A Multiplex PCR and DNA-Sequencing Workflow on Serum for the Diagnosis and Species Identification for Invasive Aspergillosis and Mucormycosis.

There has been significant increase in the use of molecular tools for the diagnosis of invasive aspergillosis (IA) and mucormycosis. However, their range of detection may be too limited as species diversity and coinfections are increasing. Here, we aimed to evaluate a molecular workflow based on a new multiplex PCR assay detecting the whole Aspergillus genus and the Mucorales order followed by a species-specific PCR or a DNA-sequencing approach for IA and/or mucormycosis diagnosis and species identification on serum. Performances of the MycoGENIE Aspergillus spp./Mucorales spp. duplex PCR kit were analyzed on a broad range of fungal strains and on sera from high-risk patients prospectively over a 12-month period. The kit allowed the detection of nine Aspergillus species and 10 Mucorales (eight genera) strains assessed. No cross-reactions between the two targets were observed. Sera from 744 patients were prospectively analyzed, including 35 IA, 16 mucormycosis, and four coinfections. Sensitivity varies from 85.7% (18/21) in probable/proven IA to 28.6% (4/14) in COVID-19-associated pulmonary aspergillosis. PCR-positive samples corresponded to 21 A. fumigatus, one A. flavus, and one A. nidulans infections. All the disseminated mucormycosis were positive in serum (14/14), including the four Aspergillus coinfections, but sensitivity fell to 33.3% (2/6) in localized forms. DNA sequencing allowed Mucorales identification in serum in 15 patients. Remarkably, the most frequent species identified was Rhizomucor pusillus (eight cases), whereas it is barely found in fungal culture. This molecular workflow is a promising approach to improve IA and mucormycosis diagnosis and epidemiology.

Novel GSK-3ß Inhibitor Neopetroside A Protects Against Murine Myocardial Ischemia/Reperfusion Injury.

Recent trends suggest novel natural compounds as promising treatments for cardiovascular disease. The authors examined how neopetroside A, a natural pyridine nucleoside containing an α-glycoside bond, regulates mitochondrial metabolism and heart function and investigated its cardioprotective role against ischemia/reperfusion injury. Neopetroside A treatment maintained cardiac hemodynamic status and mitochondrial respiration capacity and significantly prevented cardiac fibrosis in murine models. These effects can be attributed to preserved cellular and mitochondrial function caused by the inhibition of glycogen synthase kinase-3 beta, which regulates the ratio of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced, through activation of the nuclear factor erythroid 2-related factor 2/NAD(P)H quinone oxidoreductase 1 axis in a phosphorylation-independent manner.

Resistance exercise improves cardiac function and mitochondrial efficiency in diabetic rat hearts.

Metabolic disturbance and mitochondrial dysfunction are a hallmark of diabetic cardiomyopathy (DC). Resistance exercise (RE) not only enhances the condition of healthy individuals but could also improve the status of those with disease. However, the beneficial effects of RE in the prevention of DC and mitochondrial dysfunction are uncertain. Therefore, this study investigated whether RE attenuates DC by improving mitochondrial function using an in vivo rat model of diabetes. Fourteen Otsuka Long-Evans Tokushima Fatty rats were assigned to sedentary control (SC, n = 7) and RE (n = 7) groups at 28 weeks of age. Long-Evans Tokushima Otsuka rats were used as the non-diabetic control. The RE rats were trained by 20 repetitions of climbing a ladder 5 days per week. RE rats exhibited higher glucose uptake and lower lipid profiles, indicating changes in energy metabolism. RE rats significantly increased the ejection fraction and fractional shortening compared with the SC rats. Isolated mitochondria in RE rats showed increase in mitochondrial numbers, which were accompanied by higher expression of mitochondrial biogenesis proteins such as proliferator-activated receptor-γ coactivator-1α and TFAM. Moreover, RE rats reduced proton leakage and reactive oxygen species production, with higher membrane potential. These results were accompanied by higher superoxide dismutase 2 and lower uncoupling protein 2 (UCP2) and UCP3 levels in RE rats. These data suggest that RE is effective at ameliorating DC by improving mitochondrial function, which may contribute to the maintenance of diabetic cardiac contractility.

