Rapid CD4(+) T-cell loss induced by human immunodeficiency virus type 1(NC) in uninfected and previously infected chimpanzees.

To investigate the pathogenicity of a virus originating in a chimpanzee with AIDS (C499), two chimpanzees were inoculated with a plasma-derived isolate termed human immunodeficiency virus type 1(NC) (HIV-1(NC)). A previously uninfected chimpanzee, C534, experienced rapid peripheral CD4(+) T-cell loss to fewer than 26 cells/microl by 14 weeks after infection. CD4(+) T-cell depletion was associated with high plasma HIV-1 loads but a low virus burden in the peripheral lymph node. The second chimpanzee, C459, infected 13 years previously with HIV-1(LAV), experienced a more protracted course of peripheral CD4(+) T-cell loss after HIV-1(NC) inoculation, resulting in fewer than 200 cells/microl by 96 weeks postinoculation. The quantities of viral RNA in the plasma and peripheral lymph node from C459 were below the lower limits of detection prior to inoculation with HIV-1(NC) but were significantly and persistently increased after superinfection, with HIV-1(NC) representing the predominant viral genotype. These results show that viruses derived from C499 are more pathogenic for chimpanzees than any other HIV-1 isolates described to date.

Dose-response curve and optimal dosing regimen of cyclosporin A after traumatic brain injury in rats.

Acute neuropathology following experimental traumatic brain injury results in the rapid necrosis of cortical tissue at the site of injury. This primary injury is exacerbated in the ensuing hours and days via the progression of secondary injury mechanism(s) leading to significant neurological dysfunction. Recent evidence from our laboratory demonstrates that the immunosuppressant cyclosporin A significantly ameliorates cortical damage following traumatic brain injury. The present study extends the previous findings utilizing a unilateral controlled cortical impact model of traumatic brain injury in order to establish a dose-response curve and optimal dosing regimen of cyclosporin A. Following injury to adult rats, cyclosporin A was administrated at various dosages and the therapy was initiated at different times post-injury. In addition to examining the effect of cyclosporin A on the acute disruption of the blood-brain barrier following controlled cortical impact, we also assessed the efficacy of cyclosporin A to reduce tissue damage utilizing the fluid percussion model of traumatic brain injury. The findings demonstrate that the neuroprotection afforded by cyclosporin A is dose-dependent and that a therapeutic window exists up to 24h post-injury. Furthermore, the optimal cyclosporin dosage and regimen markedly reduces disruption of the blood-brain barrier acutely following a cortical contusion injury, and similarly affords significant neuroprotection following fluid percussion injury. These findings clearly suggest that the mechanisms responsible for tissue necrosis following traumatic brain injury are amenable to pharmacological intervention.

Does your clinical investigator understand the consequences of non-compliance?

The increasing number and complexity of clinical trials, coupled with the recent increase in attention and enforcement from the U.S. Food and Drug Administration, arguably makes it more important than ever before that clinical investigators understand the regulatory requirements with which they must comply. This article outlines some of these requirements, common areas of non-compliance and FDA's enforcement options in dealing with such non-compliance. Finally, the article provides some suggestions for attaining and managing investigator compliance.

Dissociation of buprenorphine-induced locomotor sensitization and conditioned place preference in rats.

The locomotor and rewarding effects of the opioid mixed agonist-antagonist buprenorphine were assessed in a conditioned place preference (CPP) experiment. Separate groups of rats were given one of three doses of buprenorphine (0.3, 1.0 or 3.0 mg/kg) or saline paired with the white compartment of a CPP apparatus. The following day, all rats received saline paired with the black compartment. After six conditioning trials, rats were given free access to all compartments of the CPP apparatus. Horizontal activity data obtained during conditioning revealed increased activity (i.e., behavioral sensitization) for the three doses on trial 6. Vertical activity data revealed significant increases on trial 6 for the 1.0 and 3.0 mg/kg doses only. Significant CPP was obtained with the 0.3 mg/kg and 1.0 mg/kg doses of buprenorphine, but not with the 3.0 mg/kg dose. These data indicate that buprenorphine elicits locomotor sensitization after repeated exposures that follows a linear dose-response relationship. In contrast, these data suggest that the rewarding effects of buprenorphine follow an inverted U-shaped dose-response curve.

