Clinically-based determination of safe DNAemia cutoff levels for preemptive therapy or human cytomegalovirus infections in solid organ and hematopoietic stem cell transplant recipients

Transplantation Centers using human cytomegalovirus (HCMV) antigenemia-based preemptive therapy will need to replace in the near future the antigenemia assay with a more standardized and automatable assay, such as a molecular assay quantifying HCMV DNA in blood (DNAemia). Thus, in view of replacing antigenemia with clinically safe cutoff values, DNAemia levels corresponding to antigenemia cutoffs guiding HCMV preemptive therapy were determined retrospectively in solid organ and hematopoietic stem cell transplant recipients (HSCTR) using an "in-house" quantitative PCR (QPCR) method. Since preemptive therapy had prevented appearance of HCMV disease in all patients tested, DNA cutoffs determined retrospectively had to be considered as safe clinically as antigenemia cutoffs used prospectively. However, in solid organ transplant recipients (SOTR), initiating preemptive therapy upon an antigenemia cutoff of 100 pp65-positive leukocytes, a DNAemia cutoff of 300,000 copies/ml blood had positive and negative predictive values of >90%, indicating that a DNAemia cutoff could achieve, in terms of prevention of HCMV disease, the same clinical results as the antigenemia cutoff. In HSCTR, initiating preemptive therapy upon first antigenemia positivity, a DNAemia cutoff of 10,000 copies/ml blood had a positive predictive value of >90%, indicating that the great majority of patients treated under the antigenemia guidance would have been treated also using this DNA cutoff. On the other hand, the negative predictive value of 28.6% indicated that two out of three HSCTR had been treated under the antigenemia guidance having the same levels of viral DNA as the untreated patients. The data suggest that a quantitative cutoff could be adopted as a guiding criterion for preemptive therapy also in HSCTR. Regression analysis allowed to determine the DNAemia (corresponding to QPCR) cutoff values for two commercial assays tested both in solid organ and HSCTR. Retrospective DNAemia cutoff values will be verified for safety in prospective trials.     

Genomic allelotyping for distinction of recurrent and de novo hepatocellular carcinoma after orthotopic liver transplantation.

Distinction between recurrent and de novo hepatocellular carcinoma (HCC) after orthotopic liver transplantation (OLT) bears important clinical and therapeutic implications. Techniques for molecular profiling of clinically suspected de novo and recurrent HCC are required since the histological/clinical discrimination of donor vs. recipient tumor origin is difficult. Multiple PCR amplification of 16 highly polymorphic short tandem repeat (STR) DNA sequences (routinely used for paternity and forensic assays) was applied in two patients who developed a second HCC after OLT. In both patients the technique provided reliable evidence that the two second HCC were recurrences of the primary tumor. Multiple STR genetic allelotyping is an effective tool for clear-cut discrimination of donor/recipient origin of a second HCC after OLT. Its application could be of great therapeutic relevance for such OLT patients.     

Postnatal development of dopamine receptor expression in rat peripheral blood lymphocytes.

Postnatal development in the expression of dopamine D1-like and D2-like receptors was investigated in peripheral blood lymphocytes of male Wistar rats aged 1, 3, 4, 8, 12 and 16 weeks of age by radioligand binding assay techniques. Sample of frontal cortex, striatum and hippocampus were also investigated as reference tissues. The dopamine D1-like receptor antagonist [3H]SCH 23390 and the dopamine D2-like receptor agonist [3H]7-OH-DPAT were used as radioligands. The affinity (K(d)) of [3H]SCH 23390 or of [3H]7-OH-DPAT binding was unchanged in lymphocytes of rats of different age groups. The density (B(max)) of [3H]SCH 23390 binding sites increased from the 1st to the 3rd week of age, remained constant from the 3rd to the 8th week of age, and then increased slightly at 12 and 16 weeks of age. The B(max) value of [3H]7-OH-DPAT binding to lymphocytes increased from the 1st to the 3rd week of age, remained constant from the 3rd to the 4th week, increased again until the 12th week and then plateaued. Dopamine D1-like and D2-like receptor maturation in frontal cortex, hippocampus and striatum revealed an increased receptor density until the 4th week of age and a relative stabilization of receptor density values between the 4th to the 12th week depending on the area considered. Comparatively postnatal maturation of lymphocyte dopamine D1-like receptors displayed a pattern different from that of brain areas investigated, whereas maturation of D2-like receptors displayed a pattern similar to that of striatum. The quantitative and/or qualitative dissimilarities between development of lymphocyte and brain dopamine receptors suggest that from a developmental point of view lymphocyte dopamine receptors probably cannot be considered as a marker of homologous brain receptors.     

