Effects of disease stage and zidovudine therapy on the detection of human immunodeficiency virus type 1 in semen.

To determine the prevalence and temporal expression of infectious human immunodeficiency virus type 1 (HIV-1) in the semen of HIV-1 seropositive men and to determine whether the detection of HIV-1 in semen is associated with disease stage, zidovudine treatment status, or other clinical factors. A microculture technique was used to detect infectious HIV-1 in semen from a cohort of 95 seropositive men. In addition, semen cultures were performed monthly for at least 6 months for 14 of the men. Information was obtained by interview and extracted from medical records to identify clinical variables associated with HIV-1 in semen. Sixty HIV-1 seropositive homosexual men participating in clinical studies at the Fenway Community Health Center, Boston, Mass, and 35 HIV seropositive bisexual or heterosexual men participating in the California Partner Study of the University of California, San Francisco. Semen HIV-1 culture results, seminal leukocyte counts, Centers for Disease Control (CDC) disease stage, peripheral CD4+ cell counts, zidovudine therapy, HIV risk category. In the cross-sectional study, HIV-1 was cultured from the semen of nine (9%) of 95 men. Factors associated with detection of HIV-1 in semen were peripheral CD4+ cell counts of 0.20 x 10(9)/L (200/microL) or less (adjusted odds ratio [OR], 23.33; 95% confidence interval [Cl], 2.89 to 175.63); symptomatic (CDC class IV) disease (adjusted OR, 6.56; 95% Cl, 1.02 to 66.76); and seminal leukocytosis (greater than 1 x 10(9) white blood cells per liter of semen) (adjusted OR, 7.02; 95% Cl, 1.28 to 39.29). Zidovudine therapy was associated with decreased detection of HIV-1 in semen (adjusted OR, 0.04; 95% Cl, 0.00 to 0.63). In the longitudinal study of 14 men who had neither peripheral CD4+ cells counts of 0.20 x 10(9)/L or less nor seminal leukocytosis, seminal HIV-1 was detected in at least one sample from six men (43%). HIV-1 is more commonly found in semen from men with advanced HIV-1 infection and seminal leukocytosis but can also be cultured from semen of men with neither of these conditions. Zidovudine therapy may decrease the prevalence and/or titer of seminal HIV-1. However, all HIV-1-infected persons should continue to assume that they are potentially infectious through sexual contact.


Fluorogold administration via microdialysis labels neurons terminating within the dialysis region.

The use of microdialysis to monitor the release of neurotransmitters in selected regions of the CNS has increased substantially in the last several years. We describe here a method for retrogradely labeling neurons that terminate within the effective sampling region surrounding the dialysis cannula. This is accomplished by direct infusion of fluorogold through the dialysis cannula. By combining this technique with immunohistochemistry, it is possible to identify neurons that could contribute to the neurotransmitter release measured by microdialysis.


The treatment of hairy cell leukemia: an update.

The treatment of hairy cell leukemia (HCL) requires continuing update. The role for splenectomy is extremely limited. Alfa-interferon therapy has been available and utilized for five years; there have been few complete remissions. The experience with Pentostatin has resulted in a majority of clinical complete remissions after several months of therapy, but persistence of a small percentage of hairy cells in the bone marrow. The most recent therapeutic agent, 2-chloro-deoxyadenosine (2-CDA) is given for seven days and results in a complete remission in the great majority of patients with no evidence of persistent bone marrow disease. If this trend persists, 2-CDA will become first line therapy for HCL as it may cure the disease.


[Genotyping for drug metabolism capacity can give valuable clinical information].

There is pronounced individual variation in the human metabolism (eg, acetylation and hydroxylation) of drugs, in many cases due to genetic factors. The genetic basis of cytochrome P4502D6 gene defects has recently been elucidated, for instance. The cytochrome P4502D6 is absent from 7 percent of the Caucasian population, usually owing to the presence of either of two different mutations in the corresponding gene. PCR amplification of genomic DNA with allele-specific primers enables rapid prediction of the patient's drug metabolism capacity and the individualization of drug treatment with respect to dosage.


Value of axillary dissection in addition to lumpectomy and radiotherapy in early breast cancer. The Breast Carcinoma Collaborative Group of the Institut Curie.

Axillary dissection in early breast cancer remains controversial because of its substantial side-effects and because its value with respect to recurrence or survival has not been unequivocally proven. Between 1982 and 1987, 658 patients were included in a prospective randomised comparison of lumpectomy alone with lumpectomy plus axillary dissection. All patients had a unilateral breast tumour not exceeding 3 cm in diameter and lymph-node involvement or metastases. Radiation therapy was given to both groups. The two groups of patients were similar with respect to mean age, TNM stage, and presence of hormonal receptors. Median follow-up was 54 months. 5-year survival of the patients was 94.2% (95% Cl: 92.1-96.4). There was a significant advantage in survival in the axillary dissection group (p = 0.014). Recurrence of tumour in the breast was similar in the two groups but visceral metastases, supraclavicular metastases, and lymph-node recurrences were less frequent in the axillary dissection group. Survival was related to the age of the patients (p = 0.005), the presence of positive nodes (p = 0.006), the histological grading (p less than 0.0001), and the presence of hormonal receptors (progesterone p = 0.0008, oestrogen p less than 0.0001). Treatment-adjusted relative risk was 2.4 (95% Cl: 1.3-4.2). The findings show that axillary dissection is justified for treatment of small breast cancers, although whether the better survival is due to axillary clearance itself or to adjuvant treatment for lymph-node involvement is unclear.


Prognostic factors and survival in children with perinatal HIV-1 infection. The Italian Register for HIV Infections in Children.

The signs that may arise after perinatal infection with human immunodeficiency virus type 1 (HIV-1) have been classified by the Centers for Disease Control, but the clinical usefulness of the classification system and the prognostic importance of each disease pattern have not been established. We sought to address these issues by analysing data from the Italian Register for HIV infection in children. We studied 1887 children born to HIV-1-seropositive mothers. 1045 were identified at birth and the others were registered later (median age 4.8 [range 0.4-72] months). HIV-1-associated signs developed in 433 (81.8%) of 529 seropositive infected children at a median age of 5 (0.03-84) months. These signs appeared significantly earlier in the 102 children who died of HIV-1-related illness than in those who are still alive (median 3 [0.03-55] vs 6 [0.03-84] months; p less than 0.001). The cumulative proportion surviving at age 9 years was 49.5% (95% confidence interval 27-65%) and the median survival time was 96.2 months. Separate analysis of the 112 seropositive infected children followed from birth and older than 15 months gave similar results. Hepatomegaly, splenomegaly, lymphadenopathy, parotitis, skin diseases, and recurrent respiratory tract infections formed the mildest disease pattern. Lymphoid interstitial pneumonitis and thrombocytopenia were signs of intermediate disease. By contrast, in multivariate analysis specific secondary infectious diseases, severe bacterial infections, progressive neurological disease, anaemia, and fever were significant and independent negative predictors of survival. Growth failure, persistent oral candidosis, hepatitis, and cardiopathy were associated in univariate analysis with significantly shorter survival. Our findings suggest that the outlook for children with perinatal HIV-1 infection is better than previously thought and that a new clinical staging system of single disease patterns is needed.


Intensity of reinfection with Ascaris lumbricoides and its implications for parasite control.

Intestinal helminths are among the most common and widespread of human infections. Because it is typical to find that most worms are aggregated in a few potential hosts it has been suggested that some individuals are predisposed to heavy infections and that morbidity could be controlled by the treatment of heavily infected individuals only. We have studied the prevalence and intensity of reinfection with the intestinal nematode Ascaris lumbricoides among people living in Dhaka, Bangladesh. 880 people were treated with pyrantel pamoate three times at six month intervals, and on each occasion they collected all their stools for 48 h after treatment. Worms expelled by each subject were counted and weighed. The prevalence of infection at round 1 of treatment was 89% and the mean burden was 18.5 worms. Reinfection was rapid and at rounds 2 and 3 the prevalence was 82% and 80%, respectively, with mean burdens of 14.0 and 11.5 worms. The intensity of reinfection was not random: more subjects than expected became heavily reinfected (greater than or equal to 15 worms) and more subjects than expected remained lightly infected (less than or equal to 14 worms) (p less than 0.001). Worms were highly aggregated at each round of treatment but although just over 10% of all subjects were heavily infected at each and every round of treatment, over 60% of all subjects were heavily infected at least once. The findings show that some individuals seem to be susceptible to heavy infection whereas others are not, that deworming has a greater effect on the intensity of infection than on the prevalence, and that mass chemotherapy is likely to be a more effective means to control morbidity than is selective treatment of heavily infected individuals only.


Effects of insulin-like growth factor on linear growth, head circumference, and body fat in patients with Laron-type dwarfism.

Patients with Laron-type dwarfism are clinically indistinguishable from those with isolated growth hormone (GH) deficiency, yet have high circulating GH concentrations associated with an inability to generate endogenous insulin-like growth factor I (IGF-I). Biosynthetic IGF-I was administered subcutaneously once daily for 3 to 10 months to 5 children with Laron-type dwarfism aged 3.3 to 14.5 years. There was a rapid stimulation of linear growth in body limbs, with a striking increase in head circumference, increased body weight, and a reduction in subcutaneous fat. Administration of IGF-I to patients with Laron-type dwarfism seems to have a beneficial effect on growth similar to that observed with long-term administration of GH in children with GH deficiency.


Localised alveolar-septal amyloidosis with hypersensitivity pneumonitis.

Localised alveolar-septal amyloidosis has been thought irreversible. A woman exposed to the dust of sea-snail shells during the manufacture of nacre buttons had clinical and immunological features typical of hypersensitivity pneumonitis; however, transbronchial lung biopsy showed alveolar-septal amyloidosis. There was no evidence of other diseases known to be associated with amyloidosis, nor were amyloid deposits found in other organs. After a year without exposure to the antigen there was no trace of either pneumonitis or amyloidosis.


Mortality from tobacco in developed countries: indirect estimation from national vital statistics.

