Relationship between antiretroviral plasma concentration and emergence of HIV-1 resistance mutations at treatment failure.

PURPOSE: The relationship between antiretroviral pharmacokinetic exposure and acquisition of human immunodeficency virus-1 (HIV-1) drug resistance mutations (DRM) is not fully understood. The aim of this study was to investigate whether antiretroviral plasma concentration could predict the emergence of DRM at treatment failure. METHODS: The study cohort comprised retrospectively selected patients with failing antiretroviral regimens for whom a protease inhibitor (PI) or non-nucleoside reverse transcriptase inhibitor (NNRTI) trough concentration measurement (TDM) had been obtained before failure, a genotypic resistance test (GRT1) had been performed before the TDM, and a genotypic resistance test (GRT2) had been performed at therapeutic failure. Drug levels were classified as undetectable/detectable or subtherapeutic/therapeutic according to limits of quantification of a high-performance liquid chromatography-ultraviolet assay or pre-defined efficacy thresholds, respectively. The number of DRM acquired at treatment failure was evaluated by comparing the results of the GRT2 and GRT1. RESULTS: A total of ten and 57 failure episodes occurred among our patients on NNRTI-based and PI-based regimens, respectively, and included in the evaluation. PI concentration was subtherapeutic in 28.1% of patients, among which the levels were undetectable in 21.1%. Twenty-five (43.9%) patients acquired at least one new PI-DRM according to the GRT2. Patients with undetectable PI levels showed a lower emergence of PI-DRM (minor + major) than those with detectable levels (8.3 vs. 53.3%, p = 0.007). Multivariate analysis confirmed that undetectable PI levels were independent negative predictors of DRM selection. NNRTI measurements were subtherapeutic in 2/10 (20%) patients. NNRTI-DRM were acquired by all patients regardless of NNRTI levels. CONCLUSIONS: A PI measurement showing undetectable drug levels prior to treatment failure predicted the lack of emergence of PI-DRM at failure. These results suggest that PI levels can help clinicians interpret the reasons for treatment failure and guide the type of interventions needed.

Mid-dosing interval concentration of atazanavir and virological outcome in patients treated for HIV-1 infection.

OBJECTIVES: We investigated the clinical significance of monitoring the mid-dosing interval atazanavir (ATV) concentration (measured 12 +/- 2 h after intake; C(12 h)) in patients taking this drug once daily in the evening. METHODS: We retrospectively selected HIV-infected patients harbouring ATV-susceptible virus who underwent therapeutic drug monitoring (TDM) of ATV C(12 h) during routine out-patient visits, and we correlated C(12 h) to the 24-week virological response and toxicity. RESULTS: A total of 115 plasma samples from 86 patients (76.7% with baseline HIV RNA<50 HIV-1 RNA copies/mL) were analysed. ATV plasma concentrations showed high inter-individual variability. ATV plasma levels were higher in samples obtained from patients taking boosted regimens (P<0.001) and not concomitantly receiving acid-reducing agents (P=0.007). In a multivariate model, ritonavir boosting, use of acid-reducing agents and liver cirrhosis showed an independent association with ATV level. Virological response at 24 weeks was observed for 94 of the 115 samples (81.7%). We identified a concentration cut-off of 0.23 mg/L which predicted virological response at 24 weeks: samples with a C(12 h)< or =0.23 mg/L showed virological failure in 41.2% of cases, whereas samples with a C(12 h)>0.23 mg/L showed virological failure in 14.3% of cases (P=0.021). In multivariate analysis, C(12 h)>0.23 mg/L was an independent predictor of virological response [odds ratio (OR) 4.23, P=0.031]. ATV levels correlated with concomitant unconjugated bilirubin levels (r=0.223, P=0.037), but a concentration cut-off predictive of moderate/severe hyperbilirubinaemia could not be identified. CONCLUSIONS: We identified a C(12 h) efficacy threshold that predicted virological response; this could be useful for morning TDM in selected subjects receiving ATV in the evening. Results must be interpreted with caution given the retrospective design of the study.

Mild hepatitis at recommended doses of acetaminophen in patients with evidence of constitutionally enhanced cytochrome P450 system activity.