Outcome after failure of allogeneic hematopoietic stem cell transplantation in children with acute leukemia: a study by the société Francophone de greffe de moelle et de thérapie cellulaire (SFGM-TC).

Allogeneic hematopoietic stem cell transplantation (SCT) contributes to improved outcome in childhood acute leukemia (AL). However, therapeutic options are poorly defined in the case of post-transplantation relapse. We aimed to compare treatment strategies in 334 consecutive children with acute leukemia relapse or progression after SCT in a recent 10-year period. Data could be analyzed in 288 patients (157 ALL, 123 AML and 8 biphenotypic AL) with a median age of 8.16 years at transplantation. The median delay from first SCT to relapse or progression was 182 days. The treatment consisted of chemotherapy alone (n=108), chemotherapy followed by second SCT (n=70), supportive/palliative care (n=67), combination of chemotherapy and donor lymphocyte infusion (DLI; n=30), or isolated reinfusion of donor lymphocytes (DLI; n=13). The median OS duration after relapse was 164 days and differed according to therapy: DLI after chemotherapy=385 days, second allograft=391 days, chemotherapy=174 days, DLI alone=140 days, palliative care=43 days. A second SCT or a combination of chemotherapy and DLI yielded similar outcome (hazard ratio (HR)=0.85, P=0.53) unlike chemotherapy alone (HR=1.43 P=0.04), palliative care (HR=4.24, P<0.0001) or isolated DLI (HR=1,94, P<0.04). Despite limitations in this retrospective setting, strategies including immunointervention appear superior to other approaches, mostly in AML.

Cytogenetics and outcome of allogeneic transplantation in first remission of acute myeloid leukemia: the French pediatric experience.

We analyzed the impact of cytogenetics on 193 children enrolled in two successive French trials (LAME89/91 and ELAM02), who received hematopoietic stem cell transplantation during CR1. Detailed karyotype was available for 66/74 (89%) in LAME89/91 and 118/119 (99%) in ELAM02. Several karyotype and transplant characteristics differed according to therapeutic protocol: unfavorable karyotypes were more frequent in ELAM02 (36% vs 18%), pretransplant chemotherapy included high-dose cytarabine in ELAM02 and not in LAME89/91, IV replaced oral busulfan in the conditioning regimen, methotrexate was removed from post-transplant immunosuppression, and matched unrelated donor and cord blood transplantation were introduced. Five-year overall survival (OS) was 78.2% in LAME89 and 81.4% in ELAM02. OS was significantly lower for the unfavorable cytogenetic risk group in LAME89/91 when compared with intermediate and favorable groups (50% vs 90.6 and 86.4%, P=0.001). This difference was no longer apparent in ELAM02 (80.9% vs 71.3% and 5/5, respectively). Survival improvement for children with unfavorable karyotype was statistically significant (P=0.026) and was due to decrease in relapse risk. Five-year transplantation-related mortality was 6.75% in LAME89/91. In ELAM02, it was 3.2% for patients with a sibling donor and 10.9% with an unrelated donor or cord blood. We conclude that the outcome of children with unfavorable karyotype transplanted in CR1 has improved.

Effect of immune modulation in relapsed peripheral T-cell lymphomas after post-allogeneic stem cell transplantation: a study by the Société Française de Greffe de Moelle et de Thérapie Cellulaire (SFGM-TC).

Peripheral T-cell lymphoma carries a poor prognosis. To document a possible graft-versus-lymphoma effect in this setting, we evaluated the impact of immunomodulation in 63 patients with peripheral T-cell lymphoma who relapsed after allogeneic transplant in 27 SFGM-TC centers. Relapse occurred after a median of 2.8 months. Patients were then treated with non-immunologic strategies (chemotherapy, radiotherapy) and/or immune modulation (donor lymphocyte infusions (DLI) and/or discontinuation of immunosuppressive therapy). Median overall survival (OS) after relapse was 6.1 months (DLI group: 23.6 months, non-DLI group: 3.6 months). Among the 14 patients who received DLI, 9 responded and 2 had stable disease. Among the remaining 49 patients, a complete response accompanied by extensive chronic GvHD was achieved in two patients after tapering of immunosuppressive drugs. Thirty patients received radio-chemotherapy, with an overall response rate of 50%. In multivariate analysis, chronic GvHD (odds ratio: 11.25 (2.68-48.21), P=0.0009) and skin relapse (odds ratio: 4.15 (1.04-16.50), P=0.043) were associated with a better response to treatment at relapse. In a time-dependent analysis, the only factor predictive of OS was the time from transplantation to relapse (hazards ratio: 0.33 (0.17-0.640), P=0.0009). This large series provides encouraging evidence of a true GvL effect in this disease.

