Inter-laboratory comparison of qualitative and quantitative detection of hepatitis C (HCV) virus RNA in diagnostic virology: a multicentre study (MS) in Italy

BACKGROUND: The importance of the standardisation of nucleic acid amplification technology (NAT) assays for the detection of hepatitis C virus RNA is well known today, as many studies carried out in different European countries attest. The results of a previous study performed in Italy (J. Clin. Virol. 1 (2003) 83) by the Italian Society of Clinical Microbiology (AMCLI) showed that the use of external reference standards and of multicentre collaborative studies significantly improves laboratory performance for the qualitative evaluation of HCV RNA. OBJECTIVES: the AMCLI organised a new study on the standardisation of both the qualitative and the quantitative evaluation of HCV RNA with NAT in order to improve the implementation of the diagnostic methods for HCV RNA detection. STUDY DESIGN: seventeen diagnostic centres of major Italian Hospitals participated in this quality control study. The study consisted of testing three panels, each made up of 10 coded samples including negative and positive samples. Positive samples contained four levels of HCV RNA (genotype 1). RESULTS AND CONCLUSION: Seven out of 510 qualitative results obtained were incorrect (1.4%), two false negative and five false positive. The results gave a sensitivity of 99.5% and a specificity of 95.8%. Regarding quantitative tests, the geometric mean (GM) and standard deviation (S.D.) could be calculated only for the three highest HCV RNA levels. The percentage of results within the range of GM +/- 0.5 log(10) varied from 91% to 100%. Some laboratories had some difficulty in the exact quantification of the lowest (3.00 log IU/ml) as well as of the highest viral levels (6.35 log IU/ml) values, very near to the limits of the dynamic range of the assays. The comparison of the results of this study with that previously carried out one confirms that a regular participation in external quality assessment (EQA) assures the achievement of a high proficiency level in the diagnosis of HCV infection.     

A proposed autacoid mechanism controlling mastocyte behaviour

Evidence is provided here supporting the existence of a novel autacoid mechanism negatively modulating mast cell behaviour in response to noxious stimuliin vivo; hence, the denomination “autacoid local inflammation antagonism” (ALIA). In particular, as lipid amides of theN-acylethanolamine type have been reported to accumulate in tissues in degenerative inflammatory conditions, we examined whether theseN-acylated lipids could exert regulatory effects on mast cell activationin vivo. The results reported show that both long- and short-chainN-acylethanolamines, when systemically administered, are effective in reducing mast cell degranulation induced by local injection of substance P in the earpinna of developing rats. These and other data suggest that the endogenous production ofN-acylethanolamines may constitute a local autocrine/paracrine response for the negative feedback control of mast cell responses to various activating signals. Such a process may be of physio-pathological relevance in the regulation of functional neuroimmune-mast cell interactions.

     

Epitope definition by proteomic similarity analysis: identification of the linear determinant of the anti-Dsg3 MAb 5H10

Cell-based bioassays have been suggested for screening of hormones and drug bioactivities. They are a plausible alternative to animal based methods. The technique used is called receptor/reporter system. Receptor/reporter system was initially developed as a research technique to understand gene function. Often reporter constructs containing viral promoters were used because they could be expressed with very 'high' magnitude in a variety of cell types in the laboratory. On the other hand mammalian genes are expressed in a cell/tissue specific manner, which makes them (i.e. cells/tissues) specialized for specific function in vivo. Therefore, if the receptor/reporter system is to be used as a cell-based screen for testing of hormones and drugs for human therapy then the choice of cell line as well as the promoter in the reporter module is of prime importance so as to get a realistic measure of the bioactivities of 'test' compounds. We evaluated two conventionally used viral promoters and a natural mammalian promoter, regulated by steroid hormone progesterone, in a cell-based receptor/reporter system. The promoters were spliced into vectors expressing enzyme CAT (chloramphenicol acetyl transferase), which served as a reporter of their magnitudes and consistencies in controlling gene expressions. They were introduced into breast cell lines T47D and MCF-7, which served as a cell-based source of progesterone receptors. The yardstick of their reliability was highest magnitude as well as consistency in CAT expression on induction by sequential doses of progesterone. All the promoters responded to induction by progesterone doses ranging from 10^-12 to 10^-6 molar by expressing CAT enzyme, albeit with varying magnitudes and consistencies. The natural mammalian promoter showed the most coherence in magnitude as well as dose dependent expression profile in both the cell lines. Our study casts doubts on use of viral promoters in a cell-based bioassay for measuring bioactivities of drugs and hormones for human therapy and suggests caution regardingtranslation in toto, of a research technique as a cell-based bioassay for drug screening.     