Acetaminophen (paracetamol) is used throughout the world for pain relief and antipyresis in both children and adults. In many countries, it can be purchased without a medical prescription and it is also a common component of a number of over-the-counter remedies for colds, influenza and the like. Fasting, malnutrition and use of alcohol and/or other drugs are thought to play causal roles in hepatotoxicity associated with recommended doses of acetaminophen although liver injury provoked by therapeutic doses has also been observed in the absence of these factors. We describe two patients who experienced subclinical hepatotoxic reactions after taking acetaminophen at therapeutic doses. The results of an antipyrine metabolism test suggest the presence of constitutional hyperactivity of the cytochrome P450-dependent mixed function oxidative system in both patients. We hypothesize that the latter contributed to the hepatotoxicity and that it may play a role in idiosyncratic reactions to this drug.

Inhibition of heme oxygenase in the central nervous system potentiates endotoxin-induced vasopressin release in the rat.

Previous in vitro studies have shown that increases in endogenous carbon monoxide (CO) generation via activation of the enzyme heme oxygenase (HO) within the rat hypothalamus are associated with the reduced release of the neuropeptides, vasopressin (AVP) and oxytocin, while evidence concerning corticotrophin-releasing hormone (CRH) is controversial. The present study investigated whether there is also a functional relationship between the HO-CO pathway and AVP and corticosterone (Cort) in vivo. Male Wistar rats were challenged with bacterial lipopolysaccharide (LPS) at doses producing significant activation of the hypothalamo-pituitary-adrenal (HPA) axis. LPS was given alone or after pretreatment with the HO inhibitor Sn-protoporphyrin-9 (SnPP9). The latter was injected either intraperitoneally (i.p.) or by intracerebroventricular (i.c.v.) route. SnPP9 given i.p. failed to modify either basal or LPS-stimulated levels of AVP and Cort. On the contrary, i.c.v. SnPP9 strongly potentiated LPS-induced AVP release and significantly enhanced basal serum Cort levels, although it failed to potentiate stimulation by LPS. The LPS + i.c.v. SnPP9 also significantly reduced the hypothalamic stores of AVP compared to controls, correlating with increased circulating levels of AVP. Taken collectively, these data are in concordance with previous in vitro observations showing that the HO-CO pathway acts centrally to attenuate endotoxin-stimulated AVP release, while having less effects on the pituitary-adrenal axis.

Peptide histidine isoleucine-like immunoreactivity release from the rat gastric fundus.

1. Longitudinal muscle strips from the rat gastric fundus were subjected to in vitro electrical field stimulation (EFS) under non-adrenergic non-cholinergic (NANC) conditions to study the release of peptide histidine isoleucine-like immunoreactivity (PHI-LI) and the correlation between PHI-LI release and NANC relaxation. 2. Different radioimmunoassay (RIA) systems employing C-terminal- and N-terminal-specific anti-PHI sera were used to determine the relative contributions of PHI and its C-terminally extended forms, peptide histidine glycine (PHI-Gly) and peptide histidine valine [PHV(1-42)], to the PHI-LI released by the rat gastric fundus. 3. In the presence of atropine (1 microM) and guanethidine (5 microM), EFS (120 mA, 1 ms, 0.25-32.0 Hz, trains of 2 min) induced frequency-dependent relaxations of 5-hydroxytryptamine (3 microM) pre-contracted strips. 4. EFS at frequencies of 8-32 Hz evoked significant increases in PHI-LI outflow. The increases in PHI-LI outflow evoked by 16-Hz EFS were abolished by tetrodotoxin (3 microM) and by a calcium-free medium, indicating an active release process from intramural nerves. 5. The EFS-induced release of PHI-LI measured with the N-terminal-specific antiserum was significantly greater than that detected with the C-terminal-specific antisera. 6. Sephadex G-25 gel permeation chromatographic analysis was performed on the PHI-LI release in response to 32-Hz EFS. A C-terminal-specific antiserum revealed one peak co-eluting with the rat PHI standard. When PHI-LI was measured with the N-terminal-specific antiserum, two peaks were found that co-eluted with the rat PHV(1-42) and rat PHI-Gly/PHI standards, respectively. 7. The present data suggest that the extended forms of PHI are the primary components of the PHI-LI released by NANC inhibitory neurones in the rat gastric fundus and support a NANC inhibitory neurotransmitter role for PHI and its extended forms in this tissue.