Hematopoietic stem cell transplantation in relapsed ALK+ anaplastic large cell lymphoma in children and adolescents: a study on behalf of the SFCE and SFGM-TC.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) is a treatment option for relapsed anaplastic large cell lymphoma (ALCL) in children, but reports on its efficacy in this disease are still limited. We analyzed data concerning 34 patients under 18 years of age prospectively registered in the French SFGM-TC database, who had undergone an allo-SCT for the treatment of ALK+ ALCL between 1993 and 2011. At transplant, 28 patients (82.4%) were in CR, whereas 6 exhibited detectable disease. Conditioning regimens were mostly myelo-ablative (n=31). With a median follow-up of 6 years, 5-year overall and event-free survival rates were 70% (SE=8%) and 58% (SE=9%), respectively. The 5-year cumulative incidence of relapse and treatment-related mortality was 18% (SE=7%) and 24% (SE=8%), respectively. Six patients had relapsed (median time, 141 days (35-235)). A durable CR had been obtained in 4/6 patients after injection of donor lymphocytes (n=1) or vinblastine-corticosteroid therapy (n=3). Ten patients had died, eight due to transplant toxicity and two due to progressive disease. Allo-SCT is an efficient treatment for pediatric patients with high-risk relapsed ALK+ ALCL. However, the overall morbidity of allo-SCT raises questions about its place, given the efficacy of targeted agents currently under development in this disease.

[Managing late-effects after allogeneic stem cell transplantation in children: recommendations from the SFGM-TC]. / Prise en charge des séquelles après greffe de cellules souches hématopoïétiques chez l'enfant : recommandations de la SFGM-TC.

In this report, we address the issue of late-effects after allogeneic stem cell transplantation in children. In an effort to harmonize clinical practices between different French transplantation centers, the French Society of Bone Marrow Transplantation and Cell Therapy (SFGM-TC) set up the fourth annual series of workshops which brought together practitioners from all member centers and took place in September 2013 in Lille.

Adenovirus infections in Bordeaux University Hospital 2008-2010: clinical and virological features.

BACKGROUND: Transversal epidemiological data on adenovirus infections in a hospital setting, including both immuno-competent and transplanted patients, are limited and rarely contain the application of molecular virology. OBJECTIVES: To describe the clinical characteristics and molecular epidemiology of adenovirus infections in Bordeaux University Hospital from 2008 to 2010 (clinical data, viral load and adenovirus species distribution). STUDY DESIGN: Adenovirus DNA quantification (qPCR) and typing (sequencing of hexon and protein VI genes and protein VI polymerase chain reaction (PCR) product analysis) were applied retrospectively to 215 clinical samples from 105 adenovirus-infected patients (2008-2010, Bordeaux University Hospital). Clinical data were recovered and analysed for 73 children and 25 adults. RESULTS: Viral loads were measured in stools, upper and lower respiratory fluids, blood, urine and digestive tract biopsies; the highest values were observed in stools and respiratory samples. Stool viral loads were comparable whatever the immune status. Adenovirus was typed in 57 patients: species Human adenovirus (HAdV) C dominated (n=36), followed by B (n=15), F (n=5) and D (n=1). We could demonstrate no association between HAdV species and load or clinical severity (observed in most patients). In the immuno-compromised, in contrast to immuno-competent patients, adenovirus infections presented no seasonal variation. Co-infections were frequent: mostly bacterial in immuno-competent children (33%) and viral in immuno-compromised people (34%). CONCLUSIONS: The species HAdV C dominates the local ecology, in both respiratory and digestive tract infections, independently of the patient's immune status. Adenovirus infections, often associated with co-infection of bacterial or viral agents, frequently lead to severe clinical consequences in hospital patients.

