclic AMP concentrations within and among treatments and times around parturition. Either logarithmic or Michaelis-Menten equations predicted similar maximum lipolysis but increased sensitivity to cyclic AMP in tissue from lactating compared with pregnant heifers. Thus, the sensitivity of response of lipolysis to cAMP may be increased in adipose tissue from first lactation cows. These relationships also may be useful in constructing and improving mechanistic models of adipose and whole animal metabolism.


Effect of total and rumen undegradable protein on the performance of cows fed low fiber diets. Twelve multiparous and 18 primiparous Holstein cows were fed a 17.3% CP, 21.0% ADF diet during wk 2 through 6 postpartum. Cows then were assigned from wk 7 through 14 to one of three low fiber (10.7% ADF) dietary treatments containing either 14.4 or 18.7% CP, the latter with or without a soybean meal enhanced with rumen undegradable protein. Treatments had no effect on milk yield or composition in multiparous cows, although milk fat percentage was not depressed in multiparous cows receiving the low fiber diets. The soybean meal diet enhanced with rumen undegradable protein increased yields of milk, 4% FCM, fat, protein, and DMI compared with the 14.4% CP diet in primiparous cows; it also increased yields of 4% FCM and fat versus the 18.7% CP, untreated diet in primiparous cows. Blood urea N concentrations were greater for high CP diets than for the low CP diet in both parity groups. Rumen acetate: propionate ratios were higher for both high CP diets than for the low CP diet in multiparous cows. Soybean meal enhanced with rumen undegradable protein improved yields of milk and its components in primiparous cows fed low fiber diets, even when high protein diets were fed.


Influence of dietary protein and supplemental niacin on lactational performance of cows fed normal and low fiber diets. Forty-seven cows (24 primiparous) were assigned to one of four normal (20.5%) ADF diets for wk 2 to 5 postpartum. Dietary treatments in a 2 x 2 factorial design were diets of 13.8 versus 18.8% CP and 0 versus 12 g/d of niacin per cow. During wk 6 to 13 postpartum, cows were fed low (11.8%) ADF diets while maintaining CP and niacin treatments. Low CP diets contained solvent-extracted soybean meal; rumen soybean meal with enhanced undegradable protein was used in high CP diets. High CP diets increased milk protein percentage in multiparous cows and yields of milk, 4% FCM, fat, protein, and SNF in primiparous cows during the normal fiber period. High dietary CP also increased yields of 4% FCM, fat, protein, and SNF in primiparous cows fed normal fiber diets. When switched to low fiber diets, primiparous cows fed high CP diets decreased more in 4% FCM and fat yields than those fed low CP. Primiparous cows fed niacin decreased more in 4% FCM than controls. High dietary CP increased DMI in primiparous cows fed normal fiber diets, but those fed low CP diets increased more in DMI when switched to low fiber diets. Supplemental niacin appeared to interact with dietary CP in multiparous cows, increasing blood glucose and decreasing blood beta-hydroxybutyrate and NEFA concentrations with the high CP, normal fiber diet. Increased dietary CP improved yields of milk and milk components in primiparous cows.


Leukotriene D4 receptor blockade inhibits the immediate and late bronchoconstrictor responses to inhaled antigen in patients with asthma. We have tested the hypothesis that leukotriene D4 (LTD4) receptor activation is involved in the development of antigen-induced bronchoconstriction. In two studies, patients with asthma received infusions of placebo or MK-571, a potent and specific LTD4 receptor antagonist (450 mg or 37.5 mg total dose, respectively). Antigen was inhaled during test-drug administration, and FEV1 was measured for 10 hours after challenge. Urine samples were collected for measurement of LTE4; plasma samples were drawn repeatedly for assay of MK-571. MK-571 infusions inhibited both immediate (0 to 3 hours) and late (3 to 10 hours) asthmatic responses. For the high MK-571 dose, the extent of inhibition, as assessed by the area under the curve of FEV1 versus time was 88% (p = 0.01) and 63% (p = 0.01), for immediate and late responses, respectively. The low MK-571 dose also inhibited both responses but to a minor extent. Mean urinary LTE4 excretion was elevated after antigen challenge and was unaffected by administration of the LTD4 receptor antagonist. The present study demonstrates that MK-571 inhibits antigen-induced asthma in a dose-related fashion; it had not effect on antigen-induced increases in urinary LTE4 excretions. The results suggest that LTD4 receptor activation plays an important role in antigen-induced asthma.


Inhibition of eosinophil density change and leukotriene C4 generation by nedocromil sodium. Nedocromil sodium (NS) has been shown to inhibit the late asthma response to inhaled antigen and to control symptoms in chronic asthma; in both processes the eosinophil is thought to be an important contributor. To understand the antiinflammatory actions of NS in asthma, its effects on three eosinophil functions were evaluated: (1) change in cell density during in vitro culture, (2) synthesis of leukotriene C4 (LTC4), and (3) generation of superoxide anion. In these studies normal density (greater than 1.095 gm/ml) purified human peripheral blood eosinophils were cultured for 24 hours in 50% conditioned medium from cow pulmonary arterial endothelial cells. After incubation, 45.2% +/- 8.0% of the eosinophils had a density less than 1.085 gm/ml. In the presence of NS (10 mumol/L), only 32.0% +/- 7.3% became less dense (p = 0.0393). In contrast, NS had no effect on changes in cell density after a 20-minute exposure of eosinophils to functional activators N-formyl-methionyl-leucyl-phenylalanine (0.1 mumol/L) or platelet activating factor (0.1 mumol/L). Furthermore, calcium ionophore-activated LTC4 secretion was found to be significantly inhibited by NS (3.8 +/- 0.6 ng/ml vs. 2.4 +/- 0.3 ng/ml with 1 mumol/L NS or 1.7 +/- 0.6 ng/ml with 10 mumol/L NS, p less than 0.025). However, NS did not significantly alter eosinophil superoxide anion generation. The effects of NS on eosinophil function suggest a mechanism by which this medication may be effective in asthma, particularly in the regulation of the late asthmatic response. Furthermore, the selective regulatory effects of NS may also provide insight into the biologic activities of eosinophils.


Inhibitory effects of proopiomelanocortin on cortical and medullary activity in the calf adrenal. The effects of the infusion of ACTH1-24 and proopiomelanocortin on the denervated adrenal gland have been investigated in conscious 3-6 weeks-old calves by means of the adrenal-clamp technique. To prevent variation in the release of endogenous ACTH the pituitary stalk was cauterized during preparatory surgery. ACTH1-24 (5 ng/min per kg, i.v.) increased the output of cortisol from the adrenal by about 500 ng/min per kg body weight and this effect was rapidly reduced by simultaneous infusion of ovine proopiomelanocortin at 5 ng/min per kg. Release of met5-enkephalin and leu-enkephalin from the adrenal was reduced by ACTH1-24 (P less than 0.05) and this effect was enhanced significantly by additional infusion of proopiomelanocortin (P less than 0.02). However met5-enkephalin represented less than one-third of the met-enkephalin containing peptide released and the output of this pool was unaffected by infusion of ACTH. Proopiomelanocortin had no effect on met5-enkephalin production nor that of the total pool of met5-enkephalin containing peptides. It is concluded that ACTH reduced the quantity of pro-enkephalin processed to met5-enkephalin.


Viral agents associated with infantile gastroenteritis in Nigeria: relative prevalence of adenovirus serotypes 40 and 41, astrovirus, and rotavirus serotypes 1 to 4. Sixty-six stool specimens from infants with diarrhoea in Nigeria were examined for the presence of viral pathogens. Rotaviruses were found in 25.8% of specimens and astroviruses in 1.5%. Serotypes were determined for 47.1% of the rotavirus positive specimens, all of which were serotype 1. RNA analysis revealed no unusual electrophoretic profiles. No enteric adenoviruses were detected. In contrast, in a parallel study conducted in the UK, rotaviruses (including serotypes 1, 2 and 4) accounted for 21.9% of infections, adenovirus serotypes 40 and 41 13.6%, and astroviruses 4.5%.


Atypical rotavirus genomic patterns identified by polyacrylamide gel electrophoresis. Two atypical "short" rotavirus genomic profiles were detected in stool specimens following the testing of 84 children with diarrhoea. Both rotaviruses were identified as Group A, and confirmation was performed by blocking assay using antiserum against simian rotavirus SA-11. This report underscores the importance of screening on the molecular level to detect atypical or unusual rotaviruses that would normally proceed unrecognised by testing with any of the commercial antigen detection kits.