Biochemical studies of soluble atrial natriuretic peptide (ANP) receptors from rat olfactory bulb and vascular smooth muscle cells.

1. Aim. The biochemical characteristics of atrial natriuretic peptide receptors (ANP-R) derived from rat vascular smooth muscle (A-10 cell line) and central nervous system (CNS; olfactory bulb) tissue were compared. 2. Method and Results. ANP-Rs from each source were solubilized with 40 to 65% efficiency utilizing the nonionic detergent Lubrol-PX. Upon solubilization, the ANP-R from each source maintained the ability to bind 125I-ANP (99-126) with a high affinity; Scatchard analysis indicated that the VSMC ANP-R displayed a Kd for the radioligand of approximately 10 pM, whereas the olfactory receptor possessed a Kd of about 165 pM. The Bmax values for the soluble VSMC and olfactory ANP-Rs were 285 and 30 fmol/mg protein, respectively. Competition binding studies indicated that the VSMC ANP-R bound ANP(99-126), ANP(103-126), and ANP(103-123) with similar affinities, whereas the olfactory ANP-R was much more sensitive to changes in the COOH-terminal structure of the competing peptide. The soluble ANP-Rs from VSMC and olfactory were chromatographically indistinguishable on phenyl-, DEAE-, and wheat germ agglutinin-agarose columns. However, the ANP-Rs could be distinguished using GTP-agarose; the olfactory ANP-R was capable of binding to the resin, whereas the VSMC ANP-R was not. 3. Conclusions. Coupled with other studies, these data suggest that the A10 VSMC ANP-R observed in this study may not be coupled to guanylate cyclase and may represent a receptor serving a clearance function, whereas a significant proportion of the olfactory CNS ANP-R appears to be associated with GTP-binding proteins, likely particulate guanylate cyclase, and probably represents a coupled form of the receptor.

Secretion of atrial natriuretic factor-(1-98) by primary cardiac myocytes.

Previous studies have demonstrated that primary cultures of cardiac myocytes maintained in a complete serum-free medium contain a precursor to atrial natriuretic factor (ANF-(1-126]. The cultured cells secrete this precursor unless maintained in the presence of glucocorticoids wherein the known circulating form derived from the C-terminal of ANF (ANF-(99-126] is secreted. The present study was designed to determine the fate of the N-terminal region of the ANF precursor during secretion from myocytes maintained in glucocorticoids. A radioimmunoassay (RIA) was developed using synthetic ANF-(1-16); the antiserum demonstrated cross-reactivity toward ANF-(1-126) and ANF-(1-98)-like peptides but did not cross-react with ANF-(99-126). Coupling this RIA with an ANF-(99-126)-specific RIA and reversed phase, size exclusion, and ion exchange high performance liquid chromatography (HPLC), it was shown that primary cultures of atrial myocytes maintained in dexamethasone contained ANF-(1-126) and secreted ANF-(99-126) and a peptide that was chromatographically indistinguishable from ANF-(1-98). Isolated perfused rat hearts were also shown by RIA and HPLC to secrete similar peptides. The primary cells were labeled with [35S]methionine, and the secreted N-terminal ANF-related material was immunoprecipitated with the ANF-(1-16) antiserum. HPLC, tryptic peptide mapping, and radiosequencing demonstrated that this peptide possessed an N-terminal structure identical to that of ANF-(1-126). When the cells were labeled with [3H] leucine and the secreted N-terminal ANF-related material was immunoprecipitated and analyzed by tryptic mapping, it was shown to possess labeled tryptic peptides consistent with the structure of ANF-(1-98). Tryptic mapping of [3H]arginine-labeled N-terminal ANF-related material demonstrated the presence of all peptides consistent with the ANF-(1-98) structure, including ANF-(92-98). These studies demonstrate that primary atrial myocytes contain ANF-(1-126) and in the presence of dexamethasone secrete both ANF-(1-98) and ANF-(99-126), the two major circulating forms of the hormone.

Characterization of solubilized atrial natriuretic peptide receptors from rat olfactory bulb and A10 cultured smooth muscle cells.