Engraftment of unrelated cord blood after reduced-intensity conditioning regimen in children with refractory neuroblastoma: a feasibility trial.

Relapsed or refractory neuroblastoma is uniformly fatal. We hypothesized that allogeneic response could provide a platform for immunotherapy in neuroblastoma. We therefore undertook a pilot trial of unrelated cord blood transplantation after reduced intensity conditioning regimen (RIC) in children with relapsed neuroblastoma to assess engraftment and tolerability in this heavily pretreated population. The RIC included CY (50 mg/kg, day -6), fludarabine (40 mg/m(2), days -6 to -2), total body irradiation (200 cGy, day -1), and rabbit anti-thymocyte globulin (2.5 mg/kg, days -3 to -1). Six patients were enrolled: four were in partial responsive relapse, one with a mixed response and one in refractory relapse. All patients tolerated the regimen well and had donor engraftment with full neutrophil and plt recovery (median time 12 and 35 days, respectively). One patient never experienced neutropenia and another did not need plt transfusions. All patients progressed after transplant (median time 55 days, 26-180 days). Natural killer (NK) cell counts were normal within 2 months, whereas T-cell recovery was slower. In conclusion, unrelated cord blood engrafts after RIC in children with refractory neuroblastoma. Future research should be aimed at transplanting patients with minimal residual disease, using less intensive immunosuppression and adding NK-cell based post transplant immunotherapy.

[Targeted therapies in pediatric oncology: a new therapeutic approach?]. / Thérapies ciblées en oncologie pédiatrique: nouvelle approche thérapeutique.

A multidisciplinary therapeutic approach has led to significant increase in survival of children with cancer, however often with a high rate of severe sequela. Better understanding in tumor cell biology and transformation process allowed to describe active tyrosine kinases (mainly growth factor receptors) as a new target for cancer treatment. This review presents 2 approaches to target receptor tyrosine kinase activity: on one hand, antibodies that target the extracellular domain, the natural ligand binding site, and on the other hand, small inhibiting molecules, such as imatinib, targeted against the activated intracellular receptor tyrosine kinase. We focus on their clinical development and current application in the treatment of childhood cancer. Targeted therapies are in full rise and new perspectives are explored, such as their association to other treatment modalities and the targeting of microenvironment. This new therapeutic approach necessitates well designed clinical trials that include relevant biomarkers to evaluate its real therapeutic potential.

[Methotrexate for the treatment of patients with chronic cutaneous sarcoidosis: 4 cases]. / Traitement de la sarcoïdose cutanée par le méthotrexate: 4 observations.

INTRODUCTION: Pathogenesis of sarcoidosis remains partially unknown. Cutaneous lesions are frequent (20 to 35% of cases). Their clinical features and follow-up data are highly variable. Numerous treatments have been proposed. The clinical features and follow up data of four patients with chronic cutaneous sarcoidosis treated with methotrexate are reported. CASE REPORT: Mean age of patients (3 female, 1 male) was 40 years old (34-49 years). One patient presented with a lupus pernio, two patients with papules and nodules, and the last with an annular lesion of the face. All patients had been previously treated with topical corticosteroids and/or hydroxychloroquine without any success. Patients were treated with methotrexate at doses ranging from 12.5 mg to 30 mg per week for at least 6 months. Complete remission of cutaneous lesions was observed in 3 of 4 patients after a mean treatment duration of 29 months (16 to 36). Methotrexate side effects were observed in one patient (elevated liver enzymes) leading to methotrexate discontinuation. DISCUSSION: Methotrexate seems to be an effective treatment of cutaneous sarcoidosis. It should be used namely in patients who failed to respond to previous treatments with topical corticosteroids or antimalarial drugs.

Compound A uptake and metabolism to mercapturic acids and 3,3,3-trifluoro-2-fluoromethoxypropanoic acid during low-flow sevoflurane anesthesia: biomarkers for exposure, risk assessment, and interspecies comparison.