Rifabutin and ethambutol do not help recurrent Crohn's disease in the neoterminal ileum. We tested the efficacy of rifabutin and ethambutol in combination in healing severe recurrent Crohn's disease in the neoterminal ileum. Sixteen patients who had undergone an ileal resection with partial colectomy and ileocolonic anastomosis and who had severe recurrent lesions in the neoterminal ileum at baseline ileocolonoscopy were included in the study. The patients received rifabutin in a dose of 5 mg/kg together with ethambutol, 20 mg/kg. Ten patients were treated for 6 months; six patients dropped out because of adverse events. As no effect was observed after 6 months, five patients completed a 12-month therapy course. Follow-up ileocolonoscopy was performed at 3, 6, and 12 months. In none of the 10 patients who completed the 6-month trial and in none of the five patients who completed 12 months of therapy was endoscopic improvement of the lesions observed. In four patients the disease was clinically progressive, and two patients underwent a reresection after 6 and 8 months, respectively. This study shows that long-term therapy with rifabutin and ethambutol does not heal Crohn's disease in the neoterminal ileum and does not prevent recurrence of symptoms and complications.


The use of antimycobacterial agents in Crohn's disease. Seventy-five years ago, a Scottish surgeon, Dalziel, clearly described Crohn's disease (CD) and suggested that it might be caused by a mycobacteria. However, mycobacteria were not isolated from CD tissue until 1978 and 1984. Since then several investigators, using sophisticated polymerase chain reaction (PCR) techniques, found Mycobacterium paratuberculosis sequences in cultures of Crohn's disease tissue in approximately two thirds of patients. Because of a possible mycobacterial etiology, Rutgeerts et al. (J Clin Gastroenterol 1992;15:24-8) treated resected ileocolonic CD patients who had early evidence of recurrence with ethambutol and rifabutin. Patients were followed by colonoscopy to observe changes in the intestinal lesions, but no improvement was noted. Rutgeerts et al. concluded that antimycobacterial therapy did not affect the course of CD. Failure of these antibiotics does not necessarily negate the mycobacterial theory of CD, however. The study population of Rutgeerts et al. was too small, and the treatment period too short. Furthermore, ethambutol shows little or no activity against the proposed Crohn's organism, Mycobacterium paratuberculosis; use of only one active agent (rifabutin) can lead to drug resistance and failure of antibiotic therapy. We hope that Rutgeerts et al. will continue these important studies, but with a more active combination of antimycobacterial drugs for a longer time.


Per-rectal portal scintigraphy with technetium-99m pertechnetate for the early diagnosis of cirrhosis in patients with chronic hepatitis. We evaluated the role of per-rectal portal scintigraphy with 99m-technetium pertechnetate (99m-Tc test) for early diagnosis of cirrhosis. Forty patients with biochemical evidence of chronic liver disease were studied. Laparobiopsy documented chronic active hepatitis (CAH) without cirrhosis in 22 of the patients and CAH with cirrhosis (CAHc) in 18 patients. Clinical or laboratory findings could not differentiate between CAH and CAHc. Twelve healthy volunteers served as controls. The results, expressed as shunt index (SI), i.e., the ratio between heart radioactivity and the sum of heart and liver radioactivity in the first 30 s of observation, were: controls 5.66 +/- 1.66, CAH 15.27 +/- 2.83 and CAHc 24.88 +/- 3.95. A significant difference between the mean SI values in the three groups studied (F = 142.71, p less than 0.0001) was observed. At values less than 17, our test showed a predictivity of 100% for cirrhosis exclusion, while at values higher than 19 the predictive positive value for a diagnosis of cirrhosis was 100%. Invasive diagnostic procedures should be performed only in patients with SI values between 17-19.


Enhanced expression of HLA class I by inhibited replication of hepatitis B virus. HLA class I display on hepatitis B virus (HBV)-infected hepatocytes is important for limiting HBV infection. However, the effect of HBV replication on HLA class I expression on host cells has not been determined. Since acyclovir is known to inhibit HBV replication of the novel cell line HB611, which was transfected with HBV genome using human hepatoblastoma cells as the recipient and continuously replicates HBV DNA, we analyzed HLA class I expression on acyclovir-treated HB611 by quantitative flow cytometry. The results demonstrated that acyclovir treatment clearly increases the level of HLA class I on HB611, and suggested that HBV replication inhibits expression of HLA class I on infected hepatocytes. This effect of HBV replication on the host cell may be a means by which HBV evades immune surveillance to maintain chronic infection.


Phosphorus-31 magnetic resonance spectroscopy of the human liver using chemical shift imaging techniques. Phosphorus-31 magnetic resonance spectroscopy of the human liver was undertaken in 28 healthy adult individuals and in 49 patients with liver disease of varying aetiology. Data localised to the liver were obtained using chemical shift imaging techniques. The mean (+/- 1 S.D.) of the peak area ratio phosphomonoesters (PME)/phosphodiesters (PDE) in healthy adult individuals, from spectra obtained with pulse angle 45 degrees and repetition time 1 s, was 0.24 +/- 0.07. The intra-examination variability of this ratio was 20%, the intra-subject variability 27% and the inter-subject variability 32%. An increase in the PME/PDE was observed in the 31P hepatic MR spectrum from primary or secondary tumours in all 17 patients studied, which invariably represented an increase in PME/ATP and, in some cases, a reduction in PDE/ATP. The spectra did not show aetiological characteristics. A non-specific elevation in PME/PDE was also observed in the 31P hepatic MR spectra of 10 (40%) of 25 patients studied who had diffuse liver diseases, such as cirrhosis and infiltrating malignancies. The spectral pattern did not distinguish between diseases of varying aetiologies, but there was a linear correlation between increasing PME/PDE and a reduction in plasma albumin concentrations (p = 0.03). In three patients with hepatic malignancy and abnormal hepatic 31P-MRS, marked spectral changes were observed after successful treatment to debulk the tumour. Only minor changes were observed in the abnormal spectrum of a fourth patient in whom treatment was unsuccessful. Hepatic 31P-MR spectroscopy may prove useful for monitoring disease processes and treatment effects in well characterised patient populations.


Interferon-gamma receptor expression in chronic hepatitis B virus infection. It is known that interferon-gamma (IFN gamma) is not effective in inducing a sustained inhibition of HBV replication in patients with chronic HBV infection in contrast to interferon-alpha (IFN alpha). To determine whether this was related to IFN gamma receptor (IFN gamma-R) underexpression, binding characteristics of IFN gamma to peripheral blood lymphocytes were studied in chronic HBV infection using radioiodinated recombinant IFN gamma. Peripheral blood lymphocytes from patients with chronic HBV infection (n = 20), normal healthy controls (n = 12) and patients with non-viral related chronic liver disease expressed a similar number of IFN gamma-R (medians (ranges): 1891 (1581-2515); 1916 (1589-2441); 1893 (1692-2104) sites/cell, respectively, p = N.S.) with a similar dissociation constant (Kd approximately 0.7-2.7 nM). There was no correlation between IFN gamma-R expression and serum transaminase, serum HBsAg and HBV-DNA titres and liver histology. IFN alpha therapy in vivo also did not enhance IFN gamma-R expression (n = 3). There is therefore, no evidence from this data that IFN gamma-R is underexpressed in patients with chronic HBV infection to account for the difference in clinical response to these two forms of therapy.


Effect of amphotericin B on hepatic cytochrome P-450 and glucose-6-phosphatase in the rat. The effect of amphotericin B on hepatic microsomal cytochrome P-450 (P-450) concentration was measured in vitro, in vivo and ex vivo in the rat. In vitro, both amphotericin B (0-500 micrograms/ml) and its vehicle, sodium deoxycholate (0-410 micrograms/ml), caused similar dose-dependent decreases of P-450 concentrations and glucose-6-phosphatase activity. Intravenous amphotericin B (3 mg/kg) given daily for 3 days decreased antipyrine clearance from control values of 1.24 +/- 0.24 ml/min to 0.67 +/- 0.12 ml/min (p less than 0.001); whereas antipyrine clearance was unchanged by sodium deoxycholate. The P-450 concentration on the third day was reduced from 0.74 +/- 0.14 nmol/mg protein in control rats to 0.33 +/- 0.09 nmol/mg protein in rats treated with amphotericin B (p less than 0.001). Sodium deoxycholate had no effect on P-450 concentration. In contrast, amphotericin B had no effect on either antipyrine clearance or P-450 concentration following enzyme induction by phenobarbital. Amphotericin B had no effect on microsomal glucose-6-phosphatase activity in vivo. Neither amphotericin B nor sodium deoxycholate induced lipid peroxidation, measured as malondialdehyde production. These results show that amphotericin B decreases hepatic cytochrome P-450 content and function in the rat. These effects can not be observed in the enzyme induced state. Amphotericin B has no effect on glucose-6-phosphatase in vivo, the key enzyme of the gluconeogenesis, indicating selective effects on hepatic microsomal function.