Atrial natriuretic peptide (ANP) receptors from A10 cultured vascular smooth muscle cells (VSMC) and rat olfactory bulbs have been solubilized and then pharmacologically and biochemically compared. The dissociation constant for 125I-ANP(99-126) was 12.7 pM for the VSMC-derived receptor and 164 pM for the olfactory receptor. Competition binding between 125I-ANP(99-126) and several unlabeled ANP analogs with the soluble olfactory receptor, demonstrated a rank order potency of ANP(99-126) = ANP(103-126) much greater than ANP(103-123). However, the rank order potency of the soluble VSMC ANP receptor was ANP(99-126) = ANP(103-126) = ANP(103-123). Therefore, the olfactory ANP receptor appears to require the complete COOH-terminal sequence of ANP as compared with the VSMC ANP receptor. When the 2 soluble receptor preparations were applied to a GTP-agarose column, a portion of the olfactory ANP receptor was retained on the column and could be eluted with 5 mM GTP, while the VSMC ANP receptor did not adsorb to the column. Since the olfactory bulb ANP receptor has been shown to contain a binding component of 116 kDa, while the VSMC ANP receptor binding component is 66 kDa, these receptors appear to be similar to the 2 receptor classes described recently in which the 120 kDa receptor that binds GTP is postulated to be coupled to guanylate cyclase, while the 60 kDa receptor does not bind GTP, is not coupled to guanylate cyclase, and may possess a hormone clearance function. Taken together, these data indicate that cyclic GMP appears to be a second messenger for ANP in the brain.

The characterization of atrial natriuretic peptide (ANP) expression by primary cultures of atrial myocytes using an ANP-specific monoclonal antibody and an ANP messenger ribonucleic acid probe.

The biochemical and morphological characteristics of primary neonatal rat atrial myocytes were examined in order to establish a model system for future studies of the biosynthesis and secretion of atrial natriuretic peptide (ANP). Preliminary studies demonstrated that the quantity of immunoactive ANP/microgram protein within rat atria increased as a function of age from 2 ng/micrograms in 19 day prenatal animals to 400 ng/micrograms in the adult. Gel filtration, reversed phase HPLC, and ion exchange HPLC indicated that there were similar quantities of immunoactive ANP in the right and left atria at various ages, and that the major molecular form of the peptide in the heart is chromatographically indistinguishable from ANP(1-126). Cultures of dissociated cells were prepared from pooled left and right atria derived from 1 day postnatal animals. A complete serum-free medium was developed which resulted in the maintenance of high levels of immunoactive ANP in the cultures. As determined by RIA, the cellular content of ANP increased in the cultures as a function of time through 7 days in vitro. The quantity of immunoactive ANP in the cultures increased approximately 2- to 3-fold between days 3 and 7. When the cultures that had been maintained for 7 days were submitted to immunocytochemistry using an ANP-specific monoclonal antibody, distinct colonies of spindle-shaped cells stained positively. In situ hybridization, utilizing an 35S-labeled ANP messenger RNA probe, demonstrated that these colonies of myocytes expressed the ANP message. Using quantitative dot-blot hybridization it was shown that the ANP mRNA level increased approximately 50-fold between days 1 and 7 in culture. These studies indicate that the serum-free culture medium allows continued accumulation of both ANP and the ANP message in culture and will provide a useful model system to characterize factors that regulate the biosynthesis and secretion of this hormone.

The conversion of atrial natriuretic peptide (ANP)-(1-126) to ANP-(99-126) by rat serum: contribution to ANP cleavage in isolated perfused rat hearts.

Atrial myocytes cultured for 7 days in serum-free medium secrete a 15K form of atrial natriuretic peptide (15K ANP), but isolated perfused rat hearts secrete the major circulating form of the hormone, a 3K peptide, 3K ANP. This difference was examined in the present study. 15K ANP was purified from rat atria, and sequencing analysis demonstrated that this atrial-derived ANP possessed an NH2-terminal sequence identical to that of pro-ANP; this is consistent with other reports suggesting that the major form of ANP in the atria is ANP-(1-126). Fresh rat serum was shown to cleave efficiently ANP-(1-126) to form a 3K immunoactive ANP-related peptide. Upon purification and sequencing the identity of this peptide was confirmed as ANP-(99-126); ANP-(99-126) was relatively resistant to further proteolysis by rat serum. To probe further the specificity of the serum conversion, synthetic ANP-(92-126) was used as a substrate; purification and sequencing of the immunoactive product peptide verified its identity as ANP-(99-126). Since purified thrombin and plasma kallikrein both cleaved ANP-(1-126) to 3K ANP-like material, inhibitors of these enzymes were tested for their ability to inhibit the serum cleavage of ANP-(1-126). D-Phe-Phe-Arg-Chloromethylketone (D-Phe-Phe-Arg-CMK) and D-Phe-Pro-Arg-CMK both inhibited serum ANP cleavage by over 90% at low micromolar concentration. When these inhibitors were added to the isolated heart perfusate, 3K ANP was still released by the atria, indicating that ANP processing occurs in the heart in a region not accessible to the inhibitors (i.e. intracellularly) or that the ANP-processing enzyme(s) is not inhibited by these CMK analogs and is, therefore, not related to serum-derived proteases.