Release of vasoactive intestinal polypeptide from the rat gastric fundus.

1. Auxotonic responses and release of vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) induced by electrical field stimulation (EFS) were studied in longitudinal muscle strips from the gastric fundus of reserpinized rats suspended between parallel platinum electrodes in Krebs solution containing atropine (1 microM), 5-hydroxytryptamine (3 microM) and bovine serum albumin (50 mg l-1). 2. EFS (supramaximal voltage, 1 ms, 0.25-32.0 Hz, trains of 2 min) induced frequency-dependent relaxations. 3. EFS at frequencies greater than or equal to 8 Hz also produced significant increases in VIP-LI release. 4. VIP-LI release induced by EFS at 16 Hz no longer occurred in the presence of tetrodotoxin (1 microM) or a Ca(2+)-free medium. 5. Detection of VIP-LI upon activation of inhibitory non-adrenergic, non-cholinergic neurones indicates that VIP meets the 'detectable release' criterion for an inhibitory neurotransmitter in the rat gastric fundus.

Evidence that hydrogen sulphide can modulate hypothalamo-pituitary-adrenal axis function: in vitro and in vivo studies in the rat.

The gas hydrogen sulphide (H2S) is normally produced in large amounts in the central nervous system during the metabolism of sulphur-containing aminoacids. H2S was recently shown to influence long-term potentiation in the rat hippocampus; this finding suggested that the gas may act as a neuromodulator in the brain. We therefore tested the effect of the gas on the release of corticotropin-releasing hormone (CRH) from rat hypothalamic explants. CRH immunoreactivity in the incubation media was taken as a marker of peptide release. We found that the addition of NaHS to incubation media was consistently associated with a concentration-dependent decrease in KCl-stimulated CRH release, whereas basal secretion was unaffected. Increased endogenous H2S production may be also obtained using an indirect precursor of H2S formation, S-adenosyl-L-methionine (SAMe). The latter mimicked the effects of NaHS, since it reduced potassium-stimulated CRH release. In vivo, SAMe showed no effect on hypothalamo-pituitary-adrenal (HPA) function under resting conditions, but inhibited stress-related glucocorticoid increase.

Anaphylaxis increases 8-iso-prostaglandin F2alpha release from guinea-pig lung in vitro.

8-Iso-prostaglandin F2alpha, release from isolated and perfused guinea-pig lung was measured by radioimmunoassay. 8-Iso-prostaglandin F2alpha release was detectable under basal conditions and increased 10-fold during antigen-induced bronchoconstriction, concomitant with the increase of thromboxane B2 and prostaglandin E2. The anti-8-iso-prostaglandin F2alpha serum used in the radioimmunoassay seems to be quite specific for this compound. Pretreatment of lungs with indomethacin (a non-selective inhibitor of cyclooxygenase) reduced 8-iso-prostaglandin F2alpha release under basal conditions and completely abolished the increase observed during lung anaphylaxis. Pretreatment of lungs with NS 398 (N-[2-cyclohexyl]-4-nitrophenyl methanesulphonamide), a selective inhibitor of cyclooxygenase-2, did not change basal or antigen-induced 8-iso-prostaglandin F2alpha release at all. We conclude that under basal conditions guinea-pig lung perfusates contain low levels of 8-iso-prostaglandin F2alpha-like immunoreactivity, which increase 10-fold during antigen-induced bronchoconstriction. This isoprostane seems to be derived from the cyclooxygenation of arachidonic acid via the constitutive form of cyclooxygenase.

Leptin stimulates prostaglandin E2 and F2alpha, but not nitric oxide production in neonatal rat hypothalamus.