Infrequent point mutations in codons 12 and 61 of ras oncogenes in human hepatocellular carcinomas. DNA from human hepatocellular carcinomas (HCC) were analysed for the presence of mutations in codons 12 and 61 of the K-ras, H-ras and N-ras genes. The relevant ras sequences were amplified in vitro using the polymerase chain reaction and point mutations detected by selective hybridisation using mutation-specific synthetic oligonucleotides. In one of the 19 HCCs a mutation in codon 61 of the K-ras gene was detected, whilst in 3/19 HCCs a mutation was found in codon 61 of the N-ras gene. The mutations were all heterozygous A-T transversions and were found in HCCs arising in patients with underlying cirrhosis. In two of these patients where the corresponding normal tissue was available only the wild-type ras gene was detected, indicating that oncogenic activation of the ras gene was a consequence of somatic mutation. In another patient the same mutation in codon 61 of the N-ras gene was found in cirrhotic liver tissue and in all four patients the same mutation was also detected in formalin-fixed, paraffin-embedded liver biopsy HCC tissue obtained at diagnosis. These results indicate that mutational activation of the ras genes at codon 61 is an infrequent but possibly early event in the development of HCC in Britain.


Hepatic expression of type A and type B receptors for tumor necrosis factor. We have examined the in-situ distribution of type A and type B receptors for tumor necrosis factor (TNF) in normal and diseased human liver biopsy specimens. In normal liver tissue, no or very small amounts of TNF receptors were found. In acute and chronic inflammatory liver diseases, a strong up-regulation of the expression of both TNF receptors was found on hepatocytes, bile duct epithelium, sinusoidal lining cells and mononuclear inflammatory cells. With immunoelectronmicroscopy, all these cells showed cytoplasmic, in addition to membranous staining, suggesting active synthesis of the receptor or, alternatively, internalization of the receptor and its ligand. This up-regulated expression of both type A and type B receptors for TNF was similar in acute and chronic active hepatitis, and was not related to the etiology of the liver disease, nor restricted to areas of liver inflammation. Our results indicate that hepatocytes, sinusoidal endothelial cells, bile duct epithelial cells and mononuclear inflammatory cells, by displaying receptors for TNFs, represent target cells for both these cytokines. Up-regulated expression of type A and type B receptors for TNFs endows these cells with augmented responsiveness for the pleiomorphic biological activities of these cytokines during liver injury.


CD3+4-8- alpha beta T cell population with biased T cell receptor V gene usage. Presence in bone marrow and possible involvement of IL-3 for their extrathymic development. Analysis of TCR of a series of CD4-8- (double negative; DN) alpha beta T cell lines induced with IL-3 revealed that their V gene usage was biased for V alpha 4 and V beta 2. This has been confirmed in the primary short-term cultures. Thus, IL-3 induced the generation of DN alpha beta T cells with predominant V beta 2 gene expression from the CD4+/CD8+ T cell-depleted spleen or bone marrow (BM) cells of both normal and nude BALB/c mice within 10 days. It was further indicated that the V beta 2+ beta-chain genes contained few junctional N regions in both IL-3-induced primary DN alpha beta T cells and continuous lines. Search for the in vivo counterpart of in vitro IL-3-induced DN alpha beta T cells revealed that BM, but not spleens, of normal BALB/c and B6 mice did contain a significant proportion of DN alpha beta T cells, and that the majority of them expressed V beta 2+ beta-chain genes with few junctional N regions. The presence of V beta 2+ DN alpha beta T cells was similarly observed in the BM of BALB/c nude mice, but their proportion varied markedly among various strains of mice, which was not linked to H-2 haplotypes. The results indicated that V beta 2+ DN alpha beta T cells in the BM represented one of the thymus-independent T cell populations, whose development was under the major histocompatibility Ag complex-unlinked genetic control. TCR of these T cells were shown to be functional as judged by the proliferative response to anti-V beta 2 antibody. Taken together, present results suggested that IL-3 could induce differentiation and/or proliferation of DN alpha beta T cells with uniquely limited repertoire, which existed preferentially in BM in vivo, and implied the possible involvement of extrathymic endogenous ligands as a positive selection force.


Expression of the Mls-1a superantigen results in an increased frequency of V beta 14+ T cells. Minor lymphocyte-stimulating (Mls) Ag are alloantigens that stimulate T cells expressing specific V beta regions. Recent studies have established that Mls Ag are examples of endogenous superantigens encoded by the products of endogenous mouse mammary tumor virus (MLV) genomes. In a mouse strain that expresses a given mammary tumor virus (Mls) Ag, reactive T cells expressing the corresponding V beta region are profoundly deficient, due at least in part to clonal deletion of the cells during their development. Expression of Mls and other endogenous superantigens, therefore, results in profound alterations in the ultimate repertoire of T cells in an animal. A role for endogenous superantigens in positive selection of T cells has not been previously established. Here we present evidence that expression of Mls-1a leads to a specific increase in the abundance of V beta 14+ T cells. Genetic studies indicate linkage of the effect to the Mls-1a gene. Neonatal tolerance studies argue against the possibility that the increase is due solely to the deletion of Mls-reactive V beta 14- T cells. The results are consistent with the Mls-1a product playing a role in the positive selection of V beta 14+ T cells.


Cognate interaction between T helper cells and B cells. VII. Role of contact and lymphokines in the expression of germ-line and mature gamma 1 transcripts. Complete reconstitution of Th cell function for B cell growth and differentiation was provided by plasma membranes (PM) derived from activated Th (PMAct) in combination with IL-4 and IL-5 (IL-4/IL-5). IL-5 has been shown to be essential for the secretion of all Ig isotypes by resting, conventional B cells activated by PMAct and IL-4. It was shown that in the presence of PMAct/IL-4/IL-5, a high frequency of resting B cells differentiated to express IgG1. IL-4 alone transiently induced the expression of germ-line gamma 1 transcripts. PMAct alone were ineffective at inducing germ-line gamma 1 transcript expression by resting B cells suggesting that Th-B cell contact was an insufficient signal to cause a detectable increase in the steady-state levels of gamma 1 germ-line transcripts. PMAct alone or PMAct/IL-4 did not induce the appearance of transcripts for secreted mu or mature gamma 1. IL-5, in combination with PMAct/IL-4, provided the necessary signal(s) required for the expression of secreted mu and mature gamma 1 transcripts. Therefore, IL-5 appeared to be an important if not essential differentiation factor for conventional B cells that have been activated by cognate help. It appeared that IL-5 promoted the secretion of Ig by inducing the synthesis of mature Ig transcripts.


Endogenous Mtv-8 or a closely linked sequence stimulates rearrangement of the downstream V kappa 9 gene. Mtv-8 is an endogenous retrovirus located 4.6 kb upstream of a V kappa region gene (called V kappa 9M) within the kappa-Ig locus. The proximity of these two genes resulted in several effects. Using a newly developed RNase protection assay for measuring transcription from a single endogenous provirus, we found that Mtv-8 transcription could be detected after juxtaposition of the kappa-enhancers to the normally silent provirus. Reciprocally, using the polymerase chain reaction we observed that the frequency of V kappa 9M rearrangement was 5- to 10-fold higher in spleens from Mtv-8-positive mice (BALB/c, C58.C, A/J, and B6) compared to spleens from mice that lacked the Mtv-8 provirus (C58, C.C58, NZB, and PERA/Ei). Molecular cloning and sequencing of the V kappa 9M gene from C.C58 mice (containing the kappa-locus from C58 mice on a BALB/c background) indicated that at least some Mtv-8-negative strains have a functional V kappa 9M gene. Together these data suggest that Mtv-8 or a closely linked gene enhances V kappa 9M rearrangement. Since Mtv-8 also reportedly produces a superantigen, it appears that Mtv-8 may influence both the T cell and B cell repertoires.