Monte Carlo simulation of light fluence in tissue in a cylindrical diffusing fibre geometry

The propagation of light emitted by a linear light diffuser in a cylindrical hollow organ was investigated by means of the Monte Carlo (MC) method. The height and radius of the cavity, scattering (mu(s)) (or reduced scattering, mu'(s)) and absorption (mu(a)) coefficients, anisotropy (g), and refractive indices of the media involved (n1, n2) are required as input data by the MC code, as are characteristics of the light diffuser (length, delivered power and emission profile). Results of our MC model were tested by measuring the light fluence rate in a tissue-simulating phantom (mu(a) = 0.5 cm(-1), mu(s) = 23 cm(-1) and g = 0.75) irradiated at 633 nm with a cylindrical diffuser. Since geometric and optical parameters determine the behaviour of light propagation in tissue, MC simulations with different sets of input parameters were carried out to provide qualitative as well as quantitative data useful in planning photodynamic therapy. Data are reported on light penetration and fluence rate build-up at mu(a) and mu'(s) values ranging between 0.1 and 5 cm(-1) and 2.5 and 50 cm(-1), respectively. Furthermore, results suggest that a shift and spread could occur in the isofluence curves along the symmetry axis, which depend on the diameter of the treated lumen as well as on the emission profile of the light diffuser. Using our data it is possible to estimate how inaccuracy in knowledge of the optical coefficients can affect (i.e. usually by increasing) the light dose scheduled at a certain depth into tissue.     

Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi

Summary The yeast Saccharomyces cerevisiae has been engineered to synthesize and secrete desulfato-hirudin (hirudin), a thrombin inhibitor from the leech Hirudo medicinalis. The synthetic gene coding for hirudin was expressed constitutively under the control of four size-variants of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) and cloned into a 2 based multicopy yeast vector. The constitutive action of the four promoter variants was confirmed by demonstrating that the expression and secretion of hirudin is growth-related. The different efficiencies of the promoter variants not only affected hirudin expression but also led to changes in several cellular parameters, such as cell growth, average plasmid copy number and plasmid stability. The observed changes show that yeast cells establish a specific equilibrium for each promoter variant. We conclude, that the adjustment of cellular parameters in response to the expression levels of a heterologous protein is regulated by two counteracting selective forces: (1) the need for complementation of the auxotrophic host marker by the plasmid-encoded selection gene which, in the case of dLEU2, requires several plasmid copies; and (2) a selective advantage of cells with a lower copy number enabling them to escape the burden of heterologous protein production.     

Quantitative analysis of beta-adrenergic blocking agents by NMR spectroscopy

The quantitative analysis by (1)H NMR of labetalol, oxprenolol and four other beta-adrenergic blocking agents is described. The method depends on the integration of selected resonances of the analyte and an internal reference which do not overlap. Procedures for the extraction of the analyte from tablets are given and corrections due to the NMR features of excipients outlined in some cases. The NMR method is reasonably precise and rapid, and the assay results compare favourably with those obtained by a UV procedure.     

Epidemiology of adverse drug reactions in intensive care units. A multicentre prospective survey

Summary Clinically relevant events possibly attributable to drug exposure have been monitored prospectively over a period of six months in 27 general intensive care units. Fifty-four events attributed to drugs were reported in 51 patients during their stay in hospital, corresponding to an overall incidence of 1.35%. The behaviour of the physicians following attribution of the events to a prescribed drug is analyzed and discussed in detail with respect to its relationship to the quality and severity of the reaction, and the classes of drugs. Twenty-four of the 4537 monitored admissions during the six months were due to life-threatening emergencies linked to the administration of drugs (14) and radio-contrast media (10) (overall incidence 0.5%). While the clinical burden attributable to adverse drug reactions in Intensive Care Units appears to be relatively small, the analysis shows that there is ample room for a greater reduction in their incidence. Coordinators: Drs M. L. Farina and G. Tognoni, Istituto di Ricerche Farmacologiche Mario Negri, Milan; Dr F. Procaccio, Neurosurgical ICU, Ospedale Ca' Granda, Niguarda, Milan.Investigators: Drs G. Barusco, Rovigo; F. Bassi, Milan; L. Bianchetti, Torino; E. Carchietti, Udine; G. Chilloni, Reggio Emilia; G. Costantini, Savigliano (CN); P. Ferrero, Aosta; E. Geat, Trento; F. Gorgerino, Torino; A. Lusini, Empoli (FI); G. Mantovani, Ferrara; S. Marchi, Bologna; P. Marcovigi, Forli; G. Marraro, Merate (CO); F. Merlo, Vicenza; E. Pagni, Bagno a Ripoli (FI); R. Pellegrino, Cuneo; C. Peruselli, Milan; A. Piovesano, Pordenone; R. Rinaldo, Cremona; R. Ruggerini, Piacenza; S. Sammartino, Torino; A. Sartore, Cittadella (PD); A. Scaglioli, Carpi (MO); G. Scopa, Terni; G. Zeffiro, Treviso; P. Zuccoli, Parma     