Prolonged cigarette smoking causes even more deaths from other diseases than from lung cancer. In developed countries, the absolute age-sex-specific lung cancer rates can be used to indicate the approximate proportions due to tobacco of deaths not only from lung cancer itself but also, indirectly, from vascular disease and from various other categories of disease. Even in the absence of direct information on smoking histories, therefore, national mortality from tobacco can be estimated approximately just from the disease mortality statistics that are available from all major developed countries for about 1985 (and for 1975 and so, by extrapolation, for 1995). The relation between the absolute excess of lung cancer and the proportional excess of other diseases can only be approximate, and so as not to overestimate the effects of tobacco it has been taken to be only half that suggested by a recent large prospective study of smoking and death among one million Americans. Application of such methods indicates that, in developed countries alone, annual deaths from smoking number about 0.9 million in 1965, 1.3 million in 1975, 1.7 million in 1985, and 2.1 million in 1995 (and hence about 21 million in the decade 1990-99: 5-6 million European Community, 5-6 million USA, 5 million former USSR, 3 million Eastern and other Europe, and 2 million elsewhere, [ie, Australia, Canada, Japan, and New Zealand]). More than half these deaths will be at 35-69 years of age: during the 1990s tobacco will in developed countries cause about 30% of all deaths at 35-69 (making it the largest single cause of premature death) plus about 14% of all at older ages. Those killed at older ages are on average already almost 80 years old, however, and might have died soon anyway, but those killed by tobacco at 35-69 lose an average of about 23 years of life. At present just under 20% of all deaths in developed countries are attributed to tobacco, but this percentage is still rising, suggesting that on current smoking patterns just over 20% of those now living in developed countries will eventually be killed by tobacco (ie, about a quarter of a billion, out of a current total population of just under one and a quarter billion).


Randomised comparison of olsalazine and mesalazine in prevention of relapses in ulcerative colitis.

Sulphasalazine extends remissions and lessens disease activity during relapses of ulcerative colitis, but it also causes many adverse side-effects. The adverse reactions are mostly attributable to the sulphapyridine carrier moiety rather than the active principle 5-aminosalicylic acid (5-ASA), so agents to deliver 5-ASA to the colon by other means have been designed. We have compared the efficacy and tolerability of two such agents, olsalazine and mesalazine, in maintenance therapy of ulcerative colitis. 100 patients with ulcerative colitis in remission were recruited at one centre and assigned randomly to treatment with olsalazine (Dipentum; 1.0 g daily) or mesalazine (Asacol, with Eudragit-S coating; 1.2 g daily). Compliance, biochemical and haematological variables, and clinical evidence of disease activity were assessed every 3 months for 12 months by observers unaware of treatment allocation. In intention-to-treat analysis, which included as treatment failures patients withdrawn for protocol violations, adverse reactions, intercurrent illness, or non-compliance as well as those with relapses of ulcerative colitis, the olsalazine group had a significantly lower rate of treatment failure than the mesalazine group (12/49 [24%] vs 23/50 [46%]; p = 0.025). Analysis restricted to 64 patients still in remission at 1 year and 18 with relapses also showed a significant difference in relapse rate (olsalazine 5/42 [12%] vs mesalazine 13/40 [33%]; p = 0.024). Both drugs were well tolerated; only 9 patients reported substantial side-effects. Olsalazine was clearly superior to mesalazine in prevention of relapses in ulcerative colitis, especially in patients with left-sided disease.


Validity of methods used to test airway responsiveness in children.

Methods to test airway responsiveness to inhaled agonists in children were originally developed for use in adults, and agonist dosage regimens do not adequately correct for the size of the child. Because small children receive a higher dose relative to their body size than do large children, the age-related decline in airway responsiveness reported in many recent studies might reflect failure to adequately size-correct test dosages rather than a genuine physiological event. Until the administered doses of inhaled agonists can be satisfactorily size-corrected, tests of airway responsiveness in children should be regarded as qualitative rather than quantitative.


Inhibitory effects of beta-alanine on the vagal efferent and afferent excitatory responses of the stomach to stimulation of vagal trunk in cats.

The effects of beta-alanine on the electrically evoked vagal efferent (hexamethonium-sensitive initial excitatory response) and afferent (hexamethonium-resistant delayed excitatory response) responses of the cat stomach were studied. beta-alanine (30 to 300 micrograms/kg, i.v.) dose-dependently inhibited both the efferent and afferent response. The IC50 values of beta-alanine on the efferent and afferent response were 296 +/- 65 micrograms/kg and 128 +/- 35 microgram/kg, respectively. Maximal inhibitory effects of beta-alanine (300 micrograms/kg, i.v.) appeared about 1 hr after the injection. Glycine and taurine (100 to 10,000 micrograms/kg) did not affect these responses. Treatment with hexamethonium (10 mg/kg, i.v.) prevented the efferent response, but augmented the afferent response. The treatment with hexamethonium abolished the inhibitory effect of beta-alanine on the afferent response. Both picrotoxin (100 and 500 micrograms/kg, i.v.) and bicuculline (2000 micrograms/kg, i.v.) antagonized the inhibitory effects of beta-alanine on the vagal efferent and afferent responses of the stomach. The present experiments clearly demonstrated that beta-alanine inhibited both the vagal efferent and afferent excitatory responses of stomach to electrical stimulation of vagal trunk in cats.


Antipunishment effects of acute and repeated administration of phencyclidine and NPC 12626 in rats.

The effects of phencyclidine (PCP) and NPC 12626 on punished responding were examined using a modified Geller-Seifter procedure in rats. Both drugs are known to antagonize N-methyl-D-aspartate (NMDA) receptor mediated neurotransmission, albeit at different sites on the NMDA receptor complex. Rats were trained to lever press for food reinforcement under a multiple schedule, with responding in one component reinforced under a fixed-interval 60-sec schedule, while each response in the other component resulted in both food and brief electric shock. Both PCP and NPC 12626 produced selective increases in punished responding, although the effects were not as large as those produced by chlordiazepoxide. Repeated daily administration of each of these drugs for 6 days resulted in increases in punished responding during different portions of the treatment. A 5 mg/kg dose of chlordiazepoxide produced increases over the last 2 days of administration. PCP (2 mg/kg) produced an increase only during the second session, whereas NPC 12626 (30 mg/kg) produced increases for all but the first and fifth days of the 6-day regimen. Both competitive and noncompetitive NMDA antagonists can have antipunishment effects in this model.


A low protein-high carbohydrate diet decreases D2 dopamine receptor density in rat brain.

The property of D2 dopamine receptors in the rat brain was evaluated after long-term dietary manipulation. Groups of rats were pair-fed with equicaloric diet containing low protein (8%)-high carbohydrate, high protein (52%)-low carbohydrate and normal protein (20%) for 36 weeks. The low protein-high carbohydrate fed rats exhibited a significant decrease in the density (Bmax) of D2 dopamine receptor in the striatum (28%) and the mesolimbic regions (36%) with no apparent change in the receptor affinity (Kd). These findings suggest that long-term consumption of a low protein-high carbohydrate diet, by decreasing D2 dopamine receptor density, may be an important determinant of central dopaminergic function.


Esterification of fatty acids by bovine intramuscular and subcutaneous adipose tissues.

Exogenous fatty acid esterification in intramuscular and subcutaneous adipose tissues from 72-hr fasted or ad libitum fed Angus cattle was investigated. Intramuscular (interfascicular) and subcutaneous adipose tissue snips were obtained from the longissimus dorsi muscle and were incubated with radioisotopically labeled fatty acids (palmitate, stearate, oleate, linoleate or linolenate) at three different concentrations (0.3 mM, 0.6 mM and 2.0 mM) to assess rates of fatty acid incorporation into glycerolipids. Rates of fatty acid esterification in vitro increased with fatty acid concentration in both intramuscular and subcutaneous adipose tissues. For all of the fatty acids investigated, triglycerides were the predominant products (60-85%). Subcutaneous adipose tissue had larger adipocytes and more actively (P less than 0.05) esterified fatty acids, with the exception of palmitate, than intramuscular adipose tissue. The rate of palmitate esterification was not different between tissues, although intramuscular adipose tissue esterified a greater proportion (P less than 0.10) of palmitate as triglyceride (85%) than did subcutaneous adipose tissue (75%). Relative rates of incorporation of fatty acids into lipids in intramuscular and subcutaneous adipose tissues were: palmitate greater than linolenate greater than linoleate greater than stearate. In general, 72-hr fasting did not significantly reduce the rates of fatty acid incorporation in bovine adipose tissues. Results of this study revealed that:i) rates of exogenous fatty acid incorporation into adipose tissue lipids were dependent on the medium fatty acid concentration and adipose tissue depot; and ii) the relative esterification rates of the various fatty acids in vitro did not necessarily reflect the proportion of these fatty acids in bovine adipose tissues.


Docosahexaenoic acid in developing brain and retina of piglets fed high or low alpha-linolenate formula with and without fish oil.

Docosahexaenoic acid (22:6n-3) can be synthesized in the liver and/or brain from alpha-linolenic acid (18:3n-3) and is required in large amounts in structural membranes of developing brain and retina. The adequacy and efficacy of formulas containing 18:3n-3 and/or fish oil in providing 22:6n-3 for deposition was investigated in piglets fed formula from birth to 15 days. The test formulas contained high (HL) or low (LL) 18:3n-3 (3.9 or 0.7% of the total formula fatty acids, respectively), or low 18:3n-3 plus fish oil (LL+FO) to provide C20 and C22 n-3 polyunsaturated fatty acids (0.8% of total fatty acids). Fatty acid analyses of synaptic plasma membrane and retina ethanolamine phospholipids (EPL), which are especially enriched in 22:6n-3, were compared to those of 15-day-old piglets fed sow milk (SM). Feeding LL resulted in lower 22:6n-3 in synaptic plasma membrane. Fatty acid levels in HL and LL+FO piglets were equivalent to SM, with the exception of lower 22:5n-3 in the synaptic plasma membrane of LL+FO and in the retina of HL and LL+FO-fed piglets. Levels of 22:4n-6 were also lower in the retina of the LL+FO group. The results suggest formula 18:3n-3 is at least 24% as effective as C20 and C22 n-3 fatty acids as a source of membrane 22:6n-3. This study shows dietary 18:3n-3, as the only n-3 fatty acid, can support deposition of comparable percentage of 22:6n-3 to natural milk. Fish oil also supported tissue levels of 22:6n-3 similar to natural milk; however, lower 22:4n-6 may indicate possible inhibitory effects on n-6 metabolism.


BMY 14802, a potential antipsychotic drug, increases expression of proneurotensin mRNA in the rat striatum.

Treatment with efficacious antipsychotic drugs, such as haloperidol, increases the concentrations of neurotensin (NT) in the nucleus accumbens and caudate nucleus of the rat. The increases in NT content produced by haloperidol have been shown to be associated with an increase in NT biosynthesis as assessed by the level of expression of proNT mRNA. The potential antipsychotic drug and sigma receptor ligand BMY 14802 has been shown to produce increases in the concentrations of NT in the nucleus accumbens and caudate that are similar to those produced by haloperidol with respect to the magnitude of the increases and the time course over which they occur. This study evaluated the effects of BMY 14802 on the expression of proNT mRNA and NT concentration in the rat striatum. Three hours after a single injection of BMY 14802 (35 mg/kg, i.p.) expression of proNT mRNA was significantly increased in the nucleus accumbens and caudate. Labeling was most intense in the dorsal caudate. NT concentrations were unaltered at this time point. Eighteen hours after injection, significant increases in NT concentration in the nucleus accumbens and caudate were detected. At this time, expression of proNT mRNA was substantially reduced compared to 3 h after treatment although labeling of the dorsal and medial caudate was still greater than that observed in controls.