Leptin, an adipocyte-derived 16 kDa polypeptide hormone, has been found to regulate food intake and thermogenesis by modulating stimulatory and inhibitory pathways in the feeding circuitry of the hypothalamus, among which corticotropin releasing hormone (CRH). Nitric oxide (NO) and prostaglandins have been shown to be involved in both CRH neurosecretion and feeding regulation. We have investigated the role of NO, prostaglandin E2 and prostaglandin F2alpha as mediators of the hypothalamic effects of leptin and their possible involvement in leptin-stimulated CRH secretion. Using primary cultures of neonatal (5- to 6-day-old) rat hypothalamic cells, we confirmed that leptin (0.1-10 nM) stimulates CRH secretion. This effect was not blocked by L-N(G)-nitro-methyl-arginine (L-NAME, 100 microM), a NO-synthase competitive inhibitor; and leptin did not stimulate NO production. Cyclooxygenase inhibition by indomethacin (10 microM) did not modify leptin-induced CRH secretion, while leptin stimulated prostaglandin E2, and prostaglandin F2alpha secretion. In conclusion, leptin-induced hypothalamic CRH secretion is not modulated by NO-synthase- or cyclooxygenase-mediated mechanisms; leptin does not stimulate NO production, but it stimulates prostaglandin E2 and F2alpha production, which could add to the growing list of mediators of leptin signaling in the hypothalamus.

A possible role for nitric oxide but not for prostaglandin E2 in basal and interleukin-1-beta-induced PRL release in vitro.

In previous experiments we have shown that nitric oxide (NO) was able to modulate CRH and ACTH release from cultured rat hypothalamic and anterior pituitary cells, in vitro. Now, we show experimental evidence of an involvement of NO in basal and interleukin-1 beta-induced prolactin (PRL) release. L-NG-nitro-arginine, an inhibitor of nitric oxide synthetase, and hemoglobin, a NO scavenger, impaired basal and interleukin-1-beta-induced PRL release, while molsidomine, a NO donor, was able to release PRL and to amplify interleukin-1-beta-induced PRL release, confirming a modulatory role for nitric oxide in pituitary hormone secretion. On the other hand, no evidence regarding a possible role of prostaglandin E2 (PGE2) in IL-1beta-induced PRL release came out from our experiments.

Effects of lipopolysaccharide on hypothalamic-pituitary-adrenal axis in vitro.

The possible involvement of lipopolysaccharide (LPS) and interleukin-1 beta (IL-1 beta) and their eventual interplay in CRH and ACTH release from cultured hypothalamic and pituitary cells respectively, have been studied. IL-1 beta was able to activate the hypothalamo-pituitary-adrenal axis at both hypothalamic and pituitary sites; LPS showed no direct action at hypothalamic level but it was able to inhibit basal and IL-1 beta-induced ACTH release: this could be responsible for a blunting of the adrenal cortex response that normally occurs in septic shock syndrome.

Involvement of nitric oxide in basal and interleukin-1 beta-induced CRH and ACTH release in vitro.

NG-Nitro-L-arginine, an inhibitor of nitric oxide synthase, does not influence CRH basal release but is able to modify ACTH basal secretion and to block interleukin-1 beta-induced CRH and ACTH release from rat hypothalamic and anterior pituitary cell cultures in vitro. The data suggest a different involvement of NO at hypothalamic and pituitary level.

Interleukin-1 beta specifically stimulates nitric oxide production in the hypothalamus.

In previous experiments we have shown the role of nitric oxide (NO) in basal and interleukin-1 beta (IL-1 beta)-induced CRH and ACTH release in vitro. Now, we have studied the possible production of NO from hypothalamic cell cultures, particularly after IL-1 beta stimulation or L-NOArg inhibition, by high performance liquid chromatographic (HPLC) assay of L-citrulline production, adding further evidence for a role of NO in IL-1 beta activity in the hypothalamus.

Effects of vindesine on hypothalamic-pituitary-adrenal axis.

Vindesine, a cell-cycle-specific agent currently employed in the treatment of some neoplasias, was able to produce a remarkable dose-dependent adrenocortical activation, but it was unable to increase plasma corticosterone in hypophysectomized rats in vivo. In addition, vindesine was able to increase ACTH release in vitro when tested on isolated pituitary cells in culture suggesting a direct involvement of the pituitary gland in the increase of adrenal secretion in vivo.

A possible prolactin-related adverse effect of certain antineoplastic anthracyclines.

Nonhormonal antineoplastic therapy can affect the endocrine system with consequent effects on the growth of hormone-sensitive tumors. Amongst anthracycline antibiotics, we have found daunorubicin and epirubicin able to acutely stimulate prolactin (PRL) secretion both in vivo, in the rat, and in vitro, from rat anterior pituitary cell cultures. Despite a similar structure, doxorubicin showed no such activity. Considering the possible role of PRL in breast cancer cell proliferation, the effects of certain anthracyclines might be viewed as an adverse drug reaction involving the anterior pituitary gland.