Autoradiographic localization and characterization of atrial natriuretic peptide binding sites in the rat central nervous system and adrenal gland.

Atrial natriuretic peptides (ANP) have recently been identified in both heart and CNS. These peptides possess potent natriuretic, diuretic, and vasorelaxant activities, and are all apparently derived from a single prohormone. Specific ANP binding sites have been characterized in the adrenal zona glomerulosa and kidney cortex, and one study reported ANP binding sites in the CNS. However, a detailed examination of the localization of ANP binding sites throughout the brain has not been reported. In this study, quantitative autoradiography was employed to examine the distribution of ANP receptors in the rat CNS. The binding of (3-125I-iodotyrosyl28) rat ANP-28 to binding sites in the rat CNS was saturable, specific for ANP-related peptides, and displayed high affinity (Kd = 600 pM). When the relative concentrations of ANP binding sites were determined throughout the rat brain, the highest levels of ANP binding were localized to the circumventricular organs, including the area postrema and subfornical organ, and the olfactory apparatus. Moderate levels of ANP binding sites were present throughout the midbrain and brain stem, while low levels were found in the forebrain, diencephalon, basal ganglia, cortex, and cerebellum. The presence of ANP binding sites in the subfornical organ and the area postrema, regions considered to be outside the blood-brain barrier, suggests that peripheral ANP levels may regulate some aspects of CNS control of salt and water balance. The possible functions of ANP binding sites in other regions of the rat brain are not known, but, like many other peptides, ANP may act as a neurotransmitter or neuromodulator at these loci.

Ascorbic acid increases the thyrotropin-releasing hormone content of hypothalamic cell cultures.

Thyrotropin-releasing hormone (TRH) is one of many COOH-terminal alpha-amidated neuropeptides. Recent work with the intermediate pituitary has indicated that ascorbate is a required cofactor for the COOH-terminal alpha-amidation of alpha-melanotropin. This is consistent with the ascorbate requirement of an enzyme found in pituitary and hypothalamus capable of converting peptides with a COOH-terminal glycine (-X-Gly) to alpha-amidated molecules (-H-NH2). Thus, it has been proposed that COOH-terminal glycine-extended TRH (TRH-Gly) may be the direct precursor to TRH. In the present study, primary hypothalamic cultures supplemented with ascorbate for 7 d contained two- to threefold more TRH immunoactivity (amide-specific) than cultures maintained without ascorbate. A dose-response experiment indicated that 20 microM ascorbate was capable of producing 50% of the maximum observable increase in culture TRH immunoactivity; this concentration is similar to the Km value for ascorbate uptake obtained in adrenal chromaffin and pituitary cells. A stereoisomer of ascorbate, D-isoascorbate, was also capable of producing an increase in TRH immunoactivity, but oxidized ascorbate was not. Recent studies have shown that the amidation enzyme from pituitary is capable of utilizing both L-ascorbate and D-isoascorbate but is incapable of utilizing oxidized ascorbate. The culture extracts were analyzed further by reversed-phase high-performance liquid chromatography; the increased TRH immunoactivity observed in extracts of cultures maintained in ascorbate comigrated with standard synthetic TRH.(ABSTRACT TRUNCATED AT 250 WORDS)

Ascorbate transport by AtT20 mouse pituitary corticotropic tumor cells: uptake and secretion studies.