Chronic stress elevates enkephalin expression in the rat paraventricular and supraoptic nuclei.

Numerous studies have implicated opioids in the regulation of hypothalamic functions. Dynorphin, which is co-expressed with vasopressin in the magnocellular neurons of the paraventricular and supraoptic nuclei, is co-regulated with vasopressin in response to hyperosmolality and appears to inhibit vasopressin and oxytocin release from the posterior pituitary. Enkephalin is present in paraventricular parvocellular neurons and its expression is elevated in response to various stresses. However, enkephalin's presence and roles in paraventricular and supraoptic magnocellular neurons are uncertain. By giving rats daily intraperitoneal injections of hypertonic saline for up to 12 days, we induced a marked increase in enkephalin expression in magnocellular neurons of the paraventricular and supraoptic nuclei, beyond what develops from drinking hypertonic saline. Our results suggest that enkephalin expression in both vasopressin and oxytocin neurons may increase in response to chronic stresses and provide another source of enkephalin in addition to the parvocellular neurons.


Induction of immediate-early gene proteins in dentate granule cells and somatostatin interneurons after hippocampal seizures.

The expression of the protein products of the immediate-early genes c-fos, Fos B, Fos-related proteins (FRAs), c-jun, jun B, jun D and krox-24 was investigated in the rat hippocampus at various times after electrically-induced hippocampal seizures. Hippocampal seizures induced all the immediate-early gene proteins in dentate granule cells with differing time-courses. In addition, Krox-24, Fos and Jun D were also induced in somatostatin-containing interneurons throughout the hippocampus and also in a small percentage of parvalbumin-containing interneurons. Thus, hippocampal seizures induce waves of immediate-early gene protein expression in dentate granule cells and a selective expression of krox-24, Fos and Jun D in hippocampal somatostatin interneurons. These results suggest that biochemical and/or morphological changes occurring in dentate granule cells and somatostatin interneurons after seizures may be regulated by immediate-early gene expression, and that these immediate-early gene proteins may be involved in seizure development in the nervous system.


A dementing illness associated with a novel insertion in the prion protein gene.

Following our previous report of an 144-bp insertion in the open reading frame of the prion protein (PrP) gene, we have now identified a larger, 216-bp, insertion in the gene. The insertion which is in frame encodes 9 extra octapeptide repeat sequences in addition to the 5 repeats normally present and represents the largest insertion so far detected in the PrP gene.


Temporal changes in tyrosine hydroxylase mRNA levels in A1, A2 and locus ceruleus neurons following electrical stimulation of A1 noradrenergic neurons.

We examined the effects of electrical stimulation (ES) of right A1 noradrenergic cells on temporal changes in tyrosine hydroxylase (TH) mRNA levels in A1, A2 and locus ceruleus (LC) neurons by in situ hybridization histochemistry and quantitative image analysis methods. The stimulation parameters used previously have been shown to increase hypothalamic norepinephrine (NE) release. Within 1 h after beginning A1 stimulation, TH mRNA levels were significantly increased and they continued to rise to reach plateau by 6 h. TH message levels at 12 h were not difference from 6 h values. A1-ES did not affect TH mRNA levels in contralateral A1 or in A2 or locus ceruleus neurons. These data suggest that changes in TH mRNA levels may serve as an index of increased A1 neuronal activity in circumstances when increases in hypothalamic NE secretion occur.


Immunobiology of experimental leishmaniasis.

Self-cure versus uncontrolled disease progression in experimental murine cutaneous leishmaniasis depends upon a delicate interplay among various activated cells of the host's immune system. Susceptibility or resistance to infection with Leishmania major is correlated with the ability of different inbred strains of mice to produce the characteristic spectra of lymphokines upon infection. Appropriate experimental interventions now allow the modulation of these responses, providing the possibility to render genetically susceptible mice resistant to infection and, vice versa, to cause genotypically "healer" strains to express a "non-healer" phenotype. These experimental manipulations have proven to be powerful tools in the dissection of the underlying immune mechanisms and cellular parameters responsible for susceptibility and resistance, and will perhaps allow the identification of molecules of parasite origin that induce deleterious immune responses to infection with Leishmania, and thus to exclude them from future vaccines. More importantly, rational immune intervention could permit the diversion of established host-damaging immune responses to host-protective immunization.


Optimal drug and behavior therapy for treatment-refractory institutionalized schizophrenics.

Institutionalized persons with a deteriorating form of schizophrenia that was refractory to neuroleptic medication were titrated downward in their haloperidol dose. Based on ratings of their clinical status, an optimal dose was reached that was an average 66 percent reduction from their initial levels. Patients then participated in a personalized, intensive behavior therapy program to remediate their extreme, persisting deficits and disturbances in behavior.


Glutamate-induced long-term potentiation of the frequency of miniature synaptic currents in cultured hippocampal neurons.

Glutamate application at synapses between hippocampal neurons in culture produces long-term potentiation of the frequency of spontaneous miniature synaptic currents, together with long-term potentiation of evoked synaptic currents. The mini frequency potentiation is initiated postsynaptically and requires activity of NMDA receptors. Although the frequency of unitary quantal responses increases strongly, their amplitude remains little changed with potentiation. Tests of postsynaptic responsiveness rule out recruitment of latent glutamate receptor clusters. Thus, postsynaptic induction can lead to enhancement of presynaptic transmitter release. The sustained potentiation of mini frequency is expressed even in the absence of Ca2+ entry into presynaptic terminals.


Heat shock in Escherichia coli alters the protein-binding properties of the chaperonin groEL by inducing its phosphorylation.

When bacterial or eukaryotic cells are exposed to high temperatures or other harsh conditions, they respond by synthesis of a specific set of heat-shock proteins. Certain heat-shock proteins such as groEL, called 'chaperonins', can prevent misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under harmful conditions. We report here a new aspect of the heat-shock response in Escherichia coli: at high temperatures a fraction of groEL becomes modified covalently, altering its interaction with unfolded proteins. The heat-modified form can be eluted with ATP from an unfolded protein more easily than normal groEL. The critical heat-induced modification seems to be phosphorylation, which is reversed on return to low temperature. Treatment of the modified groEL with phosphatases caused its apparent size, charge and binding properties to resemble those of the unmodified form. Thus during heat shock some groEL is reversibly phosphorylated, which allows its ATP-dependent release from protein substrates in the absence of its usual cofactor (groES), and probably promotes the repair of damaged polypeptides.


Somatostatin and neuropeptide Y in organotypic slice cultures of the rat hippocampus: an immunocytochemical and in situ hybridization study.

The neuronal distributions of somatostatin and neuropeptide Y and their respective mRNAs in hippocampal slice cultures were examined by immunohistochemical staining and in situ hybridization. For the in situ hybridization we used an alkaline phosphatase-labelled oligodeoxynucleotide probe for somatostatin mRNA and an 35S-labelled oligodeoxynucleotide probe for neuropeptide Y mRNA. For both neuropeptides the immunostained and hybridized neurons displayed a comparable, organotypic distribution. Most labelled neurons were located in the dentate hilus and stratum oriens of CA3 and CA1. Additional neurons were found in stratum radiatum and pyramidale of CA3, but very few in the corresponding layers of CA1. In all locations the density of somatostatin- and neuropeptide Y-reactive cells exceeded that observed in vivo. Also, the hybridization signal of the individual neurons appeared enhanced in the slice cultures. Methodologically it was noted that the non-radioactive alkaline phosphatase-labelled oligodeoxynucleotide probe gave excellent in situ hybridization results with detailed cellular resolution and no apparent problems of tissue penetration, even when used on whole-mount explants. These results demonstrate that somatostatin and neuropeptide Y-immunoreactive and mRNA containing neurons retain their organotypic distribution and basic morphological characteristics in the slice cultures. The supernormal density of these neurons and their hybridization signals indicate that a transient developmental increase in neuropeptide expression may persist in vitro.


Distribution of somatostatin immunoreactivity in the forebrain of the squirrel monkey: basal ganglia and amygdala.

The distribution of somatostatin immunoreactivity in the basal ganglia and amygdala of the squirrel monkey (Saimiri sciureus) was studied with specific polyclonal antibodies directed against somatostatin-28 and somatostatin-28(1-12). Both antibodies gave similar results with regard to the distribution of somatostatin-immunoreactive neuronal profiles. A moderately dense and highly heterogeneous network of somatostatin-positive fibers was observed throughout the striatum. A dorsoventral gradient of increasing immunoreactivity was noted in the striatum and the caudate nucleus was found to strain generally less intensely than the putamen. The immunoreactive fibers within the striatum were mostly thin and varicose and formed patches corresponding to the striosomes, as visualized on adjacent sections immunostained for calbindin. Although some somatostatin cell bodies rimmed the striosomes, most of the positive cells were rather uniformly scattered in the striatum. These medium-sized cells were significantly smaller in the caudate nucleus (93 microns2, S.D. = 26 microns2) than in the putamen (122 microns2, S.D. = 39 microns2), but their density was significantly higher in the caudate nucleus (29.7 cells/mm2, S.D. = 8.8 cells/mm2) than in the putamen (20.5 cells/mm2, S.D. = 7.0 cells/mm2). The nucleus accumbens stained moderately and positive cell bodies were evenly dispersed throughout this structure. In contrast, the olfactory tubercle displayed a heavily stained neuropil but positive neurons were encountered only in its polymorph layer. In the sublenticular region, dense fiber plexuses appeared in register with nonreactive cell clusters of the nucleus basalis of Meynert and of the nucleus of the anterior commissure. More caudally, a dense bundle of positive fibers was observed at the level of the ansa lenticularis, the inferior thalamic peduncle, and the adjoining bed nucleus of the stria terminalis. Several fibers contributing to this bundle were of the woolly type. Woolly fibers also coursed in the substantia innominata between the ventral aspect of the globus pallidus and the optic tract, and ascended in the internal medullary lamina separating the internal and external segments of the globus pallidus. Somatostatin-immunoreactive cell bodies were uniformly scattered throughout the substantia innominata. The various nuclei of the amygdala showed a wide range of immunoreactivity. The central nucleus was lightly reactive, whereas the intercalated masses displayed a moderate staining. A dorsoventral gradient of immunostaining was noted in the ventrolateral portion of the amygdala, the lateral nucleus being moderately to densely stained and the basal nucleus very lightly to lightly immunoreactive.(ABSTRACT TRUNCATED AT 400 WORDS)


Blockade of hippocampal long-term potentiation by saccharin.