Recombinant tumor necrosis factor-alpha potentiates neutrophil degranulation in response to host defense cytokines neutrophil-activating peptide 2 and IL-8 by modulating intracellular cyclic AMP levels. The neutrophil-activating peptide 2 (NAP-2) and IL-8 are closely related in structure and function. In order to further determine their potential biologic roles in inflammation, we studied their interaction with TNF-alpha-primed human polymorphonuclear neutrophil granulocytes at the levels of effector functions and signal transduction. After short term priming (5 min) by TNF-alpha, suspended cytochalasin B-treated PMN responded to NAP-2 or rIL-8 by substantial augmentation of the degranulation response. After priming with 3 ng/ml TNF-alpha marker release from both azurophilic and specific granules was near maximum. NAP-2 and rIL-8 cooperated with TNF-alpha in very similar ways, as indicated by the almost identical increases in release rates that were induced by equipotent doses of either secondary stimulus. At the signal transduction level, pharmacologic elevation of intracellular cAMP led to the inhibition of NAP-2- or rIL-8-induced degranulation in primed and unprimed PMN, indicating a role for this second messenger as a negative feedback signal. Direct measurement of intracellular cAMP revealed that TNF-alpha by itself did not affect its levels. Instead, TNF-alpha reduced both the scale as well as the duration of the cAMP burst generated in response to secondary stimuli NAP-2 or rIL-8. Thus, there is evidence that TNF-alpha priming of neutrophils for enhanced NAP-2- or rIL-8-promoted degranulation involves the antagonistic down-modulation of stimulus-induced rises in cAMP.


Retinoic acid and phorbol ester synergistically up-regulate IL-8 expression and specifically modulate protein kinase C-epsilon in human skin fibroblasts. Phorbol ester (TPA) and retinoic acid (RA) are two potent immunomodulatory agents whose actions are mediated through distinct signal transduction pathways involving protein kinase C (PKC) and nuclear RA receptors, respectively. We have investigated the interactions between these two pathways in the regulation of expression of the inflammatory cytokine IL-8 in human skin fibroblasts. TPA (as previously reported) and RA both induced IL-8 mRNA and protein in a time- and dose-dependent manner. IL-8 mRNA induction by TPA (10 nM) was maximal (15-fold) within 6 h, and returned to baseline within 24 h of treatment, although maximal induction (10-fold) by RA (1 microM) did not occur until 24 h posttreatment. Induction of IL-8 by TPA was blocked by 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine, which inhibits PKC and cAMP-dependent protein kinases (PKA), but not by N-(2-ganidinoethyl)-5-isoquinoline sulfonamide, which preferentially inhibits PKA, consistent with the participation of PKC in the induction of IL-8 by TPA. In contrast, induction of IL-8 by RA was inhibited by both 1-(5-isoquinoline sulfonamide and N-(2-gamidinoethyl)-5-isoquinoline sulfonamide, suggesting the participation of PKA in the induction of IL-8 by RA. However, activation of PKA by addition of cAMP analogues was not sufficient to induce IL-8 expression. Induction of IL-8 by RA also did not appear to be mediated indirectly through induction of IL-1, because addition of IL-1R antagonist did not block IL-8 induction by RA. RA and TPA added in combination synergistically enhanced expression of IL-8 mRNA, measured at 6 (2-fold) and 24 h (10-fold) posttreatment. To investigate the mechanism of this synergy, the effect of TPA and RA on fibroblast PKC activation and PKC isozyme levels were determined. TPA, either alone or together with RA, but not RA alone, stimulated phosphorylation of an endogenous 80-kDa PKC substrate. Dermal fibroblasts expressed three PKC isozymes (alpha, (delta, and (epsilon). TPA, but not RA, down-regulated PKC-alpha, neither TPA or RA affected the level of PKC-delta, and both TPA and RA down-regulated PKC-epsilon. This latter effect was enhanced 2-fold by addition of RA and TPA together. These data suggest that modulation of PKC-epsilon may be a common participant in the regulation of IL-8 expression by TPA and RA.


Phase I-II trial of foscarnet for prevention of cytomegalovirus infection in autologous and allogeneic marrow transplant recipients. The safety and efficacy of foscarnet for prevention of cytomegalovirus (CMV) infection was evaluated in 19 CMV-seropositive bone marrow transplant (BMT) recipients. All patients received intermittent intravenous (iv) foscarnet: 40 mg/kg every 8 h from 7 days before to day 30 after BMT, then 60 mg/kg once a day until day 75. The main toxicity was transient renal dysfunction, with a greater than 50 mumol/L increase in serum creatinine above baseline in 5 of the 7 autograft recipients and in 6 of the 12 allograft recipients. Only 4 allograft recipients developed CMV infection during foscarnet prophylaxis, and no patient showed evidence of CMV disease. Because 3 allograft recipients receiving concomitant iv amphotericin B showed rapid impairment of renal function, foscarnet prophylaxis should not be given to allograft recipients requiring amphotericin B; otherwise, foscarnet prophylaxis at this dose appears safe after BMT.


Pharmacokinetics of stavudine in patients with AIDS or AIDS-related complex. The pharmacokinetics of stavudine (d4T; 2',3'-didehydro-3'-deoxythymidine) were studied in patients with AIDS-related complex or AIDS enrolled in a dose-ranging phase I/II study. Twenty-two patients were studied after the first oral dose of 0.67, 1.33, 2.67, or 4 mg/kg of body weight; 17 of them underwent an additional steady-state pharmacokinetic evaluation after thrice-daily dosing of the above doses. Stavudine absorption was rapid, with mean peak concentrations of 1.2-4.2 mg/L over the four dose levels studied. From 34% to 41% of an oral dose was excreted as unchanged drug in the urine. The mean values for plasma elimination half-life ranged from 1 to 1.6 h. The absolute bioavailability of a 4 mg/kg oral dose exceeded 80%. There was no change in pharmacokinetic parameters measured after the first dose and after chronic dosing. Stavudine is a new dideoxynucleoside with more complete and less variable oral absorption than existing nucleosides used for treatment of human immunodeficiency virus infection.


UV light-induced reactivation of herpes simplex virus type 2 and prevention by acyclovir. UV B light is a potent stimulus for inducing reactivation of latent herpes simplex virus (HSV) infections. Patients were enrolled in a double-blind placebo-controlled crossover trial to determine whether acyclovir can prevent UV light-induced HSV-2 recurrences. Twenty-four patients with a history of recurrent infection of perigenital sites (e.g., buttock, thigh) were exposed one to four times with 4 minimum erythema doses of UV light. Patients were given acyclovir 200 mg orally five times daily or matched placebo beginning 1 day before each exposure and continuing for 5 days after exposure. There were 13 UV-induced recurrences among 36 placebo treatments and 3 after 38 acyclovir treatments (P = .004). The mean time to recurrence (+/- SE) was 4.8 +/- 0.3 days. HSV-2 lesions developed primarily at the site of UV exposure. The cutaneous distribution and timing of UV-induced recurrences was consistent with a neural localization (dorsal root ganglia) of latent viral infection. This UV light model permits direct examination of events leading to HSV-2 recurrences in humans and can be used to evaluate approaches to prevention.


Factors associated with detection of human papillomavirus E4 and L1 proteins in condylomata acuminata. The E4 and L1 gene products of human papillomavirus (HPV) types 6 and 11 are detected in variable amounts in condylomata acuminata. To study factors associated with detection of these proteins, biopsy specimens containing HPV-6 or -11 were analyzed for E4 protein, L1 protein, and HPV copy number. Seventeen of 50 women biopsied were pregnant. Nine men were also biopsied. Both the E4 and L1 proteins were found more frequently in lesions from pregnant women than from nonpregnant women or men. Both proteins were more often detected in lesions from women than from men. E4 gene products were more often detected in lesions in which L1 protein was detected and a higher HPV copy number was present. Detection of E4 gene products correlates with the detection of L1 protein in condylomata acuminata caused by HPV-6 or -11.


Multistate outbreak of hepatitis A associated with frozen strawberries. A multistate outbreak of hepatitis A was traced to frozen strawberries processed at a single plant. Among 827 students and 60 teachers at an elementary school in Georgia during a 2-week period, 15 developed hepatitis A. Three months later, among 174 residents and 467 staff in an institution for the developmentally disabled in Montana during a 3-week period, 13 developed hepatitis A. Primary attack rates were 10% in the school and 8% in the institution. Cohort analysis in the school implicated consumption of strawberry shortcake in hepatitis A virus (HAV) infection (relative risk, 7.6; 95% confidence interval, 1.04-55.6). In the institution, such analysis implicated desserts and uncooked strawberries as the most biologically plausible vehicle of HAV transmission. Molecular analysis of HAV from patients in the two outbreaks revealed that the viral genomes were genetically identical and distinct from other known US strains. Contamination of food products before retail distribution is rare but should be considered in investigating common-source outbreaks of hepatitis A.