Chronic Oleoylethanolamide Treatment Decreases Hepatic Triacylglycerol Level in Rat Liver by a PPARγ/SREBP-Mediated Suppression of Fatty Acid and Triacylglycerol Synthesis

Oleoylethanolamide (OEA) is a naturally occurring bioactive lipid belonging to the family of N-acylethanolamides. A variety of beneficial effects have been attributed to OEA, although the greater interest is due to its potential role in the treatment of obesity, fatty liver, and eating-related disorders. To better clarify the mechanism of the antiadipogenic effect of OEA in the liver, using a lipidomic study performed by ¹H-NMR, LC-MS/MS and thin-layer chromatography analyses we evaluated the whole lipid composition of rat liver, following a two-week daily treatment of OEA (10 mg kg⁻¹ i.p.). We found that OEA induced a significant reduction in hepatic triacylglycerol (TAG) content and significant changes in sphingolipid composition and ceramidase activity. We associated the antiadipogenic effect of OEA to decreased activity and expression of key enzymes involved in fatty acid and TAG syntheses, such as acetyl-CoA carboxylase, fatty acid synthase, diacylglycerol acyltransferase, and stearoyl-CoA desaturase 1. Moreover, we found that both SREBP-1 and PPARγ protein expression were significantly reduced in the liver of OEA-treated rats. Our findings add significant and important insights into the molecular mechanism of OEA on hepatic adipogenesis, and suggest a possible link between the OEA-induced changes in sphingolipid metabolism and suppression of hepatic TAG level.     

Role of Cachexia and Fragility in the Patient Candidate for Cardiac Surgery

Frailty is the major expression of accelerated aging and describes a decreased resistance to stressors, and consequently an increased vulnerability to additional diseases in elderly people. The vascular aging related to frail phenotype reflects the high susceptibility for cardiovascular diseases and negative postoperative outcomes after cardiac surgery. Sarcopenia can be considered a biological substrate of physical frailty. Malnutrition and physical inactivity play a key role in the pathogenesis of sarcopenia. We searched on Medline (PubMed) and Scopus for relevant literature published over the last 10 years and analyzed the strong correlation between frailty, sarcopenia and cardiovascular diseases in elderly patient. In our opinion, a right food intake and moderate intensity resistance exercise are mandatory in order to better prepare patients undergoing cardiac operation.     

What Are the Maternal Factors that Potentially Intervenes in the Nutritional Composition of Human Milk?

Background: To evaluate the potential factors associated with the nutritional composition of human milk of puerperal women. Methods: cross-sectional study, conducted between March 2016 and August 2017, with 107 women, selected in a Tertiary Health Care Tertiary Health Facility of the Unified Health System (SUS) in the Municipality of Rio de Janeiro. Data were collected two months after delivery. The dependent variable of the study was the nutritional composition of human milk. We divided the independent variables into hierarchical levels: distal (age, schooling, parity and pregestational nutritional status), intermediate (number of prenatal visits and gestational weight gain) and proximal (alcohol consumption, smoking, diabetes mellitus and hypertension). For data analysis, we applied the multiple linear regression, centered on the hierarchical model. Only the variables associated with the nutritional composition of breast milk remained in the final model at a 5% level of significance. Results: The nutritional composition of human milk yielded by women with pregestational overweight, smokers and hypertensive had higher amounts of lipids and energy. Conversely, women with gestational weight gain below the recommended had lower amounts of these components. Conclusion: The evaluation of factors associated with the nutritional composition of human milk is extremely important to assist post-partum care practices. In this study, we observed that lipid and energy contents were associated to pregestational nutritional status, gestational weight gain, smoking and hypertension.