Population spikes, population excitatory postsynaptic potentials and intracellular excitatory postsynaptic potentials were recorded in the CA1 area of guinea-pig hippocampal slices in response to low frequency stimulation of the stratum radiatum. Tetanic stimulation of the same afferents during an application of saccharin (10 mM, 10 min) failed to induced a long-term potentiation of the population spike, population excitatory postsynaptic potential and intracellularly recorded excitatory postsynaptic potential. A post-tetanic application of saccharin did not prevent long-term potentiation of the population spike from developing. Saccharin did not change the input resistance, the membrane potential or the ability to induce action potentials in the CA1 neurons. The slope of the intracellular excitatory postsynaptic potentials recorded in normal medium, in normal medium containing 2-amino-5-phosphonovalerate, or in Mg(2+)-free medium containing 6-cyano-7-nitroquinoxaline-2,3-dione was not significantly altered by saccharin. The depolarizations of CAI neurons produced by superfusion of N-methyl-D-aspartate or during a brief tetanic stimulation of the stratum radiation were also not altered by the drug. It therefore appears that saccharin blocks the induction of long-term potentiation by a mechanism that does not involve a blockade of N-methyl-D-aspartate receptors. Application of fluid samples collected from rabbit neocortical surface during a tetanic stimulation of the neocortex caused neurite growth in PC-12 cells, suggesting that growth-related substances were present in the collected samples. If these samples were superfused onto hippocampal slices, long-term potentiation developed. If however, the samples were co-applied with saccharin, neither neurite growth in PC-12 cells nor long-term potentiation in hippocampal slices was observed, raising the possibility that growth-related substances are involved in long-term potentiation.


Contribution of an alpha 1-adrenergic receptor subtype to the expression of the "ventral tegmental area syndrome".

Bilateral electrolytic lesions of the rat ventral tegmental area, a mesencephalic structure containing the cell bodies of ascending dopaminergic neurons, induce a behavioural syndrome characterized by a permanent locomotor hyperactivity. Acute intraperitoneal injections of prazosin, an alpha 1-adrenergic receptor antagonist, at a dose (0.5 mg/kg) which does not affect locomotor activities of control animals, abolished locomotor hyperactivities of lesioned rats. Antagonists of other monoaminergic receptors (propranolol, ritanserin, yohimbine), and also another antagonist of alpha 1-adrenergic receptors, 2-(2',6'-dimenthoxyphenoxyethyl)-aminomethyl-1,4-benzodioxan (WB4101) were ineffective. Comparisons of autoradiograms of brain slices incubated in the presence of 1 nM [3H]prazosin or 10 nM [3H]WB4101 indicated clear topographical differences. [3H]Prazosin labelling is present in the septum and in layer III of the cerebral cortex but absent in the striatum. [3H]WB 4101 labelling is diffuse in the superficial layers of the cerebral cortex and present in the striatum. In addition, intraperitoneal injection of WB4101 displaces, only weakly, [3H]prazosin binding in layer III of the cerebral cortex (-18%) while it decreases by 50% [3H]prazosin binding in the more superficial cortical layers. These observations strongly suggest that the binding site labelled by [3H]prazosin is different from alpha 1A- and alpha 1B-adrenergic receptor subtypes labelled by [3H]WB4101. Finally, it is proposed that the prazosin-induced blockade of the locomotor hyperactivity exhibited by ventral tegmental area lesioned animals is linked to the previously demonstrated regulatory role of noradrenergic neurons on cortical dopamine transmission.


Early and widespread normalization of dopamine-neuropeptide Y interactions in the rat striatum after transplantation of fetal mesencephalon cells.

Graft-to-host interactions were examined at cellular level, by measuring changes in the immunoreactivity of striatal interneurons expressing neuropeptide Y after dopamine denervation and transplantation of fetal mesencephalon neurons into the striatum of adult rats. Mesencephalic cell suspensions were implanted unilaterally into the dorsal part of the striatum in rats two weeks after intranigral injection of 6-hydroxydopamine. One month and three to four months later, rats showing abolition of amphetamine-induced turning were perfused. Serial brain sections containing intrastriatal grafts were treated for tyrosine hydroxylase and neuropeptide Y immunocytochemistry, and neuropeptide Y-immunoreactive neurons were quantified in various parts of the striatal surface and compared with the striatum of controls and age-matched rats with lesions. Biochemical analyses of dopamine and dihydroxyphenyl acetic acid tissue levels and [3H]dopamine uptake were also performed on striatal samples from similar groups of normal, lesioned and transplanted rats. As early as one month post-grafting, a complete reversal of the increase in the number of neuropeptide Y-immunoreactive neurons occurring after 6-hydroxydopamine lesion was observed in dopamine-grafted animals, although a partial restoration of the tyrosine hydroxylase immunostaining and a recovery of 8% dopamine tissue level were observed in the striata of grafted as compared to normal rats. This effect on the host immunoreactivity was found to be specific to dopamine grafts, since no reversal was observed in sham-spinal cord-transplanted rats. Moreover, similar degrees of normalization were recorded either in the total striatum, or in the area immediately adjacent to the graft, or even in the zone most sensitive to dopamine denervation in terms of neuropeptide Y immunoreactivity. No more pronounced functional effects were observed three to four months after transplantation. These data suggest that grafted dopamine neurons are able to induce rapid and extensive host responsiveness, possibly by means of mechanisms involving synaptic and diffuse release of dopamine and adaptive changes in the host brain. These data may provide a cellular basis for interpreting larger behavioural recoveries than those expected to occur with dopamine grafts in view of the partial restoration of the dopaminergic innervation.


Mechanisms of glutamate activation of axon-to-Schwann cell signaling in the squid.

Membrane potentials from Schwann cells associated with giant axons of the small squid (Alloteuthis and Loliguncula) and the large squid (Loligo) were monitored with glass microelectrodes following 100 Hz/15 s axonal stimulation, or the application of 10(-7) M glutamate and ion substitutions, in the presence or absence of 10(-7) M d-tubocurarine. Glutamate or stimulation caused the membrane of the Schwann cell to depolarize to approximately -32 mV. This was rapidly replaced by a transient hyperpolarization to approximately -55 mV; the potential returning to the resting level (-40 mV) in approximately 7 min. In the presence of d-tubocurarine only the initial depolarization was evident. Nominally zero [Na+]o or treatment with 10(-7) M tetrodotoxin (in normal [Na+]o) blocked the stimulation- and glutamate-induced depolarization while low Clo- hyperpolarized the Schwann cell without effect on the glutamate- or stimulation-induced depolarization. Nao+ depletion or pretreatment with tetrodotoxin in normal Nao+ did not affect the development of the Schwann cell hyperpolarization. These results do not support the hypothesis that the glutamate-induced depolarization is the trigger leading to the Schwann cell hyperpolarization. Preliminary experiments to test the possibility that inositol phosphate second messenger and an increase in [Ca2+]i are triggered by glutamate receptor activation showed that nominally 0 Cao2+/75 mM Mgo2+ only slightly reduced the hyperpolarizing response to stimulation or glutamate while intracellular Bapta (20-30 microM) blocked the hyperpolarization but not the depolarization. [3H]Myoinositol incorporation into axon-Schwann cell plasma membranes was high.(ABSTRACT TRUNCATED AT 250 WORDS)


Neuroprotective effects of graded reoxygenation following chronic hypoxia in neuronal cell cultures.

The present study was undertaken to investigate the comparative effects of rapid vs graded correction of chronic hypoxia in vitro. Cerebral cortical cell cultures obtained from fetal mice were exposed to 5% O2 for 24 h and returned immediately to room air for the following 24 h (Group I); comparable cultures were exposed to 5% O2 for 24 h followed by 10% O2 for an additional 24 h before return to room air (Group II). At the conclusion of the experimental protocol (time 0), partial pressure of oxygen in the bathing medium of Group I cultures was significantly higher than that of Group II and non-hypoxic controls (151 mmHg vs 124 and 132 mmHg, respectively; P less than 0.05). Throughout the recovery period, Group II cultures evidenced improved neuronal survival (e.g. 35,800 vs 17,700 neurons/culture well at time 0, P less than 0.01), decreased lactate dehydrogenase efflux into the bathing medium, relative preservation of neuronal morphology, as well as higher specific and clonazepam-displaceable benzodiazepine binding and GABA uptake. Glutamate binding was not differentially affected and glutamine synthetase activity, a predominantly glial marker, was only modestly increased after graded reoxygenation. These results demonstrate that gradual reoxygenation after prolonged hypoxia in vitro (i) improves neuronal survival compared to rapid reoxygenation and (ii) delays the manifestations of metabolic dysfunction even though the length of hypoxic exposure is increased. The findings are also consistent with the concept that a period of relative hyperoxia may contribute to hypoxia-induced neuronal injury.


Tyrosine hydroxylase and neuropeptide Y are increased in ciliary ganglia of sympathectomized rats.

We have examined the expression of tyrosine hydroxylase and neuropeptide Y in ciliary ganglia of normal adult rats and of adult rats in which the environment of these neurons was altered by sympathectomy at birth. Following neonatal 6-hydroxydopamine treatment, the proportion of tyrosine hydroxylase-immunoreactive and neuropeptide Y-immunoreactive neurons in ciliary ganglia was significantly increased. In ciliary neurons of both control and sympathectomized rats, neuropeptide Y immunoreactivity was preferentially co-localized with tyrosine hydroxylase. Immunoblot analysis confirmed the presence of tyrosine hydroxylase and its increase following sympathectomy. In situ hybridization studies revealed that many ciliary neurons contain mRNA for tyrosine hydroxylase and for neuropeptide Y. Like tyrosine hydroxylase immunoreactivity, the number of ciliary neurons containing tyrosine hydroxylase mRNA and the amount of mRNA per cell were increased in 6-hydroxydopamine-treated rats. In contrast, neuropeptide Y mRNA levels were the same in control and 6-hydroxydopamine-treated rats. Nerve growth factor is a candidate for mediating the effects of sympathectomy and most ciliary neurons in control and sympathectomized rats expressed immunoreactivity for the low-affinity nerve growth factor receptor. In addition, ciliary neurons from 6-hydroxydopamine-treated animals possessed increased nerve growth factor receptor immunoreactivity. These studies indicate that both tyrosine hydroxylase and neuropeptide Y in the ciliary ganglion are regulated by alterations in their environment. Their expression was enhanced by chemical sympathectomy which does not affect ciliary neurons directly but, rather, removes sympathetic innervation of shared targets, including the iris. In situ hybridization analysis suggests that the increased tyrosine hydroxylase and neuropeptide Y levels result from different mechanisms and provides evidence that neuropeptide levels can be regulated without changes in mRNA levels.