Effects of virus load in the pathogenesis of lentivirus-induced lymphoid interstitial pneumonia. To better define the relationship between ovine lentivirus (OvLV) infection and respiratory disease, pulmonary leukocytes and postmortem lung specimens from 42 sheep seropositive or at risk for OvLV infection were obtained. The lungs were examined for lesions of lymphoid interstitial pneumonia (LIP), and animals were categorized into five groups by severity of LIP and OvLV serologic status. The presence of OvLV in alveolar macrophages was established by proviral DNA amplification using the polymerase chain reaction (PCR), and the proportion of infected cells was determined by a quantitative focal immunoassay (FIA) and by immunohistochemistry. The concentration of OvLV p25 in serum was measured by capture ELISA. In contrast to animals with mild or no pulmonary lesions, sheep with moderate or severe LIP (17/42) were all seropositive, 71% had antigenemia (greater than 2 ng/mL), and 82% had proviral DNA in 1.5 x 10(5) alveolar macrophages. Of sheep positive by PCR, those with moderate or severe LIP (79%) had an average of 3 infected cells/10(3) alveolar macrophages by FIA. These results implicate alveolar macrophages as important target cells in the pathogenesis of OvLV-induced respiratory diseases.


Gonococcal porin vaccine evaluation: comparison of Por proteosomes, liposomes, and blebs isolated from rmp deletion mutants. The gonococcal major outer membrane protein, Por (protein I), is a potential vaccine candidate. A large-scale animal trial compared the immunogenicity of Por inserted in liposomes (mimicking its in vivo structure) with previously used vaccines containing Por. Por was purified from Rmp (protein III)-negative gonococcal mutants and made into five different formulations: proteosomes, proteosomes absorbed to alum, liposomes, gonococcal membrane blebs, and gonococcal membrane blebs absorbed to alum. Por liposomes induced the greatest amount of Por antibodies; proteosomes and both preparations of blebs induced minimal amounts of Por antibodies. Proteosomes absorbed to alum induced slightly lower amounts of Por antibodies than did liposomes, especially when protein IA was used. Whole-organism absorption studies revealed that a significantly greater percentage of liposome-induced Por antibodies recognized exposed portions of the protein than did proteosome- or proteosome/alum-induced antibodies.


Acquisition of Clostridium difficile by hospitalized patients: evidence for colonized new admissions as a source of infection. The frequency of introduction and spread of specific Clostridium difficile strains among hospitalized patients were assessed by serial cultures of patients admitted to a medical-surgical ward with endemic C. difficile-associated diarrhea. Stool cultures were obtained from 634 (94%) of 678 consecutive admissions to the ward (ward admissions), and all C. difficile isolates were typed by restriction endonuclease analysis. Sixty-five ward admissions introduced C. difficile to the ward, and 54 initially culture-negative admissions acquired C. difficile on the ward. Ward admissions hospitalized within the prior 30 days in the medical center were more likely to be culture-positive for C. difficile at admission to the study ward than those not previously hospitalized at the institution (16% vs. 7%, P less than .001). Nosocomial acquisition of a C. difficile strain was preceded by a documented introduction of that strain to the ward by another asymptomatic ward admission in 16 (84%) of 19 instances, suggesting that C. difficile-colonized new admissions are a major source of nosocomial C. difficile infections.


Enhanced release of elastase and oxidative inactivation of alpha-1-protease inhibitor by stimulated human neutrophils exposed to Pseudomonas aeruginosa pigment 1-hydroxyphenazine. The in vitro effects of the Pseudomonas aeruginosa-derived phenazine pigments pyocyanin and 1-hydroxyphenazine (1-hp) on neutrophil elastase release and myeloperoxidase-induced inactivation of alpha-1-protease inhibitor (alpha 1-PI) were investigated. 1-hp (6-25 microM), but not pyocyanin, caused a dose-dependent enhancement of elastase release by FMLP:cytochalasin B (CB)-activated human neutrophils. 1-hp (0.78-6.25 microM) also increased the oxidative inactivation of the elastase inhibitory capacity of alpha 1-PI exposed to FMLP:CB-activated neutrophils. Methionine, a scavenger of hypochlorous acid, completely protected alpha 1-PI from inactivation by stimulated neutrophils in the presence or absence of 1-hp. Similar protective effects were observed with sodium azide, an inhibitor of myeloperoxidase. P. aeruginosa-derived 1-hp may promote an elastase-antielastase imbalance in vivo by increasing the release of neutrophil elastase and by enhancing the oxidative inactivation of alpha 1-PI, thereby contributing to the development of tissue destruction in P. aeruginosa-infected patients.


Molecular epidemiology of envelope glycoprotein H of human cytomegalovirus. The complete envelope glycoprotein H (gH) coding sequences of 10 clinical strains of cytomegalovirus (CMV) were determined and compared with those of laboratory strains AD169 and Towne. Their translated peptide sequences segregated into two groups, exemplified by AD169 and Towne. Peptide variation was mostly group-specific and was clustered in the first 37 amino acids, including the signal sequence; in the rest of the molecule, there were scattered amino acid substitutions, usually in single residues. Compared with CMV envelope glycoprotein B, gH is more highly conserved among strains and may be expected to have limited immunologic diversity.


Effect of foscarnet therapy on human immunodeficiency virus p24 antigen levels in AIDS patients with cytomegalovirus retinitis. Circulating human immunodeficiency virus (HIV) p24 antigen levels were measured in 22 AIDS patients who had detectable serum antigen at baseline after induction and maintenance therapy of foscarnet for cytomegalovirus retinitis in phase I/II multicenter trials. The HIV p24 antigen levels decreased from a baseline value of 199 +/- 236 (mean +/- SD) and 140 pg/mL (median) to 106 +/- 218 and 28 pg/mL after 14 days of foscarnet induction therapy (60 mg/kg every 8 h). During chronic foscarnet maintenance, there was a sustained decrease in mean HIV p24 antigen levels below pre-foscarnet therapy baseline concentrations for a median of 16 weeks after foscarnet induction. These results provide evidence for a sustained clinical antiretroviral effect of chronic foscarnet maintenance therapy, consistent with a recent report that foscarnet-treated AIDS patients live longer than ganciclovir-treated patients.


Cytomegalovirus-induced reactivation of Toxoplasma gondii pneumonia in mice: lung lymphocyte phenotypes and suppressor function. Active cytomegalovirus (CMV) infection is associated with immunosuppression and predisposes to the development of life-threatening superinfections in immunocompromised patients. In a mouse model of virus-induced immunosuppression, acute murine CMV (MCMV) infection induced reactivation of dormant Toxoplasma gondii infection, producing Toxoplasma pneumonia. Changes in lung lymphocyte numbers and phenotypes appeared to be integral to the pathogenesis of MCMV-induced reactivation of T. gondii pneumonia. Numbers of lung CD4+ cells decreased during acute MCMV infection in mice with dormant T. gondii infection as well as in previously uninfected mice. Dually infected mice subsequently developed reactivation of Toxoplasma pneumonia. The pneumonia was characterized by a large influx of T lymphocytes, predominantly CD8+ cells, into the lungs. These lung lymphocytes markedly suppressed the ability of immune splenocytes to proliferate in response to T. gondii antigens and concanavalin A in vitro. These results suggested that the initial fall in the numbers of lung CD4+ cells observed after MCMV infection may have induced reactivation of T. gondii infection in the lungs. The subsequent pneumonia appeared to be a manifestation of a massive influx of T lymphocytes, especially CD8+ cells, into the lungs.


The clinical picture of six Egyptian cases of cutaneous leishmaniasis. Cutaneous leishmaniasis (CL) is a skin disease encountered in the East Mediterranean Region including Egypt. In this paper, it was intended to throw some light on the clinical picture of six parasitologically proven human CL. Also, the results of treating three cases of them with Pontostam or Cryosurgery. The whole results were discussed.


[Molecular biological studies on expression of placental CRF gene and its product]. Corticotropin releasing factor, a neuropeptide secreted by the hypothalamus, was recently found to be also secreted by the placenta. However, the biological role of placental CRF is obscure. In this study, we performed Northern blots and in situ hybridizations to determine the time course and site of placental CRF production. Then we measured maternal and fetal CRF, ACTH, cortisol and DHAS levels during the course of pregnancy. The results of these studies indicated that placental CRF mRNA is 1.5 kb in size and its expression increases dramatically near term. It was shown that placental CRF is transcribed in the cytotrophoblast layer. Both maternal and fetal CRF, ACTH and cortisol increased near term. The DHAS level was significantly higher on the fetal side than in the maternal plasma. It was assumed that on the maternal side, placental CRF activates the basal level of the pituitary-adrenal axis to produce more corticosteroids. The increased corticosteroids may compete with progesterone receptors and cause the removal of a progesterone block of uterine muscle contraction. On the fetal side, placental CRF activates the pituitary-adrenal axis to produce corticosteroids and DHAS. The increased corticosteroids may again compete with progesterone receptors. The increased DHAS directly affects the uterine cervix to cause ripening of it.