Assessment of the acrylic resin Technovit 7200 VLC for studying the gingival mucosa by light and electron microscopy.

Technovit 7200 VLC is an acrylic resin formulated for embedding undecalcified hard tissues which are prepared for light microscopy according to a cutting-grinding technique. To employ this resin for embedding and cutting soft tissues by ultramicrotomy, we carried out a qualitative study on biopsies of canine gingival mucosa using light and transmission electron microscopy. For a critical evaluation of this resin, some biopsies were embedded in Agar 100, an epoxy resin widely used in morphological studies. At the light microscopic level the samples embedded in Technovit 7200 VLC showed good morphology and excellent toluidine blue staining of different cell types and extra cellular matrix. At the ultrastructural level, nuclei, cytoplasmic organelles, collagen fibrils and ground substance appeared well preserved and showed high electron density. The acrylic resin was stable under the electron beam and its degree of shrinkage appeared to be very low. We conclude that Technovit 7200 VLC can be employed for ultramicrotomy for both light and electron microscopic investigation of soft tissues.

A procedure for preparing undecalcified and unembedded bone sections for light microscopy.

We have developed a procedure for light microscopic investigation of undecalcified and unembedded bone sections. Biopsy samples of human metatarsus and femur and rat femur were fixed in aldehydes and sectioned with a cutting machine equipped with a diamond saw blade. Free sections 100-150 microns thick, stained with toluidine blue and von Kossa, did not show artifacts following the cutting, and the spatial relations of mineralized and nonmineralized components remained intact. Compact and trabecular bone, bone marrow and all cell types appeared well preserved and easily recognizable. Our procedure provides a simple and rapid method for preparing bone sections which undergo no chemical treatment other than fixation. This method is a useful alternative to standard histological protocols for studying bone specimens.

[Cost methodologies in dialysis in relation to the Italian National Health Service]. / Metodologie dei costi in dialisi in rapporto all'azienda salute italiana.

This work analyses the economic aspects of dialysis in Italy in relation to government resources allocated to the health service in general. The authors illustrate the procedures used to estimate the resources required by the dialytic programme. The costs of dialytic programmes in different cities and at different periods in the history of the Italian health service are compared. A concrete example is outlined of the economic management of dialysis and the authors demonstrate how the results were obtained using cost analysis.

[High-efficiency membranes and the dialysis solution]. / Membrane ad alta efficienza e bagno di dialisi.

During this work through the total bacterial count and of the pseudomonas and the determination of the positive substances to Limulus Amebocyte Lysate test, bacterial pollution of the liquid bicarbonate concentrate has been determined together with the influence that preparation conditions and the entity of interval between the said preparation and utilization of concentrates have on it. Through the evaluation of the cardiac rate and the systemic arterial pressure, we have studied the influence of the bacterial pollution on the clinical of the patient. Analysing the obtained data we can point out that the liquid bicarbonate concentrate present an elevated degree of contamination which feels the effects of the production and storage procedures. The remark of inverse correlation between systemic arterial pressure and endotoxin level of the dialysate seems to reconfirm the importance of the bacterial contamination on the clinical of the patient.

[Safety in dialysis rooms. Biologic risks]. / La sicurezza in sala dialisi. Il rischio biologico.

The search for quality in the health service cannot lead aside the safety of its operators and users, subject to the well defined parameters of Law 626. This study makes a preliminary examination of the accidents occurring in our Health District which comprises three hospitals, 600 beds and 1,800 employees. A total of 172 accidents have been reported. The percentages can be broken down between the various sectors: 73% of accidents involve nurses, 9% involve doctors and 1% administrative personnel. The greatest risk in hemodialysis is the biological factor (through accidental cuts or pricks which account for 67% of the accidents reported) and involves humans (both patients and personnel), monitors and environments as the sources of pathogens. The most frequently isolated germs are E. coli and Pseudomonas. It has been shown that prevention is above all based on the accuracy with which procedures are followed. The risk of hepatitis C has not yet been resolved, as is affinned in a review reported in the study. The HIV risk gives the greatest cause for concern, even if only 0.2% after exposure compared to 15-36 for HbsAg. Compliance with universal rules for prevention and post-exposure procedures provides an adequate guarantee for prevention.