Patients' and family members' perspectives on the benefits and working mechanisms of family nursing conversations in Dutch home healthcare

The aim of this study is to propose a model of the benefits and working mechanisms of family nursing conversations in home healthcare from the perspective of participating patients and their family members. Family nursing conversations in this study are intended to optimise family functioning, improve collaboration between family and professional caregivers and ultimately prevent or reduce overburden of family caregivers. In a qualitative grounded theory design, data were collected in 2017 using intensive interviewing with participants of family nursing conversations in home healthcare. A total of 26 participants (9 patients and 17 family members) from 11 families participated in a family nursing conversation and the study. Seven nurses who received extensive education in family nursing conversations conducted them as part of their daily practice. Interviews occurred 4–6 weeks after the family nursing conversation. The model that was constructed in close collaboration with the families consists of three parts. The first part outlines working mechanisms of the conversation itself according to participants, i.e. structured and open communication about the care situation and the presence of all of the people who are involved. The second part consists of the benefits that participants experienced during and immediately after the conversation – an increased sense of overview and improved contact among the people involved – and the related working mechanisms. The last part consists of benefits that emerged in the weeks after the conversation – reduced caregiver burden and improved quality of care – and the related working mechanisms. Insight into the benefits and working mechanisms of family nursing conversations may assist healthcare professionals in their application of the intervention and provides directions for outcomes and processes to include in future studies.     

Dual Action of NAMI-A in inhibition of solid tumor metastasis: selective targeting of metastatic cells and binding to collagen.

NAMI-A is a ruthenium complex endowed with a selective effect on lung metastases of solid metastasizing tumors. The aim of this study is to provide evidence that NAMI-A's effect is based on the selective sensitivity of the metastasis cell, as compared with other tumor cells, and to show that lungs represent a privileged site for the antimetastatic effects. The transplantation of Lewis lung carcinoma cells, harvested from the primary tumor of mice treated with 35 mg/kg/day NAMI-A for six consecutive days, a dose active on metastases, shows no change in primary tumor take and growth but a significant reduction in formation of spontaneous lung metastases. Transmission electron microscopy examination of lungs and kidney shows NAMI-A to selectively bind collagen of the lung extracellular matrix and also type IV collagen of the basement membrane of kidney glomeruli. The half lifetime of NAMI-A elimination from the lungs is longer than for liver, kidney, and primary tumor. NAMI-A bound to collagen is active on tumor cells as shown in vitro by an invasion test, using a modified Boyden chamber and Matrigel, and it inhibits the matrix metallo-proteinases MMP-2 and MMP-9 at micromolar concentrations, as shown in vitro by a zimography test. These data show NAMI-A to significantly affect tumor cells with metastatic ability. Binding to collagen allows NAMI-A to exert its selective activity on metastatic cells during dissemination and particularly in the lungs. These data also stress the wide spectrum of daily doses and treatment schedules at which NAMI-A is active against metastases.     

Association of CYP2C8, CYP3A4, CYP3A5, and ABCB1 polymorphisms with the pharmacokinetics of paclitaxel.

PURPOSE: To retrospectively evaluate the effects of six known allelic variants in the CYP2C8, CYP3A4, CYP3A5, and ABCB1 genes on the pharmacokinetics of the anticancer agent paclitaxel (Taxol). EXPERIMENTAL DESIGN: A cohort of 97 Caucasian patients with cancer (median age, 57 years) received paclitaxel as an i.v. infusion (dose range, 80-225 mg/m(2)). Genomic DNA was analyzed using PCR RFLP or using Pyrosequencing. Pharmacokinetic variables for unbound paclitaxel were estimated using nonlinear mixed effect modeling. The effects of genotypes on typical value of clearance were evaluated with the likelihood ratio test within NONMEM. In addition, relations between genotype and individual pharmacokinetic variable estimates were evaluated with one-way ANOVA. RESULTS: The allele frequencies for the CYP2C8*2, CYP2C8*3, CYP2C8*4, CYP3A4*3, CYP3A5*3C, and ABCB1 3435C>T variants were 0.7%, 9.2%, 2.1%, 0.5%, 93.2%, and 47.1%, respectively, and all were in Hardy-Weinberg equilibrium. The population typical value of clearance of unbound paclitaxel was 301 L/h (individual clearance range, 83.7-1055 L/h). The CYP2C8 or CYP3A4/5 genotypes were not statistically significantly associated with unbound clearance of paclitaxel. Likewise, no statistically significant association was observed between the ABCB1 3435C>T variant and any of the studied pharmacokinetic variables. CONCLUSIONS: This study indicates that the presently evaluated variant alleles in the CYP2C8, CYP3A4, CYP3A5, and ABCB1 genes do not explain the substantial interindividual variability in paclitaxel pharmacokinetics.     

Addressing Challenges to Providing Peer-Based Recovery Support

As more systems of care deploy peer-based recovery support (P-BRS) programs, challenges to the effective use of P-BRS have emerged. These include external challenges, embedded in the organization and culture of traditionally organized services, and individual challenges, associated with the nonprofessional status of individual peer support staff members. The Living Centers, recovery resource centers providing P-BRS, have developed methods for addressing these challenges. These include organizing the P-BRS as stand-alone programs, having peer support staff and clients organize the P-BRS, emphasizing organizational values and culture as the basis for staff training, and implementing measures designed to encourage accountability among peer support staff. In the future, research into the types of barriers to P-BRS that may exist in traditionally organized behavioral health services and the types and content of training that contribute to the provision of P-BRS will facilitate the use of these services.