[Studies on the suppression mechanism of follicle-stimulating hormone (FSH) release by porcine follicular fluid]. To study the mechanism of inhibin action on the release of FSH, the effect of porcine follicular fluid (pFF) on the LH-RH binding to the anterior pituitary cells as well as cyclic AMP (cAMP) and diacylglycerol (DAG) content in the pituitary cells were examined in monolayer cultures of the rat anterior pituitary cells. The suppression of the binding of labeled LH-RH to the pituitary cells was not affected by pFF. Neither the LH-RH receptor concentration nor the affinity of LH-RH with the binding sites was affected by pFF according to the Scatchard analysis. A dose-related decrease in FSH content in the conditioned media was observed in the pFF-treated culture wells, whereas luteinizing hormone levels remained unaltered by pFF. There was no significant difference between pFF-treated and untreated groups in cAMP or DAG content in the rat anterior pituitary cells. These data may indicate that inhibin has no effect on the LH-RH receptor, suggesting the presence of its own receptor on the gonadotropes. Messenger system(s) other than A-kinase and C-kinase systems in the cells may be involved in the suppression of FSH by inhibin.


Verotoxin glycolipid receptors determine the localization of microangiopathic process in rabbits given verotoxin-1. Infection with verotoxin-producing Escherichia coli has been implicated in the cause of hemolytic-uremic syndrome. Cases of thrombotic thrombocytopenic purpura and verotoxin infections have been also described. In this study we sought to determine the following: (1) whether verotoxin induces microvascular lesions in the rabbit, (2) the organ distribution of such lesions, and (3) the distribution of verotoxin glycolipid receptors in the various organs. Rabbits challenged with verotoxin-1 purified from E. coli O157:H7 had anorexia, lethargia, and limb paralysis; renal function, however, was normal. Central nervous system lesions found included pericellular and perivascular edema, focal hemorrhage, vascular lesions, and severe alterations of Purkinje cells. Histologic changes were also seen in the colon, with mucosal and submucosal edema and hemorrhage, and in the lung, with interstitial fibrosis and focal lymphohistiocitic infiltration. No lesions were detected in kidney, heart, liver, and spleen. Screening of various tissues for the presence of the verotoxin receptors revealed galabiosyl ceramide in the central nervous system and globotriosyl ceramide in the gastrointestinal tract, lung, and spleen. No receptors for verotoxin were found in the heart, liver, and kidney. These results indicate that organ localization of the disease in rabbits is dependent on the distribution of verotoxin receptors.


Effect of cell proliferation on H-ferritin receptor expression in human T lymphoid (MOLT-4) cells. We have previously demonstrated the presence of receptors specific for human recombinant H ferritin (HrHF) on human T lymphoid cells (MOLT-4), and changes in receptor number and binding affinity with growth and cell cycling have now been examined. Specific binding of HrHF was maximal in MOLT-4 cells harvested during exponential growth with the cells in the DNA synthesis phase of the cell cycle. Specific binding decreased progressively to the plateau phase of growth with the cells in the resting phase of the cell cycle. Scatchard analysis of the competitive binding data for HrHF demonstrated that this decrease in binding was associated with a reduction in receptor number, from 42,140 per cell to 10,306 per cell. Receptor binding affinity increased only minimally over this period, from 7.1 x 10(7) L/mol to 14.9 x 10(7) L/mol. These results indicate that growth- and cell cycle-induced changes in H-ferritin receptor expression are primarily associated with changes in receptor number rather than receptor binding affinity. The present study demonstrates that the expression of this receptor is associated with the proliferative status of the cell and suggests that the H-ferritin receptor may mediate the putative regulatory role of H-ferritin.


An Na, K ATPase inhibitor from ultrafiltrate obtained by hemodialysis of patients with uremia. Ultrafiltrate obtained by hemodialysis of patients with uremia who were not taking cardiac glycosides was used as a source of Na, K adenosine triphosphatase inhibitor for purification and further study. Inhibitory activity was measured by a linked-enzyme assay and by effect on rubidium 86 uptake in guinea pig aortic strips. Two approaches were used in purification: dialysis with a 500 dalton membrane followed by gel filtration with Sephadex G-25, and removal of protein by acidification and boiling followed by Sephadex G-10. The first procedure failed to separate the inhibitor from the salt fraction, whereas the second separated the inhibitor from the salt peak but resulted in partial coelution of the inhibitor with endogenous pyruvate, which interferes with the linked-enzyme assay. Pooled, concentrated G-10 elution fractions from the early part of the inhibitor peak, which were free of pyruvate, produced a dose-response relationship by enzymatic assay that was close to parallel with that for ouabain. Like ouabain, these fractions also inhibited 86Rb uptake in guinea pig aorta. Despite these properties, our previous work has demonstrated that the inhibitor, unlike some other ouabain-like or digitalis-like substances obtained from blood or urine, has no apparent role in body fluid homeostasis.


Effect of atrial natriuretic hormone on metoclopramide-induced stimulation of aldosterone. The effect of atrial natriuretic hormone (ANH) on metoclopramide-induced stimulation of aldosterone was studied in eight healthy young men after 3 days of controlled diet (150 mEq sodium, 100 mEq potassium). Baseline values were obtained after subjects had remained sitting for 1 hour. Subjects then received 2-hour infusions of placebo, dopamine (2 micrograms/kg/min), ANH (0.6 pmol/kg/min), and ANH plus dopamine. One hour after the beginning of each infusion, a 10 mg intravenous bolus of metoclopramide was given. Prolactin levels increased 10-fold after metoclopramide with placebo infusion, and about 50% of this stimulation was abolished by preinfusion with dopamine. ANH preinfusion did not suppress prolactin release. Urinary sodium excretion increased prominently during dopamine infusion. ANH at this dose had no effect on natriuresis. The dopamine dose given had almost no effect on metoclopramide-induced aldosterone secretion, whereas ANH infusions, which resulted in approximate doubling of plasma ANH levels, suppressed aldosterone. This study supports a role of ANH in aldosterone regulation, even at nonnatriuretic doses, and suggests that ANH is acting not only through the renin-angiotensin system but under certain conditions has significant physiologic action directly on glomerulosa cells.


Stimulation of alpha 2-adrenoreceptors blunts the phosphaturic response to parathyroid hormone. Acute renal denervation is phosphaturic and enhances the phosphaturic response to parathyroid hormone (PTH). Stimulation of alpha-adrenoreceptors inhibits the renal accumulation of 3,5-cyclic adenosine monophosphate (cAMP) in response to PTH. However, the effect of this blunted cAMP response by stimulation of alpha-adrenoreceptors on the phosphaturic response to PTH is not well understood. Therefore, the effect of alpha-adrenergic stimulation on the phosphaturic response to PTH was studied by infusion of PTH in the presence and absence of epinephrine plus propranolol. Sprague-Dawley rats were acutely thyroparathyroidectomized, and the left kidney was denervated. Stimulation of alpha-adrenoreceptors significantly blunted the phosphaturic response to PTH, decreasing the change in fractional excretion of phosphate (delta FEp) (21.2% +/- 1.3%, n = 8), as compared with the response to PTH alone (FEp, 30.0% +/- 2.4%, n = 7). In subsequent studies, yohimbine (an alpha 2-adrenoreceptor antagonist) or prazosin (an alpha 1-antagonist) were infused to dissociate alpha 1 from alpha 2-adrenoreceptor activity. Coadministration of yohimbine tended to restore the blunted phosphaturic response to PTH induced by alpha-adrenergic stimulation (delta FEp, 25.8% +/- 2.4%, n = 9), whereas addition of prazosin did not affect the blunted response (delta FEp, 16.1% +/- 4.5%, n = 7). We conclude that stimulation of alpha-adrenoreceptors blunts the phosphaturic response to PTH, most likely due to enhanced alpha 2-adrenoreceptor activity.