BACKGROUND: Sevoflurane is degraded during low-flow anesthesia to fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether ("compound A"), which causes renal necrosis in rats but is not known to cause nephrotoxicity in surgical patients. Compound A is metabolized to glutathione S-conjugates and then to cysteine S-conjugates, which are N-acetylated to mercapturic acids (detoxication pathway), or metabolized by renal beta-lyase to reactive intermediates (toxification pathway) and excreted as 3,3,3-trifluoro-2-fluoromethoxypropanoic acid. This investigation quantified compound A metabolites in urine after low-flow sevoflurane administration, to assess relative flux via these two pathways. METHODS: Patients (n = 21) with normal renal function underwent low-flow (11 min) sevoflurane anesthesia designed to maximize compound A formation. Inspiratory, expiratory, and alveolar compound A concentrations were quantified. Urine mercapturic acids and 3,3,3-trifluoro-2-fluoromethoxypropanoic acid concentrations were measured by gas chromatography and mass spectrometry. RESULTS: Sevoflurane exposure was 3.7 +/- 2.0 MAC-h. Inspired compound A maximum was 29 +/- 14 ppm; area under the inspired concentration versus time curve (AUCinsp) was 78 +/- 58 ppm x h. Compound A dose, calculated from pulmonary uptake, was 0.39 +/- 0.35 mmol (4.8 +/- 4.0 micromol/kg) and correlated with AUCinsp (r2 = 0.84, P < 0.001). Mercapturic acids excretion was complete after 2 days, whereas 3,3,3-trifluoro-2-fluoromethoxypropanoic acid excretion continued for 3 days in some patients. Total (3-day) mercapturates and fluoromethoxypropanoic acid excretion was 95 +/- 49 and 294 +/- 416 micromol, respectively (1.2 +/- 0.6 and 3.6 +/- 5.0 micromol/kg). CONCLUSION: Compound A doses during 3.7 MAC-h, low-flow sevoflurane administration in humans are substantially less than the threshold for renal toxicity in rats (200 micromol/kg). Compound A metabolites quantification may provide a biomarker for compound A exposure and relative metabolism via toxification and detoxication pathways. Compared with previous investigations, relative metabolic flux (fluoromethoxypropanoic acid/mercapturates) through the toxification pathway was sixfold greater in rats than in humans. Species differences in dose and metabolism may influence compound A renal effects.

Dose-dependent metabolism of fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (compound A), an anesthetic degradation product, to mercapturic acids and 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid in rats.

The volatile anesthetic sevoflurane is degraded in anesthesia machines to fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (FDVE), to which humans are exposed. FDVE is metabolized in rats and humans to two alkane and two alkene glutathione S-conjugates that are hydrolyzed to the corresponding cysteine S-conjugates. The latter are N-acetylated to mercapturic acids, or bioactivated by renal cysteine conjugate beta-lyase to metabolites which may react with cellular macromolecules or hydrolyze to 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid. FDVE causes nephrotoxicity in rats, which evidence suggests is mediated by renal uptake of FDVE S-conjugates and metabolism by beta-lyase. Although pathways of FDVE metabolism have been described qualitatively, the purpose of this investigation was to quantify FDVE metabolism via mercapturic acid and beta-lyase pathways. Fischer 344 rats underwent 3-h nose-only exposure to FDVE (0 +/- 0, 46 +/- 19, 98 +/- 7, 150 +/- 29, and 220 +/- 40 ppm), and urine was collected for 24 h. Urine concentrations of the mercapturates, N-acetyl-S-(1,1,3,3, 3-pentafluoro-2-fluoromethoxypropyl)-L-cysteine and N-acetyl-S-(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl)vinyl)-L- cysteine, the beta-lyase-dependent metabolite 3,3, 3-trifluoro-2-(fluoromethoxy)propanoic acid, and its degradation product trifluorolactic acid, were determined by GC/MS. There was dose-dependent urinary excretion of the alkane mercapturate N-acetyl-S-(1,1,3,3,3-pentafluoro-2-fluoromethoxypropyl)-L- cysteine and 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid, while excretion of the alkene mercapturate N-acetyl-S-(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl)vinyl)-L- cysteine plateaued at higher FDVE exposures. The alkane:alkene mercapturic acid excretion ratio was between 2:1 and 4:1. Trifluorolactic acid was only rarely observed. Urine excretion of the beta-lyase-dependent metabolite 3,3, 3-trifluoro-2-(fluoromethoxy)propanoic acid was 10-fold greater than that of the combined mercapturates. Results show that FDVE cysteine S-conjugates undergo facile metabolism via renal beta-lyase, particularly in comparison with detoxication by mercapturic acid formation. The quantitative assay developed herein may provide a biomarker for FDVE exposure and relative metabolism via toxification and detoxifying pathways, applicable to animal and human investigations.