Ascorbate is an important cofactor in the biosynthesis of alpha-amidated endocrine and neural peptides. Peptidylglycine alpha-amidating monooxygenase (PAM) is the enzyme responsible for the generation of mature COOH-terminal alpha-amidated peptides from COOH-terminal glycine-extended peptides, and this enzyme requires ascorbate for full activity in vitro. Also, cultured intermediate pituitary lobe cells contain PAM and require ascorbate for the COOH-terminal alpha-amidation of alpha MSH. Since pituitary cells are not capable of synthesizing ascorbate, the ability of the cells to accumulate the cofactor must play an important role in the biosynthesis of alpha-amidated peptides. The AtT20 corticotropic pituitary tumor cell line also contains PAM and a potential site for COOH-terminal alpha-amidation of the pro-ACTH/endorphin-derived hinge peptide and was, thus, used for the study of cellular ascorbate transport. Radiolabeled L-[1-14C]ascorbate ([1-14C]ascorbate) was incubated with the cells under various conditions, and the accumulation of radioactivity by the cells was followed. Reverse phase HPLC was used to identify the integrity of the labeled ascorbate, both intra- and extracellular, during the course of the experiments. The uptake of [1-14C]ascorbate was saturable (Km = 31.5 microM), sodium and temperature dependent, and stereoselective. The products of ascorbate autooxidation, dehydroascorbate and 2,3-diketogulonic acid, did not inhibit [1-14C]ascorbate uptake. To study the presence of ascorbate in the secretory granules, cells were incubated with [1-14C]ascorbate and then induced to secrete with isoproterenol or 8-bromo-cAMP. A 2- to 6-fold stimulation of ACTH secretion over the basal secretion rate was observed; however, the secretion of intracellular [1-14C]ascorbate did not change significantly with stimulation, suggesting that very little of the cellular ascorbate was contained within secretory granules.

Adherence of Candida to cultured vascular endothelial cells: mechanisms of attachment and endothelial cell penetration.

To elucidate the pathogenesis of hematogenous Candida infections, we developed an in vitro model of Candida adherence to and penetration of human endothelial cells. We enhanced or inhibited adherence in order to probe mechanisms of attachment. Adherence of Candida albicans showed a linear relation to Candida inoculum (range, 10(2)-10(5) cfu, r = .99, P less than .01) and exceeded that of less virulent Candida species and that of Saccharomyces cerevisiae (P less than .01). Candida immune serum blocked attachment (greater than 95% inhibition; P less than .001), however, this activity was abolished by immunoprecipitation of immune serum with C. albicans mannan (P less than .001) and was unaffected by immunoprecipitation with S. cerevisiae mannan or by adsorption with particulate chitin. Adherence was diminished by exposing C. albicans to heat (greater than 99% inhibition; P less than .01), UV light (98% inhibition; P less than .01), or sodium periodate (greater than 72% inhibition; P less than .01). An extract from heat-exposed C. albicans blocked adherence (greater than 51% inhibition; P less than .001). Transmission electron microscopy demonstrated that viable or killed Candida organisms were attached to endothelial cells, were enveloped by membrane processes from the endothelial cell surface, and were incorporated into the endothelial cells within phagosomes. Cytochalasin B blocked incorporation without blocking surface attachment.

Molecular forms of immunoactive atrial natriuretic peptide released from cultured rat atrial myocytes.

Primary cultures of atrial myocytes were prepared from newborn rats and maintained for 8 days in complete serum-free medium. The culture content of immunoactive atrial natriuretic peptide (ANP) increased from 10 to 25 ng/culture during this time. The cells released immunoactive ANP at a rate of 2 to 3% of culture content per hour in a linear fashion for at least 6 hours. When analyzed by gel filtration the major immunoactive material released by and contained within the cells displayed a molecular weight of approximately 15,000 daltons. The medium and cellular ANP-related peptides were further shown to be indistinguishable by reversed-phase HPLC. When the 15,000 dalton material was incubated with rat serum it was converted to ANP-related material possessing a molecular weight of approximately 3,000 daltons. These results suggest that under basal conditions, atrial myocytes release a large molecular weight form of ANP that is converted in the circulation to a low molecular weight form of ANP, which has been previously identified in plasma.

Acetylation of alpha MSH and beta-endorphin by rat neurointermediate pituitary secretory granule-associated acetyltransferase.