Detection of beta and delta globin gene mutations by PCR and direct DNA sequencing in an individual with normal HbA2 beta thalassemia.

Normal HbA2 beta thalassemia in a Greek individual was shown to be due to co-inheritance of beta and delta thalassemias. The genetic defects were characterized by enzymatic amplification of the beta and delta globin genes and direct genomic sequencing. Two children with a typical high HbA2 beta thalassemia trait had inherited the beta thalassemia allele whilst a third child had low-normal HbA2 associated with delta+ thalassemia. Segregation patterns confirmed that the delta+/beta zero thalassemia defects were present in trans.


Plasmid replication in Xenopus eggs and egg extracts: a 2D gel electrophoretic analysis.

We have examined the replication patterns of ribosomal DNA plasmids in vivo and in vitro using Xenopus eggs. Plasmids carrying different parts of the Xenopus ribosomal DNA sequence were allowed to replicate either in vitro in an egg extract or in vivo after microinjection into unfertilized eggs. The replication intermediates were analyzed by the 2D gel electrophoretic technique of Brewer and Fangman (1), using original or modified electrophoresis conditions. With standard electrophoresis conditions, the patterns obtained for restriction fragments larger than 5 kb were unreliable because of artefactually distorted Y arcs and unrecognizable bubble arcs. Interpretable patterns could nevertheless be obtained using suitably modified electrophoresis parameters. Under these conditions, replication was found to initiate and terminate at multiple, random locations on each plasmid both in vivo and in vitro. However, only one or very few of these potential initiation sites are used during the replication of an individual plasmid molecule. We discuss the possible artefacts and misinterpretations that can result when the 2D electrophoresis parameters are not adapted to the size of the fragment examined. We also discuss the relevance of the random replication mode to the mechanisms and the control of DNA replication in eukaryotes.


A physical and genetic map of the Spiroplasma citri genome.

A physical and genetic map of the Spiroplasma citri genome has been constructed using several restriction enzymes and pulsed field gel electrophoresis. A number of genes were subsequently localized on the map by the use of appropriate probes. The genome size of the spiroplasma estimated from restriction fragments is close to 1780 kbp, the largest of all Mollicutes studied so far. It contains multisite insertions of Spiroplasma virus 1 (SpV1) sequences. The physical and genetic map of the S. citri genome shares several features with that of other Mollicutes, especially those in the Mycoplasma mycoides cluster. This supports the finding that S. citri and these Mycoplasma spp. are phylogenetically related.


The murine Hox-2.4 promoter contains a functional octamer motif.

Like other HOX genes the murine Hox-2.4 gene is thought to be involved in regional specification along the antero-posterior axis. In addition it has been reported to have oncogenic potential. We studied expression Hox-2.4 in murine EC cells and determined the transcription start site. Studies of DNA-protein interactions in the promoter region showed that the Hox-2.4 promoter contains a CCAAT box and a perfect octamer motif, which is capable of binding Oct-factors. Cotransfection of Oct expression vectors influences the transcriptional activity of the promoter, suggesting that the Oct-gene family may be involved in regulating HOX genes.


A conserved region in intron 1 negatively regulates the expression of the PCNA gene.

The Proliferating Cell Nuclear Antigen (PCNA) gene is a growth-regulated gene, whose expression is under the control of both transcriptional and posttranscriptional mechanisms. In previous work, it was shown that the 73 bp immediately upstream of the CAP site and intron 4 are major regulatory elements. We show here that intron 1 also plays a role in determining the levels of PCNA mRNA. Specifically, we show: 1) deletion of intron 1 increases the expression of PCNA mRNA in serum-deprived cells; 2) a 35 bp sequence in intron 1, containing a reverse CCAAT element specifically binds proteins from nuclear extracts; 3) this intron 1 sequence inhibits the expression of a co-tranfected human PCNA gene in transient expression assays suggesting that it competes for positive transcription factors; 4) mutations in the CCAAT region of the 35bp intron 1 probe abrogate both its protein-binding capacity and its ability to inhibit the expression of a co-transfected wt PCNA gene; and 5) the CCAAT region of human intron 1 is highly conserved in the mouse gene. We conclude that the reverse CCAAT region of intron 1 is a negative regulatory element of PCNA gene expression, and hypothesize that its inhibitory effect is abolished when certain protein(s) bind to it and that inhibition is restored if these proteins are competed out by an homologous sequence.


Antihypertensive therapy. Current issues and challenges.

Choosing antihypertensive agents that protect patients against cardiovascular and other complications is a growing trend in the treatment of mild to moderate hypertension. Calcium channel blockers and angiotensin-converting enzyme (ACE) inhibitors are favored because they have neutral or positive effects on lipid levels and insulin resistance. The alpha 1 blockers, especially doxazosin mesylate (Cardura), are enjoying a resurgence in popularity because they have a beneficial effect on lipid levels. In terms of preserving patients' quality of life, the ACE inhibitors in particular have been shown to have a positive impact. It has been shown that systolic hypertension in elderly patients should definitely be treated, but the most appropriate agent has yet to be defined. Therapy should be tailored to the individual. The following questions should be considered when choosing an antihypertensive agent: (1) What are its side effects (especially metabolic ones)? (2) Does it require only once- or twice-a-day dosing? (3) Does it cause regression of left ventricular hypertrophy? (4) Does it prevent death from coronary artery disease? (5) How will it affect quality of life? (6) How much does it cost? The goal of therapy should be to provide adequate blood pressure control throughout the day, enhance compliance, and protect the heart, brain, and kidneys without adversely affecting metabolic state.


Supraventricular tachyarrhythmias in the intensive care unit.

Several types of supraventricular tachyarrhythmias occur commonly during intensive care. The specific type can usually be diagnosed using standard electrocardiographic techniques. Several new drugs have significantly improved the ability to successfully manage these arrhythmias.


Anxiolytic potential of 5-HT3 receptor antagonists.

The 5-HT3 receptor antagonists such as ondansetron have been shown to have anxiolytic-like activity in a wide range of preclinical tests: in the mouse black: white test, the rat social interaction test, the rat elevated X-maze and the marmoset human threat test. Ondansetron, like other 5-HT3 receptor antagonists appears to have important advantages over existing anxiolytic therapies. Thus, the 5-HT3 receptor antagonists are not sedative, they do not have addictive liabilities, there are no problems of withdrawing from chronic treatment and they can be used following benzodiazepine withdrawal. Such compounds even inhibit the behavioural syndrome associated with withdrawal from drugs of abuse, nicotine, alcohol, cocaine, opiates. The preclinical data, therefore, indicates the 5-HT3 receptor antagonists as novel anxiolytics of the future, and there is new clinical work in support of this suggestion.


The role of Mg2+ on the formation of the ternary complex between agonist, beta-adrenoceptor, and GS-protein and an interpretation of high and low affinity binding of beta-adrenoceptor agonists.

In the absence of GTP or its analogues, the beta-adrenoceptor agonist (H), the receptor (R) and the stimulatory guanine nucleotide-binding regulatory protein (GS-protein) form a slowly dissociable complex (HRGSGDP). It is presumed that the agonist in this ternary complex is bound to the receptor with high affinity while its binding to the receptor in the HR-complex is of low affinity. We have studied the effect of Mg2+ on the formation of these two complexes by the displacement of (-)-[3H]CGP-12177 with (-)-isoprenaline in cell membranes prepared from guinea-pig lung parenchyma. In the absence of Mg2+, only monophasic displacement curves were obtained with a dissociation constant not differing from that obtained in the presence of Gpp(NH)p and Mg2+. In the presence of Mg2+ and absence of Gpp(NH)p, a shallow binding curve was obtained that fitted a two site model best. One of the dissociation constants agreed well with that obtained in the presence of Gpp(NH)p and Mg2+ but the other one was 27 times smaller. It was concluded that Mg2+ is necessary for the coupling between the HR-complex and the GS-protein. The two dissociation constants obtained in the absence of Gpp(NH)p represent the dissociation constants of the HR- and HRGSGDP-complexes and may correspond to those named KL and KH in the literature. According to our study, there may be no difference in affinity between the agonist and receptor in the HR- and HRGSGDP-complexes. The two dissociation constants obtained in the absence of GTP or its analogues refer to two consecutive reactions.


Evidence that drug binding to non-adrenergic [3H]-idazoxan binding sites (I-receptors) occurs to interacting or interconvertible affinity forms of the receptor.

Characterization of [3H]idazoxan binding to guinea pig kidney membranes showed that approximately 90% bound to nonadrenergic I-receptors and approximately 10% to alpha 2-adrenoceptors. I-Receptors could be studied separately by including 3 microM rauwolscine to the assay. During these conditions 22 different compounds out of 29 (including imidazoline and guanidinium compounds) generated biphasic or shallow competion curves (Hill coefficients down to 0.57), for which computer modelling suggested that drugs bound to two sites with different affinities. However, the proportion of sites varied considerably depending on which drug was used as competitor; the variation being from approximately 50/50% to approximately 8/92% and analysis of variance clearly indicated that the variation of proportions of sites could be attributed to an effect induced by the drugs which indicated that the sites were dynamically formed or modulated by the presence of the drug. A few drugs (guanabenz, (-)-medetomidine, phentolamine, clemastin, prazosin and idazoxan itself) yielded steep uniphasic curves (Hill coefficients near unity) which were resolved only into one site fits. One drug (detomidine) yielded supersteep competition curves (Hill coefficient 1.29), the data being reminiscent of positive cooperativity. In another set of experiments attempts were made to block one of the affinity forms of the I-receptor with histamine, a compound which had grossly different affinities for the two I-receptor sites, and competition curves were then obtained using other drugs which also seemed to distinguish between the two sites. Computer analysis of experimentally obtained as well as simulate,d computer generated, competition curves clearly showed that the kidney I-receptors did not exist in two independent non-interacting forms. Instead the data suggested that the I-receptor was composed of interacting or interconverting receptor entities with different affinities for drugs. Monovalent cations such as Cs+ or NH4+ were found to be powerful inhibitors of [3H]idazoxan binding to the kidney I-receptors, the ions decreasing both the apparent Bmax and affinity of the ligand for the receptor. The cations also decreased the affinities of UK-14,304 for both the high and low affinity forms of the I-receptor, but the apparent proportions of sites were not at all affected by the ions. Moreover, ligand binding to kidney I-receptor were not at all affected by non-hydrolyzable guanine nucleotides. It is suggested that the binding data reflects functional properties of the I-receptor protein.


Quantification of SCH 39166, a novel selective D1 dopamine receptor antagonist, in rat brain and blood.