Duration of cytochrome P-450 2E1 (CYP2E1) inhibition and estimation of functional CYP2E1 enzyme half-life after single-dose disulfiram administration in humans.

Disulfiram (DSF) is a mechanism-based inhibitor of cytochrome P-450 2E1 (CYP2E1), resulting in loss of CYP2E1 protein and activity, which may be useful in preventing CYP2E1-mediated xenobiotic toxicity. The duration of inhibition after a single DSF dose is, however, unknown. The purpose of this investigation was to determine this duration, and CYP2E1 formation and degradation rates, in humans. Oral chlorzoxazone (CLZ) was used as the selective in vivo probe for CYP2E1. Healthy subjects received CLZ to determine baseline CYP2E1 activity (CLZ plasma clearance and 6-hydroxychlorzoxazone fractional metabolic clearance). One week later, DSF (500 mg orally) was administered at bedtime, and CLZ administered the following morning and 3, 6, 8, 10, and 13 days after DSF. A terminal DSF metabolite, 2-thiothiazolidine-4 carboxylic acid, was also measured in each 24-h urine sample. The mean CLZ clearance and 6-hydroxychlorzoxazone fractional metabolic clearance on the first day declined to 10.2 and 5.5% of baseline values, indicating rapid and profound CYP2E1 inhibition. CYP2E1 activity returned to half that of control on day 3, and to baseline values on day 8. Assuming zero-order synthesis and first-order degradation, the in vivo CYP2E1 synthesis rate and degradation half-life was estimated to be 11 +/- 5 nmol/h and 50 +/- 19 h, respectively. Significant amounts of 2-thiothiazolidine-4 carboxylic acid were present only on day 1, suggesting that the return of in vivo CYP2E1 activity was not caused by inhibitor washout, but by enzyme resynthesis. Results regarding CYP2E1 disposition may be useful for modeling the effects of CYP2E1 inducers and inhibitors. For prevention of CYP2E1-mediated bioactivation, depending on protoxicant disposition, a second DSF dose might be necessary to completely prevent toxicity.

Intraindividual variability in male hepatic CYP3A4 activity assessed by alfentanil and midazolam clearance.

Clinical investigations using isoform-selective probes to phenotype cytochrome P450 activity and interaction studies using isoform-selective inhibitors to determine P450 involvement in drug metabolism assume minimal interday variability in P450 activity. CYP3A4 is the most abundant human P450 isoform and metabolizes approximately half of all therapeutic agents. This investigation evaluated interday variability in hepatic CYP3A4 activity in males, using the clearances of midazolam and alfentanil as metabolic probes. Midazolam (1 mg) followed 1 hour later by alfentanil (20 micrograms/kg) were administered by intravenous bolus to 9 nonsmoking male volunteers (ages 30 +/- 8 years). Drug administration was repeated 12 and 20 days later. Venous plasma midazolam and alfentanil concentrations were determined by gas chromatography/mass spectrometery. Drug clearances were determined by noncompartmental and multiexponential analysis. There were no significant interday differences in plasma drug concentrations or clearances (3.9 +/- 1.4, 3.9 +/- 1.7, and 4.2 +/- 1.7 ml/kg/min for alfentanil, respectively, and 6.6 +/- 2.0, 7.9 +/- 2.4, and 7.9 +/- 2.5 ml/kg/min for midazolam, respectively, on days 1, 13, and 21 [mean +/- SD]). Interday variability in clearance was 13% +/- 6% and 19% +/- 12% for alfentanil and midazolam, respectively. Interday variability in the clearance of these probes, and presumably hepatic CYP3A4 activity, was small compared with interindividual variability. Consideration of interday variability in the hepatic metabolism of CYP3A4 substrates does not appear significant in the design of clinical trials.