ACTH(1-8) and ACTH(9-13)NH2 were used as potential enzyme inhibitors to begin examining the relationship between the acetylation of ACTH- and beta-endorphin-related peptides. ACTH(1-8) was a potent inhibitor of the acetylation of both ACTH- and beta-endorphin-related peptides, whereas ACTH(9-13)NH2 was an effective inhibitor only of the acetylation of ACTH-related substrates. This inhibition pattern indicated that there may be an unusual interaction between some ACTH- and beta-endorphin-related peptides as substrates for the acetyltransferase. Utilizing HPLC to separate ACTH- and beta-endorphin-related peptides present in the same reaction mixture, ACTH(1-14) and beta-endorphin(1-27) at Km and saturating concentrations were used as substrates to examine the ability of one peptide substrate to affect the acetylation of the other. It was observed that the acetylation of ACTH(1-14), even at Km concentration, was relatively unaffected by the presence of beta-endorphin(1-27). However, the acetylation of beta-endorphin(1-27) was significantly reduced by the presence of ACTH(1-14). This preferential acetylation of ACTH-related peptides over the acetylation of beta-endorphin-related peptides might have physiological importance under some conditions.

Molecular forms of immunoactive atrial natriuretic peptide in the rat hypothalamus and atrium.

Acid extracts of rat hypothalamus and atrium were prepared by a procedure previously shown to minimize proteolytic degradation of peptides. The majority of the immunoactive material in the atrial extracts had a molecular weight of approximately 9,000 to 15,000 daltons, while that in the hypothalamic extracts had a molecular weight of about 1,500 to 1,800 daltons. The major molecular weight forms of atrial natriuretic peptide from each extract were further distinguishable when analyzed by RP-HPLC. These results suggest that small peptides such as atriopeptins I, II, and III, may not be authentic post-translational processing products in the atrium, and that the hypothalamus and atrium may differentially cleave pro-atrial natriuretic peptide to form tissue-specific products.

Comparison of intraluminal and intravenous mediators of colonic response to eating.

Eating a 1000-kcal mixed meal stimulates an increase in distal colonic motility. Fat is the dietary component which is the major stimulant of colonic spike activity. In this study the colonic spike activity increased similarly after the mixed meal [19.1 +/- 2.4 spike potentials (SP)/30 min] and after the fat meal (19.4 +/- 5.4 SP/30 min). Fat stimulated a concentration-dependent increase in colonic motility only when in contact with the gastroduodenal mucosa. Intravenous administration of Liposyn (100 kcal/hr) did not stimulate an increase in colonic spike activity (3.3 +/- 1.3 SP/30 min) despite greater increase in plasma total fatty acid levels than after the oral ingestion of fat. In contrast both the oral ingestion and the intravenous administration of an amino acid mixture (Aminosyn) inhibited the gastrocolonic response after the 1000-kcal mixed meal. Thus, these studies demonstrate: (1) fat stimulates colonic motility only through direct mucosal contact, and (2) a mixture of amino acid inhibits colonic motility through either mucosal contact or by circulating in the plasma. The exact neurohumoral mechanisms involved in both of these effects is unknown at present.

Detection of fungemia obscured by concomitant bacteremia: in vitro and in vivo studies.

Our recent clinical experience suggested that bacteremia may interfere with the detection of concomitant fungemia when standard blood culture methods are used. To determine the extent to which bacteria may interfere with fungal isolation from blood cultures, an in vitro model simulating blood cultures taken during concomitant fungemia and bacteremia was created. Each of six bacteria (Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa) was combined with each of three pathogenic yeasts (Candida albicans, Candida tropicalis, and Torulopsis glabrata) in vented blood culture bottles containing enriched brain heart infusion broth and fresh normal human blood. Blood culture bottles were analyzed at 1, 2, and 7 days of incubation. Gram strains and subcultures onto chocolate and MacConkey agars failed to detect fungi in 37.0, 66.7, and 100% of samples, respectively. However, subcultures onto Sabouraud dextrose agar failed in only 13% of the samples (occurring only with P. aeruginosa). In a rabbit model of concomitant fungemia with C. albicans and bacteremia with P. aeruginosa, no yeasts were recovered from blood cultures despite 100% detection of P. aeruginosa. Therefore, the usual microbiological techniques may be inadequate to detect fungemia when concomitant bacteremia is present.