A gas chromatographic method for measuring concentrations of a novel D1 antagonist SCH 39166 [(-)-trans-6,7,7a,8,9-13b-hexahydro-3-chloro-2-hydroxy-N-methyl-5- H-benzo[d]naphto(2,1-6)azepine] in rat brain and plasma was developed. The method was applied to descriptive pharmacokinetics of two subcutaneous doses of SCH 39166 (0.25 mg/kg and 2.5 mg/kg). For comparison, concentrations of the "prototype" D1 antagonist SCH 23390 (0.25 mg/kg, SC) [R-(+)-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1-H-3-benzazepine] were also measured in plasma and brain. SCH 23390 (0.25 mg/kg, SC) had a very short elimination half-life of about 30 min in plasma, and disappeared in a slightly slower manner from striatum and cortex. SCH 39166 (0.25 and 2.5 mg/kg, SC), however, had a longer elimination half-life of about 1.5-2.5 h in plasma and brain. Interestingly, the 2.5 mg/kg dose of SCH 39166 produced only two-to five-fold increases in maximum concentrations in plasma and brain compared to the 0.25 mg/kg dose. The reason for this is not clear. The ability of these two doses of SCH 39166 to induce catalepsy in the bar test was also evaluated. It was found that SCH 39166 in these two doses, unlike SCH 23390, was not cataleptic. In conclusion, these pharmacokinetic features of SCH 39166 in the rat should be useful when designing experiments with this novel selective D1 antagonist. Furthermore, the longer elimination half-life of SCH 39166 makes it a more useful probe in pharmacodynamic comparisons of D1 receptor antagonists and classical as well as atypical neuroleptics.


Changes in dopamine D1, D2 and D3 receptor mRNA levels in rat brain following antipsychotic treatment.

The effects of administration of antipsychotic drugs (1-32 days, twice per day) on the rat brain mRNA levels of dopamine D1, D2 and D3 receptors has been assessed by a novel procedure utilising solution hybridisation with oligonucleotides. Saline and sulpiride (10 mg/kg/injection) had no effect on D1, D2 and D3 receptor mRNA levels. Haloperidol (1.5 mg/kg/injection) elicited increases in D1, D2 and D3 receptor mRNA levels of 100%, 100% and 300% respectively, after 32 days and loxapine (2 mg/kg/injection) elicited increases of 450%, 150% and 550%, respectively. These results indicate that the up-regulation of dopamine receptors may be associated with the occurrence of tardive dyskinesia but not the clinical mode of action of antipsychotics.


Effects of irreversible dopamine receptor inactivation on locomotor activity and grooming in the 17- and 90-day-old rat.

Ontogenetic differences in the behavioral recovery of R(-)-propylnorapomorphine (NPA) treated rats were assessed following irreversible DA receptor antagonism by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). In the first two experiments, 17- and 90-day-old rats were given EEDQ (7.5 or 15.0 mg/kg, IP) or vehicle after half the rats were initially treated with the selective DA D-1 and D-2 antagonists SCH 23390 and sulpiride. (The sulpiride/SCH 23390 treatment protects DA receptors from EEDQ-induced inactivation.) NPA's (0.1 or 1.0 mg/kg) effects on locomotor activity and grooming were assessed 1, 2, 4 and 8 days after the EEDQ pretreatment. In a third experiment, the effects of habituating the 17- and 90-day-old rats to the testing chamber were assessed 1, 2 and 4 days after EEDQ pretreatment. In this experiment, some groups received successive treatments of saline or NPA prior to behavioral testing. To assess the possible effects of drug-sensitization other groups received saline on days 1 and 2 and NPA on day 4. In 90-day-old rats, EEDQ eliminated, for up to 4 days, the ability of NPA to enhance locomotor activity and depress grooming. Prior treatment with DA antagonist drugs was sufficient to protect DA receptors from EEDQ-induced inactivation, since these groups exhibited normal behavioral responses after challenge with NPA. In contrast, EEDQ did not eliminate, and may have enhanced, NPA's effect on the locomotor activity and grooming of 17-day-old rat pups. Habituating the rats to the testing chamber decreased the locomotor activity of the mature rats, but not the 17-day-old rat pups.(ABSTRACT TRUNCATED AT 250 WORDS)


Benzodiazepine-induced sedation and cortisol suppression. A placebo-controlled comparison of oxazepam and nitrazepam in healthy male volunteers.

The sedative and cortisol suppressing properties of oxazepam (45 and 60 mg) and nitrazepam (10 and 15 mg) were examined in eight healthy male subjects. The most clear differences between oxazepam and nitrazepam were those seen with respect to the time course and until maximal effect (Tmax) of the different measurements. Nitrazepam showed maximal sedation after 1 h, maximal benzodiazepine level (RRA), and reaction time prolongation after 2 h, and maximal cortisol suppression after 3 h. Oxazepam showed maximal sedation after 2 h, maximal benzodiazepine levels, reaction time prolongation and cortisol suppression after 3 h. After administration of oxazepam (both doses) a transient return to baseline levels of cortisol was demonstrated. Whereas the degree of sedation correlated significantly within drug groups with the concurrent benzodiazepine levels, the Tmax of sedation was recorded 1 h earlier than the peak blood concentration (RRA) for both nitrazepam and oxazepam. The time course for cortisol suppression for the two compounds differed clearly from the other measurements and was not related to the peak blood concentration.


Behavioural tests of the dopamine depletion hypothesis of neuroleptic-induced response decrement.

This study was carried out to test the hypothesis that neuroleptic-induced within-session response decrements reflect a fatigue process, resulting from dopamine depletion, that is present before the session begins but is masked briefly by activational cues present at the start of the session. Response decrementing effects of pimozide were examined in rats lever pressing on random interval schedules of food reinforcement. An initial experiment was carried out to rule out a pharmacokinetic explanation of the response decrement. In a second experiment, the response decrement was not exacerbated by an immediately preceding period of intense forced motor activity (wheel running). Experiments 3 and 4 tested two further predictions: that the pimozide-induced response decrement should be overcome by removing the animal to its home cage and then replacing it in the apparatus (thereby reinstating the activational cues present at the start of the session); and that response impairments should be present from the outset if the animal is confined in the apparatus prior to the start of the session (thereby allowing activational cues to dissipate). Neither prediction was confirmed. Overall, the results provide no support for the dopamine depletion hypothesis.


Low dose neuroleptanxiolysis in anxiety states.

1. In order to prove that neuroleptanxiolysis represents a therapeutical alternative in the treatment of patients suffering from anxiety we conducted four investigations. 2. In the first study it was experimentally proved with 45 outpatients suffering from anxiety that fluspirilene (1.5 mg per week) is superior to bromazepam (6 mg/day), especially in patients with a high degree of somatic anxiety. 3. In the second study the tolerance of fluspirilene (1.5 mg per week) was investigated in 1261 patients with anxiety states and psychoreactive disorders under controlled and open conditions for a period of six weeks. Side effects were found in 11.5% of the patients. All side effects had in common that they occurred already within the first few weeks of treatment. 4. In the third study investigating the dose-effect relationship 106 patients received either 0.5, 1.0, or 1.5 mg fluspirilene per week for a period of 6 weeks. The main result of this study was the verification of a clear dose-effect relationship. 5. The fourth study compared 155 patients who had received long-term treatment with fluspirilene (max 1.5 mg/week) and 121 patients with long-term benzodiazepine treatment. No differences were found with regard to the frequency and intensity of extra-pyramidal disturbances. 6. The therapeutical relevance of the findings was emphasized in the general discussion.


Plasmatic somatostatin as a marker of positive symptoms of schizophrenia.

1. Somatostatin displays a regulatory function on several aminergic neurotransmitters, including dopamine. In addition, decreased CSF levels of the peptide has been found in Schizophrenia and other neuropsychiatric disorders. 2. In the present work, we have investigated levels of plasmatic somatostatin in a sample of 50 schizophrenic patients compared with a normal control group. 3. Somatostatin was increased in the patient group (p less than 0.01) as a whole but statistical analysis revealed that the increase was associated with the presence of positive symptoms (Factorial Analysis) with a significant correlation, specially with delusion and hallucination scores in the Andreasen rating scales.


Central dopamine involvement in experimental gastrointestinal injury.

1. Rats were prepared with intracerebral cannulas for microinjection of test compounds into various brain regions. 2. Selective dopamine D1 agonists (SKF38393, SKF75670C) and a D1 antagonist (SCH23390) were injected into the cell body regions of the nigrostriatal, mesolimbic and mesocortical dopamine tracts or into a terminal field of these tracts (caudate nucleus, central nucleus of the amygdala and medial prefrontal cortex) prior to gastric ulcer induction by cold-restraint stress or duodenal ulcer induction by cysteamine. 3. The dopamine D1 agonists reduced both stress gastric ulcers and duodenal lesions most significantly when given into either the cell body region or a terminal field of the mesolimbic DA tract with much less effects seen for the nigrostriatal tract. 4. No effects were seen upon infusion of the agonists into the mesocortical cell body or terminal field regions. 5. The D1 antagonist worsened both stress-induced gastric lesions and duodenal lesions if given into mesolimbic regions and, to a much lesser extent when injected into the nigrostriatal tract. 6. No effect of the D1 antagonist was seen upon administration into the mesocortical tract. 7. Central dopamine D1 receptors, particularly in the mesolimbic DA tract, appear to be involved in mediating the gastrointestinal consequences of exposure to stress.


The effect of ritanserin on treatment-resistant neuroleptic induced akathisia: case reports.

1. The authors report three cases of neuroleptic induced akathisia resistant to treatment with anticholinergics, benzodiazepines and betablockers. 2. All three patients were treated with Ritanserin 10 mg bid and improved rapidly and substantially. 3. Discontinuation of Ritanserin led to a recurrence of akathisia.


Plasma concentrations of regulatory peptides in obesity following modified sham feeding (MSF) and a liquid test meal.

Plasma concentrations of regulatory peptides were monitored in groups of obese and normal-weight subjects following modified sham feeding and a liquid fatty meal. Following modified sham feeding a significant increase in immunoreactive cholecystokinin (CCK) in plasma was recorded in both groups. In the obese subjects, however, the concentrations following sham feeding were significantly lower than in normal-weight subjects, and the initial part of the response was negative. Basal and modified sham feeding stimulated immunoreactive pancreatic polypeptide (PP) concentrations in plasma did not differ between the groups. After the liquid fatty meal plasma CCK concentrations increased similarly in both groups. In contrast immunoreactive neurotensin and somatostatin concentrations following the meal were lower in the obese group, and a changed concentration-time pattern for somatostatin was observed in the obese group. Postprandial concentrations of PP and immunoreactive gastrin were not different in the groups. The results indicate that the plasma concentration patterns of CCK, somatostatin and NT are disarranged in obesity. The changes may promote rapid propulsion and absorption of ingested food, and facilitate deposition of fat in adipose tissue in obesity and thus may be of pathophysiological importance.