Lack of single-dose disulfiram effects on cytochrome P-450 2C9, 2C19, 2D6, and 3A4 activities: evidence for specificity toward P-450 2E1.

Disulfiram and its primary metabolite diethyldithiocarbamate are effective mechanism-based inhibitors of cytochrome P-450 2E1 (CYP2E1)1 in vitro. Single-dose disulfiram diminishes CYP2E1 activity in vivo and has been used to identify CYP2E1 participation in human drug metabolism and prevent CYP2E1-mediated toxification. Specificity of single-dose disulfiram toward CYP2E1 in vivo, however, remains unknown. This investigation determined single-dose disulfiram effects on human CYP 2C9, 2C19, 2D6, and 3A4 activities in vivo. In four randomized crossover experiments, volunteers received isoform-selective probes (oral tolbutamide, mephenytoin, dextromethorphan, or i.v. midazolam) on two occasions, 10 h after oral disulfiram or after no pretreatment (controls). Plasma and/or urine parent and/or metabolite concentrations were measured by HPLC or gas chromatography-mass spectrometry. CYP2C9, 2C19, 2D6, and 3A4 activities were determined from the tolbutamide metabolic ratio, 4'-hydroxymephenytoin excretion, and dextromethorphan/dextrorphan ratios in urine and midazolam systemic clearance, respectively. Midazolam clearance (670 +/- 190 versus 700 +/- 240 ml/min, disulfiram versus controls), dextromethorphan/dextrorphan metabolic ratio (0.013 +/- 0.033 versus 0.015 +/- 0.035), 4'-hydroxymephenytoin excretion (122 +/- 22 versus 128 +/- 25 micromol), and tolbutamide metabolite excretion (577 +/- 157 versus 610 +/- 208 micromol) were not significantly altered by disulfiram pretreatment, although the tolbutamide metabolic ratio was slightly diminished after disulfiram (60 +/- 17 versus 81 +/- 40, p <.05). Results show that single-dose disulfiram does not cause clinically significant inhibition of human CYP2C9, 2C19, 2D6, and 3A4 activities in vivo. When single-dose disulfiram is used as an in vivo probe for P-450, inhibition of drug metabolism suggests selective involvement of CYP2E1. Single-dose disulfiram should not cause untoward drug interactions from inhibition of other P-450 isoforms.

[Cutaneous malacoplakia: a pediatric case]. / Malakoplakie cutanée: un cas pédiatrique.

INTRODUCTION: Cutaneous malakoplakia is an inflammatory disease characterized by granulomatous accumulation of distinctive phagocytic macrophages. It occurs mainly in visceral or orificial areas; the condition is rarely purely cutaneous, and appears to be extremely rare in childhood. CASE REPORT: A facial cutaneous crusted lesion was diagnosed as cutaneous malakoplakia in an immunocompetent child. The lesion had been excised twice and it had recurred, and the diagnosis was made possible only with a third biopsy, after a 2-year chronic expansion. This third biopsy revealed a dense granulomatous inflammation with numerous phagocytic histiocytes containing abundant fine granules and round Michaelis-Gutmann bodies, both staining with PAS, Perls and von Kossa. Biopsy cultures revealed only growth of two different streptococcus (group B) strains. The lesion resolved after a 4-month period of antibiotic therapy, including roxithromycin, ampicillin and trimethoprim-sulfamethoxasole. DISCUSSION: Diagnosis of malakoplakia is mainly made by histopathologic examination of tissue excision or biopsies. There are no specific clinical features. Most reported cases of this uncommon phagocytic reaction to common bacteria have developed in the genitourinary areas (71 p. 100); purely cutaneous localisation, as in our patient, are rare (4 p. 100). Intracytoplasmic granules may result from phagolysosomes and incomplete bacterial killing, with subsequent deposit of iron and calcium in the phagocytic macrophages. A number of reported cases have affected immunocompromised patients with either congenital immunodeficiency or secondary immunodeficiency. The most effective treatment option is based on a protracted antibiotherapy, using drugs that easily permeate the macrophages, e.g. quinolones and trimethoprim-sulfamethoxasole. Lesion may recur after surgical excision.