Direct effect of endothelin-1 on the granuloma cells of the porcine ovary. Specific binding sites for endothelin-1 (ET-1), a novel potent vasoconstrictor peptide, as well as the effects of ET-1 on cytosolic free Ca2+ concentration ([Ca2+]i), intracellular total inositol phosphate (IP) generation and steroidogenesis were studied in cultured porcine granulosa cells. Scatchard analysis of a binding study using 125I-labelled ET-1 indicated the presence of a single class of high-affinity binding sites with almost equal affinity for ET-1 and ET-3: the apparent dissociation constant was 0.59 nmol/l and the maximal binding capacity was 1.84 pmol/mg protein. Affinity-labelling of 125I-labelled ET-1 to the membranes using disuccinimidyl tartarate as a cross-linker revealed one major and one minor band with the apparent molecular weights of 32 kDa and 49 kDa respectively. ET-1 dose-dependently (1-100 nmol/l) induced rapid and transient increases in [Ca2+]i in fura-2-labelled cells. ET-1 also dose-dependently stimulated total IPs in cells prelabelled with myo-[3H]inositol. ET-1 had a slight stimulatory effect on the secretion of progesterone but not of oestradiol from porcine granulosa cells. The present data clearly demonstrate the presence of a non-selective ET receptor (ETB) in porcine granulosa cells coupled with phosphoinositide hydrolysis and [Ca2+]i mobilization, and suggest that ET-1 may play some role in the production of progesterone by porcine granulosa cells.


Effects of steroid hormones on cyclic adenosine 3',5'-monophosphate levels in the rat lung. The possible role of cyclic adenosine 3',5'-monophosphate (cAMP) in mediating the action of steroid hormones was investigated using the rat lung. Male rats were adrenalectomized and treated with olive oil, dexamethasone, corticosterone, deoxycorticosterone (DOC) or progesterone. At the end of 10 days, 100 micrograms isoprenaline/kg was injected intraperitoneally 5 min before the animals were killed to stimulate cAMP production. Adrenalectomy significantly decreased cAMP levels in the rat lung. Dexamethasone and corticosterone pretreatment reversed the effect of adrenalectomy whereas progesterone pretreatment but not DOC pretreatment significantly decreased lung cAMP levels. Cyclic AMP levels in normal female rats, whether pregnant or not, were not significantly different from those in male rats. We concluded that the absence of glucocorticoid, as after adrenalectomy, decreased the cAMP levels in rat lungs and that this could be reversed by either dexamethasone or corticosterone replacement. Progesterone reduced the cAMP content in rat lungs by acting as a glucocorticoid antagonist or by acting directly via progesterone receptors.


Hormone-induced resistance of rat Leydig cells to the cytotoxic effects of ethane-1,2-dimethane sulphonate. Several studies have shown that the cytotoxic agent ethane-1,2-dimethane sulphonate (EDS) specifically destroys Leydig cells in the adult rat testis. It has also been reported that when rats are pretreated with human chorionic gonadotrophin (hCG), administration of EDS does not result in the complete destruction of the Leydig cell population. It has been suggested that hCG pretreatment 'protects' Leydig cells against the cytotoxic action of EDS. In the present study the underlying principles for this resistance to the cytotoxic effects of EDS have been investigated. Within 48 h of the start of daily hCG treatment the number of nuclear profiles of Leydig cells (henceforth called relative number of Leydig cells) had increased from 1014 +/- 40 to 1368 +/- 30 cells per 1000 Sertoli cell nuclei. Previous experiments have indicated that these newly formed Leydig cells probably develop from differentiating Leydig cell precursors. When EDS is administered concomitantly with the third injection of hCG (2 days after the start of hCG treatment), the relative number of Leydig cells surviving EDS treatment was 388 +/- 52 per 1000 Sertoli cells. Hence, there is a similarity between the increase in the relative number of Leydig cells after 2 days of hCG treatment and the relative number of EDS-resistant Leydig cells. The Leydig cells that survived EDS administration showed characteristics which also occur in developing Leydig cells in the immature testis. It is concluded that, in rats pretreated with hCG for 2 days before EDS administration, new Leydig cells with some immature characteristics are formed. One of these characteristics is that these cells are insensitive to EDS.


Control and consequences of adrenergic activation of red blood cell Na+/H+ exchange on blood oxygen and carbon dioxide transport in fish. The catecholamines, adrenaline and noradrenaline, are released into the circulation of fish during a variety of physical and environmental disturbances that share the common feature of a requirement for enhanced blood oxygen transport. Indeed, the dominant factor controlling the mobilization of catecholamines from chromaffin tissue is a depression of blood oxygen content usually coinciding with a reduction of hemoglobin-O2 (Hb-O2) binding to 50-60% saturation. The elevation of plasma catecholamine levels, under such conditions, activates a beta-adrenergic cyclic AMP-dependent Na+/H+ exchanger on the red blood cell (rbc) membrane. The adrenergic responsiveness AMP-dependent Na+/H+ exchanger on the red blood cell (rbc) membrane. The adrenergic responsiveness of the rbc Na+/H+ exchanger to catecholamines varies both within and between species. Such inter- and intra-specific differences may reflect, in part, the availability of cell surface beta-adrenoceptors that are functionally coupled to adenylate cyclase. The activation of rbc Na+/H+ exchange and the accompanying profound adjustments of intracellular and extracellular acid-base status, nucleoside triphosphate (NTP) levels, and cooperativity of Hb-O2 binding have important consequences on both O2 and CO2 transfer and transport in the blood that vary markedly at the sites of oxygenation (the gill) and deoxygenation (the tissues) thereby enabling simultaneous amelioration of O2 loading and unloading. At the gill, oxygen transfer is enhanced owing to increases in Hb-O2 affinity and capacity while at the tissues, oxygen delivery is facilitated by a reduction of Hb-O2 affinity. This reduction in affinity at the tissues is a consequence of the combined effects of increased cooperativity of Hb-O2 binding and a rise in venous PCO2 (PvCO2) caused by the titration of HCO3- by H+ extruded by the rbc Na+/H+ exchanger. This elevation of PvCO2 may contribute to the rise in arterial PCO2 (PaCO2) observed after adrenergic activation of rbc Na+/H+ exchange that is caused primarily by impairment of rbc CO2 excretion related to modification of the intracellular acid-base status.


What good is recombinant DNA if you're not a molecular biologist? Site directed tumorigenesis combines many techniques of modern biology: DNA manipulation and splicing, gene structure and function and transgenic animals with the more "classical" disciplines of cancer research and cell biology. Is this of any use to someone interested in how a whole mammal functions? Or is this merely an attempt to explain the forest by studying the leaves? Recently, site directed tumorigenesis was used to develop a clonal cell line that may help answer some important questions in reproductive endocrinology. This essay outlines some of these questions, briefly explains how this cell line was produced and tries to describe some of the ways in which these cells will help us answer the questions.


Characterization of mineral-binding 40-kDa glycoprotein extracted from young adult rabbit alveolar bone. A forty-kilodalton (40-kDa) protein was extracted from alveolar bone of young adult rabbit with 0.5 M EDTA after extraction with 4 M GuHCl, and purified by gel-filtration, anion-exchange and hydroxyapatite columns using a high-pressure liquid chromatography system under denaturing conditions. The purified 40-kDa protein was not susceptible to bacterial collagenase and thrombin, but was cleaved by cyanogen bromide. The protein was stained blue with Stains-all. Among various lectins, concanavalin A and lentil lectin agglutinin bound to this protein, but peanut agglutinin, Ricinus communis agglutinin, phytohemagglutinin-E and wheatgerm lectin agglutinin did not. Lectin binding assays showed that the protein is a glycoprotein containing large amounts of mannose and/or glucose residues, but is not a fragment of proteoglycan. The amino acid composition of the protein shows a characteristically high content of acidic amino acids. Therefore, the mineral-binding 40-kDa glycoprotein is considered to be osteonectin/secreted protein acidic and rich in cysteine (SPARC), in terms of similarities to bovine and porcine osteonectins with regard to molecular weight and contents of glycoses and amino acids.