Meta-analysis for 2 x 2 tables: a Bayesian approach.

This paper develops and implements a fully Bayesian approach to meta-analysis, in which uncertainty about effects in distinct but comparable studies is represented by an exchangeable prior distribution. Specifically, hierarchical normal models are used, along with a parametrization that allows a unified approach to deal easily with both clinical trial and case-control study data. Monte Carlo methods are used to obtain posterior distributions for parameters of interest, integrating out the unknown parameters of the exchangeable prior or 'random effects' distribution. The approach is illustrated with two examples, the first involving a data set on the effect of beta-blockers after myocardial infarction, and the second based on a classic data set comprising 14 case-control studies on the effects of smoking on lung cancer. In both examples, rather different conclusions from those previously published are obtained. In particular, it is claimed that widely used methods for meta-analysis, which involve complete pooling of 'O-E' values, lead to understatement of uncertainty in the estimation of overall or typical effect size.


[Imidazopyridines, an alternative to benzodiazepines].

Imidazopyridines are a new class of molecules comprising so far a pure hypnotic agent--zolpidem, and an anxiolytic alpidem. The biochemical mode of action and the clinical characteristics are discussed with particular reference to benzodiazepines.


IL-4 decreases IFN-gamma-induced endothelial ICAM-1 expression by a transcriptional mechanism.

Intercellular adhesion molecule-1 (ICAM-1, CD54) is a cell surface ligand for LFA-1 (CD11a/18). Several cytokines, such as IL-1, TNF-alpha or IFN-gamma, have been shown to increase the ICAM-1 expression on cultured endothelial cells. Here we show that IL-4 decreases the IFN-gamma-induced ICAM-1 expression on an endothelial cell line (EA.hy 926), whereas IL-4 alone has practically no effects on ICAM-1 expression. The down-regulative effect was also seen at the mRNA level suggesting that this regulation process is transcriptional. At the same time IL-4 was unable to alter the TNF-alpha-induced ICAM-1 up-regulation. Both IFN-gamma and TNF-alpha are able to decrease the proliferation of the endothelial cell line, but IL-4 could not modify this response when added together with the other cytokines. Taken together we show in this paper that IL-4 decreases IFN-gamma but not TNF-alpha-induced up-regulation of ICAM-1 expression on EA.hy 926 cells.


Development of the non-palindromic adaptor polymerase chain reaction (NPA-PCR) for the amplification of alpha- and beta-chain T-cell receptor cDNAs.

We have developed a highly efficient new method for the amplification of alpha- and beta-chain human T-cell receptor (TCR) cDNAs. This method is designated non-palindromic adaptor polymerase chain reaction (NPA-PCR). cDNA was synthesized from total RNA isolated from mononuclear leucocytes, using either an oligo (dT)15-NotI or a C alpha-NotI or a C beta-NotI primer and RNase H-negative reverse transcriptase. The double-stranded cDNA was ligated with the non-palindromic adaptors EcoRI-XmnI [d(ATTCGAACCCCTTCG)] and XmnI G strand [d(pCGAAGGGGTTCG)] (phosphorylated), which resulted in the addition of the EcoRI-XmnI site in both 5' and 3' ends. These two non-palindromic adaptors, EcoRI-XmnI and XmnI G strand, are complementary to each other and both are required for ligation. The EcoRI-XmnI adaptor was removed from the 3' end by treatment with NotI restriction nuclease, whereas it was retained at the 5' end. The non-palindromic adaptor EcoRI-XmnI was used as the 5' amplification primer. C alpha or C beta constant region primers were used as 3' amplification primers. The amplified cDNAs were cloned and the plasmids were used to transform DH5 alpha competent cells. Over 1000 white colonies per 0.1-0.25 micrograms of total RNA or per 10,000 to 50,000 human peripheral blood mononuclear cells were obtained after amplification of either the alpha- or the beta-chain TCR cDNAs. Between 40 and 62% of the colonies (range from five donors) were positive after screening with either a C alpha or a C beta probe, located 5' to the C alpha and C beta amplification primers. A total of 50 amplified alpha- or beta-chain cDNA positive clones from two normal donors were randomly chosen and sequenced, and the sequences obtained were typical of alpha beta TCR. Two new J alpha gene segments were identified. Approximately 30% of the alpha-chain positive clones have 5' untranslated region, and most of the remaining alpha- or beta-chain TCR clones started from the initiation codon or near the 5' end. NPA-PCR has several advantages over existing PCR methods for the amplification of cDNAs with unknown or variable 5' end, such as the T-cell antigen receptors and the immunoglobulins. Among these advantages is that only one 5' end extension primer is required. Because of the large number of TCR V alpha and V beta families, a large number of different 5' end primers are required for amplification of alpha beta TCR cDNAs by conventional PCR.


T cells cloned from human rheumatoid synovial membrane functionally represent the Th1 subset.

The presence of activated T cells in the synovial membrane of patients with rheumatoid arthritis (RA) suggests a role for these cells in the pathogenesis of the disease. Recent evidence indicates that human T cells may fall into functional categories dependent on their cytokine profile and cytotoxic capacity. The human Th1 subset is cytolytic and produces high levels of IFN-gamma whereas the Th2 type of T cell produces IL-4. In order to investigate whether Th1 or Th2 type cells are present in the inflammatory synovial membrane in RA, a panel of synovial membrane derived T-cell clones (n = 19) was generated and studied functionally. Anti-CD3-induced cytotoxicity assays were performed to demonstrate the cytotoxic potential of clones. Except for two, all clones were cytolytic in this test. Clone cells were activated to initiate cytokine production and assessment of the cytokine levels showed that all clones produced large amounts of IFN-gamma (18 out of 19 clones: over 50,000 pg/ml) whereas IL-4 was absent or present in minimal amounts (17 out of 19 clones: less than 1000 pg/ml). The production of IL-1, IL-2 and IL-6 was variable. The functional characteristics of the clones studied indicate that they may resemble the Th1 subtype of T cells. Our data suggest a relation between Th1-type functions the chronic inflammation characteristic of RA.


Oral health in preschool children living in Sweden. Part II--A longitudinal study. Findings at three years of age.

Scientific epidemiological studies of dental health in children three years of age are relatively few in Sweden. The aim of this study was to describe the oral health of three-year-old children living in Sweden, with special reference to immigration and failure to attend health examinations. All of 671 children requested to take part in an earlier investigation (Wendt et al. 1991) were invited for a new dental examination at three years of age. A total of 632 children were examined. At the age of three years 71.7 per cent of the children were caries free. Of the children with caries, 33.5 per cent were immigrants and of the total number of immigrants, 50.5 per cent had caries compared to 21.9 per cent of the non-immigrant children. Among those children, who failed to attend the earlier investigations at one or two years of age, 61.5 per cent had caries at the age of three. Compared to studies on dental health in three-year-old children from the 70's and 80's (Hugoson et al. 1986), this study shows that dental health in three-year-old children has not improved significantly during the last decade. Furthermore, this study supports the suggestion that special preventive dental programmes should be developed for immigrant children and that extra attention should be paid to children who fail to attend health examinations and their families.


Unraveling the knots in plant development.

Homeobox genes, first discovered from studies of homeotic mutations in Drosophila, have recently been found in plants. The proteins encoded by homeobox genes thus join the ranks of other animal transcription factors that have plant developmental counterparts, suggesting that even though plant and animal development are very different, regulatory mechanisms that direct development may be shared among all higher eukaryotes. The role of homeobox genes in plants remains elusive; nonetheless, gain-of-function mutations of one homeobox gene, Knotted, profoundly affect development. The phenotype suggests that ectopic expression of Knotted in leaves causes cells to take on alternative fates.


Detection, classification and 3D reconstruction of biological macromolecules on hypercube computers.

In this work we present results of the mapping on hypercube computers of some of the key steps involved in the procedure for 3D structural determination from transmission electron microscopy images. The goal is the introduction of parallel processing tools in the field of electron microscopy image processing. We show how the rich topology of the hypercube, combined with an efficient programming strategy, allows for order-of-magnitude increase in computational capacity for such time-consuming tasks as calculation of multidimensional FFT's, cross-correlation coefficients, fuzzy partitioning functionals and the filtered back-projection 3D reconstruction method.


Inhibition of cellular autophagy in proximal tubular cells of the kidney in streptozotocin-diabetic and uninephrectomized rats.

To examine the significance of anti-catabolism in renal hypertrophy, cellular autophagy was investigated by electron microscopic morphometry in proximal tubular cells (PTCs) of the outer cortex of the rat kidney after the induction of diabetes mellitus by streptozotocin (STZ) and after unilateral nephrectomy. Adult male Sprague-Dawley rats were divided into three groups and killed by retrograde perfusion fixation, 1, 2 and 3 days after the induction of diabetes (group D; n = 24), after unilateral nephrectomy (group N; n = 24) and after combined treatment (group DN; n = 24). Untreated, age-matched litter mates served as controls (group C; n = 24). By comparison with these controls, the left kidney to initial body weight ratio was increased by 8, 23, and 15% in group D animals, by 8, 23, and 24% in group N animals, and by 10, 21, and 25% in group DN animals at the first, second and third day, respectively. Quantitative evaluation of large test areas showed that the volume and numerical densities of autophagic vacuoles (AVs) in PTCs were significantly lower in these hypertrophed kidneys than in the controls. The average reduction in AV volume density was about 65% in group D animals, about 50% in group N animals and about 75% in group DN animals. These data show that autophagic degradation of cytoplasmic components in PTCs is inhibited in renal hypertrophy independently of the growth stimulus, i.e. uninephrectomy or diabetes. Since insulin per se inhibits cellular autophagy in PTCs, the expected effect of insulin dificiency seems to be counteracted by as yet undefined stimuli that may be related to metabolic work load.


Cellular autophagy in proximal tubules of early diabetic rats following insulin treatment and islet transplantation.

The influence of insulin treatment (group 1) and allogenic islet transplantation (group 2) on renal cellular autophagy were evaluated in adult Lewis rats in the early phase of streptozotocin-induced diabetes mellitus--a condition in which autophagy is inhibited and renal mass is increased. Three days after insulin treatment or islet transplantation (IT), the right kidney was resected and cortical tubular tissue was examined by quantitative electron microscopy. In group 1, the volume and numerical densities of autophagic vacuoles (AVs) increased by 70% and 80% respectively in the proximal tubular cells compared with saline-injected controls. The additive effect of unilateral nephrectomy (Ux) on cellular autophagy was investigated 1 or 2 days after Ux. Compared with the resected right kidney, the volume and numerical densities of AVs in the remnant left kidney decreased by 49% and 43% in the insulin-treated rats, and by 43% and 39% in the saline-injected diabetic animals. In group 2, the volume and numerical densities of AVs increased by 45% and 44% in parenchyma regressing from diabetic hypertrophy after IT, compared with sham-operated controls. After Ux, the volume and numerical densities of AVs decreased by 49% and 43% in IT rats, and by 41% and 53% in the still diabetic sham-operated animals. The data show that inhibition of cellular autophagy in the proximal tubules of the early diabetic kidney can be reversed by insulin replacement, despite the fact that insulin per se inhibits cellular autophagy in the nondiabetic kidney.(ABSTRACT TRUNCATED AT 250 WORDS)


Vitamin A deficiency and inflammation: the pivotal role of secretory cells in the development of atrophic, hyperplastic and metaplastic change in the tracheal epithelium in vivo.

We showed previously that the proliferation of hamster airway secretory cells decreases during vitamin A deficiency (VAD) but later increases when submucosal inflammation develops (Virchows Arch [B] 59:231-242, 1990). This observation has important biological implications since two morphological extremes (atrophy and quiescence versus hyperplasia and hyperproliferation) are reported in the literature for VAD tracheal epithelium in vivo. In the present study, histological slides of tracheal rings from 35-day-old control and VAD hamsters (Virchows Arch [B] 45:197-219, 1984) were reviewed again. Rings from VAD hamsters were selected based on the absence or presence of a florid submucosal inflammation. Quantitative analyses were made on the cartilaginous part of rings from the anterior third of the trachea. When inflammation was absent, a mucociliary pseudostratified epithelium was, for the most part, maintained. The mitotic rate (MR, 6 h colchicine blockade) of secretory cells was markedly reduced (29-fold) but that of basal cells was not changed significantly. Moreover, cell density was not changed by VAD but ciliated cells and secretory cells were decreased and basal cells were increased, proportionally. We call this "minimal morphological change." Thinning (atrophy) of the minimally changed epithelium was associated with focal cell sloughing. Small scattered foci of epidermoid metaplasia (multiple layers of highly keratinized cells which were extremely flat, so that the epithelium was thin and attenuated) were also seen. We call this "atrophic epidermoid metaplasia." When inflammation was present, hyperplastic changes (stratification and epidermoid metaplasia) predominated and cells were in mitosis at all epithelial levels (low, middle, superficial) except in the most superficial (terminally differentiated) squames. The tracheal epithelium was thickened and hypercellular. The cells were piled up at the stratified lesions, and epithelial height, cell density and epithelial MR were significantly increased compared with the non-inflamed VAD epithelium. The effects of VAD and inflammation on cell proliferation were analyzed further by studying 7 h bromodeoxyuridine (BrdU) labelling patterns of cells in VAD tracheal epithelium, with and without submucosal inflammation. In addition, inflammation was induced in "minimally changed epithelium" by mild mechanical injury. The BrdU labelling patterns confirmed that DNA synthesis by secretory cells is reduced markedly by VAD. However, this suppression is overidden by the influx of inflammatory cells (the nature of the stimulus is unknown). The results indicate that the morphological contrasts (atrophy and hyperplasia) seen in the trachea during VAD in vivo are related to extremes in proliferation rates of tracheal secretory cells, regulated by VAD alone (minimal replication) and by inflammation (maximal replication).


Immunocytochemical observation of paraquat-induced alveolitis with special reference to class II MHC antigens.

The expression of class II major histocompatibility complex (MHC) antigens on alveolar epithelial cells and macrophages was investigated immunocytochemically in paraquat-induced alveolitis in the rat lung. Until 2 days after paraquat injection, class II MHC antigens were expressed on the type II alveolar epithelium without any inflammatory cellular infiltration. From the 4th to the 7th day after paraquat injection, class II MHC antigen-positive macrophages increased in the alveolar spaces, whereas the expression on the type II alveolar epithelium became obscure. Over 10 days after the injection, interstitial fibrosis progressed and the intra-alveolar inflammatory infiltrates decreased. Epithelial cells lining the thickened fibrous septa no longer expressed class II MHC antigens. These results suggest that chemical stimuli can induce class II MHC antigen expression on the type II alveolar epithelium in the early stage of cellular injury, followed by inflammatory infiltration and interstitial fibrosis.


An ultrastructural study of proliferative nephritis induced experimentally by a monoclonal antibody against mesangial cells: replacement of mesangial cells by cells of the monocyte-macrophage system.

An experimental nephritis accompanied by transient proteinuria can be produced by an intravenous injection of the monoclonal antibody, 1-22-3, raised against isolated rat glomeruli. The present study deals with the ultrastructural changes in the glomeruli in rats after the injection of this antibody. At 2 h after injection, all the mesangial cells had completely degenerated and neutrophils invaded most mesangial areas. Monocytes occupied the vacant mesangial areas at 24 h and gradually increased in number over the next 4 days. At 4 and 6 days, macrophage-like cells, possibly derived from monocytes, underwent frequent mitosis, resulting in a remarkable proliferation of these cells. The interpretation of these cells as macrophages was strongly supported by the fact that they contained previously injected latex particles in large numbers. From 2 to 4 weeks after injection, the macrophage-like cells gradually transformed into cells indistinguishable from normal mesangial cells. In the present experimental nephritis where all mesangial cells were initially destroyed, cells of the monocyte-macrophage system appear to play a leading role in the pathogenesis of the ensuing proliferative glomerulonephritis, and represent the source of the replacing mesangial cells.


HLA-DR antigen- and S-100 protein-positive dendritic cells in esophageal squamous cell carcinoma--their distribution in relation to prognosis.

The distribution of immunohistochemically labeled HLA-DR antigen- and S-100 protein-positive dendritic cell (DR+DC and S100+DC) was investigated in 59 human esophageal squamous cell carcinomas (SCCs). A dense infiltration of both DR+DC and S100+DC was detected in 11, only DR+DC in two, and only S100+DC in one. In the remaining 45 tumors infiltrating DC were sparse. By means of double immunostaining or the mirror section method, three different types of DC, namely S-100-negative and HLA-DR-positive DC(S100-DR+DC), S-100-positive and HLA-DR-negative DC(S100+DR-DC) and double-positive DC(S100+DR+DC) were clearly identified. With regard to postoperative survival, these patients with tumors in which there was a dense infiltration of DR+DCs and/or S100+DCs showed a significantly better survival rate than those in which DC were sparse (DR+DCs - P less than 0.001; S100+DCs - P less than 0.01). These results indicate that DC infiltration may be a prognostic factor in esophageal SCCs.


Colonic cell proliferation and growth fraction in young, adult and old rats.

Colonic epithelial proliferation was investigated in three groups of rats, aged 3, 60 and 121 weeks. As reported in previous work, the crypts were markedly longer in the young rats, and the number of labelled cells per crypt was significantly greater. There was an upward movement of the marker positions derived from the distribution of labelled cells within the crypt of the young rats. This was a consequence of the increased crypt length, so that the growth fraction, as expressed as a percentage of crypt length, was the same. The proliferative changes between the young rats and the other aged rats were therefore effected by altering the size of the crypts, while maintaining the kinetic organisation. There was no evidence of any proliferative changes or changes in the growth fraction when the colons of the old rats were compared with those of the 60 week old rats.


Endothelial cell protrusions in the rat aortic wall. Immunocytochemical evidence for an alternative transendothelial passage of plasma proteins.

Focal morphological changes in the endothelial lining were observed in the aortic wall of control rats. They consisted of endothelial cytoplasmic projections and vacuolar structures protruding towards the luminal space and containing electron-dense material. Some of these structures were observed to open into the subendothelial space. Endogenous albumin was detected in these compartments by applying protein A-gold immunocytochemistry to thin tissue sections of glutaraldehyde-fixed, Lowicryl-embedded aortic segments. The labelling was mainly distributed along the plasma membrane of the projections as well as over the dense content of the endothelial protrusions. The presence of endogenous albumin in these endothelial structures, together with their opening into the subendothelial space, suggests a role for these structures in an alternative transendothelial transport of albumin.


Fimbriae and membranes on Haemophilus parasuis.

In the electron microscope an additional layer (glycocalix) of the cell wall and fimbriae on Haemophilus parasuis were shown in thin sections of the infected CAM which have their origin on the CM of the Haemophilus parasuis-cells. No fimbriation was seen after conventional cultivation.


On the stimulation of soluble and particulate guanylate cyclase in the rat brain and the involvement of nitric oxide as studied by cGMP immunocytochemistry.

The localization of the particulate and soluble guanylate cyclase in the rat brain was studied using cGMP-immunocytochemistry. The cGMP was fixed to tissue protein using a formaldehyde fixative, and an antibody against cGMP was used which was raised against a cGMP-formaldehyde-thyroglobulin conjugate. We used the atrial natriuretic factor (ANF) as a model compound to stimulate the particulate enzyme and sodium nitroprusside (SNP) to stimulate the soluble enzyme. Sequential immunostaining for cGMP and glial fibrillary acidic protein (GFAP) showed that the great majority of the ANF-responsive, cGMP-producing cells were astrocytes. These ANF-responsive cells were found in discrete parts of the CNS; not all astrocytes in these regions were responsive to ANF. SNP stimulated cGMP in abundantly present neuronal fibres throughout the CNS; few neuronal cell bodies showed increased cGMP production after SNP. Moreover, SNP also raised cGMP in astrocytes, however, not all astrocytes showed the response to SNP. These results suggest that cells might be present in the CNS which contain both the soluble and the particulate guanylate cyclase. It was demonstrated that in the immature cerebellum, the cGMP was raised in glial structures in response to N-methyl-D-aspartate (NMDA), ANF, SNP, and kainic acid. The response to NMDA and kainic acid was sensitive to inhibition of the nitric oxide synthesis from L-arginine by NG-methyl-L-arginine. Surprisingly the response to ANF localized in the molecular layer and the granular layer was also sensitive to inhibition by NG-methyl-L-arginine, whereas the response to ANF in the deep nuclei was not. A small depolarization induced by 10 to 20 mmol/l K+ induced an increase in cGMP in chopped hippocampus tissue which showed a biphasic temporal characteristic. The initial, fast (30 sec), peak was shown to be localized in varicose fibres throughout the hippocampus, whereas the slower response (10 min) was localized in astrocytes. These studies demonstrate that the different enzymes which synthesize cGMP are differently localized. However, there is also a time dependency in the activation of the guanylate cyclases, which becomes apparent in different structures at different times. The possible role of cGMP as a regulator of ion homeostase is discussed.