Membrane lipids modifications in human gliomas of different degree of malignancy. A great deal of experimental evidence shows that the growth of tumors is accompanied by significant changes at the expense of the cell surface. For the present paper, we set out to analyse the composition of membrane lipids (cholesterol, phospholipids, neutral and acidic glycosphingolipids, sulphatides) in human cerebral astrocytomas, which is a group of tumors that offer a valid model for the study of the various grades of cellular transformations in vivo. The results obtained in the present study permit us to draw a series of conclusions in relation to the malignancy grade of glial human tumors. In particular, an increase in the malignancy is accompanied by: 1) a reduction of the total lipids, a reduction that involves all the principal classes of lipids of the plasma membrane, for which it has been possible to demonstrate a correlation of an exponential nature, which is significant with the increasing of the histological grading; 2) a gradual accumulation, in the area of glycosphingolipids, of lattosylceramide and GD3, molecules that can constitute a valid marker of the malignancy grade; moreover, the glycolipid composition of astrocytomas of high degree differs from that of tumors of a low grade because of the presence of more complex glycolipids (trihexosylceramide and tetraosylceramide); 3) a gradual increase, in the area of phospholipids, of PC/PE and PC/SM ratios, indices of the microviscosity of the membrane. The data obtained suggest that the profound modifications of membrane lipids, which are gradually accompanied by a progressive increase in the malignancy of the tumor, can, on the one hand, be responsible for functional variations connected with neoplastic growth, and, on the other hand, constitute valuable biochemical parameters which are useful, together with histological studies, in the diagnosis of these tumors.


Interaction of basic fibroblast growth factor with bovine growth plate chondrocytes. The basic fibroblast growth factor (bFGF) family of peptides influences a wide range of cellular actions. To better understand the possible role of bFGF in the growth plate, we have characterized the interaction of this growth factor with isolated bovine growth plate chondrocytes. Basic FGF interacts with two classes of binding sites on these cells. One is consistent with high-affinity bFGF receptors and the other with low-affinity heparin-like binding sites on the chondrocyte surface. Radiolabeled bFGF binding studies revealed approximately 4 x 10(6) binding sites per cell, with a Kd of approximately 42 nM. Graded concentrations of heparin or NaCl competed with [125I]-labeled bFGF in a dose-dependent fashion, reducing [125I]-labeled bFGF binding by 75 and 97%, respectively. The data suggest the presence of a high-capacity, low-affinity class of binding sites with the properties of a heparin-like moiety. Affinity cross-linking of [125I]-labeled bFGF to chondrocytes labeled two principal species with apparent molecular masses of 135 and 160 kDa. Labeled bFGF was specifically displaced from both species by subnanomolar concentrations of unlabeled bFGF. These high-affinity, low-capacity binding sites are characteristic of classical bFGF receptors. Binding of [125I]-labeled bFGF to these sites was also influenced by heparin, consistent with coregulation of binding to the two classes of binding sites. The data suggest that bFGF participates in the regulation of skeletal growth at the growth plate and that this regulation may involve bFGF interaction with at least two distinct classes of binding sites.


Vitamin D receptor expression in human brain tumors. Vitamin D receptor (VDR) has important effects not only on physiological processes related to Ca2+ metabolism but also on cell growth and differentiation. VDR is a member of the Steroid-Thyroid Receptors Superfamily (STRS). Work in our and other laboratories has shown that several other members of the STRS (androgen, estrogen, glucocorticoid, and progesterone receptors) are present in astrocytomas and glioblastomas. We now report the finding of VDR-like mRNA in human anaplastic astrocytomas and glioblastomas. VDR mRNA levels, as determined by a method, developed in our laboratory, based on the polymerase chain reaction, are significantly higher in glioblastomas compared to both low and high grade astrocytomas. We discuss the biological and clinical implications of our results.


Pituitary metastases as presenting lesions of malignancy. Metastatic tumors of the pituitary gland and parasellar region are unusual and are generally observed both in an advanced phase of the disease and at autopsy. The occurrence of symptomatic lesions is however quite rare. Though some clinical features may suggest the presence of a metastasis, the diagnosis is unlikely both clinically and radiologically and more common pituitary lesions are generally suspected. The Authors present two very unusual cases in which the pituitary lesions represented the onset symptoms of an otherwise unknown malignancy. Moreover the increased survival of cancer patients and the routine utilization of CT scan and MRI will probably induce, in the next years, a more frequent discovery of pituitary metastases. The Authors suggest that the diagnosis of pituitary metastases should be more closely considered even in the absence of a known primary tumor.


Resection, including transplantation, for hepatoblastoma and hepatocellular carcinoma: impact on survival. Long-term survival in children with primary hepatic malignancies can not be expected without complete tumor resection. In the last ten years we have treated 21 children with hepatocellular carcinoma (HCC) and 21 children with hepatoblastoma (HEP), with tumor extirpation our surgical goal. Operative treatment included partial hepatectomy ([PH] 20), either primary (10) or delayed (following chemotherapy) (10), total hepatectomy and orthotopic liver transplantation ([OLT] 13), or upper abdominal exenteration and multiple organ transplantation (2). Two patients had both PH and subsequent total hepatectomy and OLT. Overall survival was 48% (20/42), with 9 patients dying of progressive disease prior to removal of their tumor. HEP patient survival was 67% (14/21), including 2 of 6 who underwent primary PH, 7 of 8 who had delayed PH, and 5 of 6 who underwent OLT. Survival for the children with HCC was 29% (6/21), including 1 of 4 after primary PH, 1 of 2 following delayed PH, 3 of 7 following OLT, and 1 of 2 after exenteration and multiple organ transplantation. Preoperative chemotherapy facilitated removal of 10 initially unresectable tumors (8 HEP, 2 HCC) at a second-look procedure. Total hepatectomy and OLT markedly improved survival in patients with disease unresectable by standard methods. Partial hepatectomy, either primary or delayed, should be attempted in all children with hepatic malignancies. Total hepatectomy and OLT appears to be a viable adjunct in the treatment of childhood malignancies, and should be used for otherwise unresectable tumors as part of a carefully planned protocol.


S 14671: a naphtylpiperazine 5-hydroxytryptamine1A agonist of exceptional potency and high efficacy possessing antagonist activity at 5-hydroxytryptamine1C/2 receptors. The interaction at 5-hydroxytryptamine (5-HT) receptors of the novel naphtylpiperazine, S 14671 (1-[2-(2-thenoylamino)ethyl]-4[1-(7- methoxynaphtyl)]piperazine), was compared to that of the 5-HT1A ligands, 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), WY 50,324 [N-(29(4-(2-pyrimidinyl)-1-piperazinyl)ethyl)tricyclo(3.3.1.1(3,7) )- decane-1-carboxamide], (+)-flesinoxan, buspirone and BMY 7378 [(8-[2-[4-(2-methoxyphenyl)- 1-piperazinyl]ethyl]-8-azaspirol[-4-]-decane-7,9-dione 2HCl]. S 14671 showed a very high affinity for 5-HT1A sites (pKi, 9.3) as compared to the reference ligands (pKi values, 9.2, 8.7, 8.7, 7.9 and 8.7, respectively). S 14671 bound in an apparently competitive manner and, in distinction to the reference compounds, possessed a Hill Coefficient (1.4) significantly superior to 1. Although showing low affinity at 5-HT1B and 5-HT3 sites, S 14671 displayed significant affinity at both 5-HT1C and 5-HT2 sites; pKi, 7.8 in each case. Furthermore, S 14671 acted as an antagonist of 5-HT-stimulated phosphoinositide turnover in rat choroid plexus (5-HT1C) and cortex (5-HT2). In vivo, upon s.c. administration, S 14671 acted as a high efficacy agonist in models of 5-HT1A receptor-mediated activity: induction of flat-body posture, spontaneous tail-flicks, hypothermia and corticosterone secretion and inhibition of morphine-induced antinociception. In every test, S 14671 was the most potent compound: it was active at doses as low as 5 micrograms/kg s.c. Relative potency across all tests was S 14671 greater than 8-OH-DPAT greater than WY 50,324 greater than (+)-flesinoxan greater than buspirone with BMY 7378 too weak for comparison to be meaningful. The action of S 14671 in 5-HT1A tests was blocked by BMY 7378 and the 5-HT1A antagonist, (-)-alprenolol, but unaffected by the 5-HT1C/2 antagonist, ritanserin, and the 5-HT3 antagonist, ondansetron. Activation of postsynaptic 5-HT1A receptors was confirmed in 5,7-dihydroxytryptamine-lesioned rats, in which the potency of S 14671 to elicit spontaneous tail-flicks was potentiated. Activation of presynaptic receptors was demonstrated by inhibition of the electrical activity of the dorsal raphe nucleus with the following order of relative potency: S 14671 greater than 8-OH-DPAT greater than WY 50,324 greater than BMY 7378 greater than buspirone. Spiperone, which acts as a pure 5-HT1A antagonist at raphe 5-HT1A receptors, blocked the action of S 14671. In conclusion, S 14671 is a structurally novel ligand manifesting high efficacy and exceptional potency at both pre- and postsynaptic 5-HT1A receptors.(ABSTRACT TRUNCATED AT 400 WORDS)