The viral safety of plasma-derived products with respect to hepatitis C virus (HCV) is assured by selection of donors, screening of individual donations for antibodies to HCV and the incorporation of ...effective viral inactivation-removal steps into manufacturing processes. As antibody screening of single donations is not sufficient to completely eliminate HCV RNA positive plasmas from plasma pools, testing for HCV RNA by gene amplification techniques may be necessary to identify positive donations. Using modern molecular biology techniques, we developed a specific, sensitive and reproducible method for routine PCR screening for HCV RNA in plasma pools.
In the present study the in vitro and in vivo effect of Met-enkephalin (MENK) on nitric oxide (NO) release by mouse peritoneal macrophages was evaluated. While in vitro MENK was ineffective unless ...combined with suboptimal concentrations of recombinant murine interferon gamma, in vivo all the doses (2.5, 5 or 10 mg/kg bw) bimodaly modulated NO release. Only the stimulative (2.5 and 10 mg/kg bw) and not the suppressive (5 mg/kg bw) dose of MENK was opioid receptor-mediated as demonstrated by abolishing the effect by naloxone. The stimulative effect of the low (2.5 mg/kg bw) dose, that was observed only if MENK was injected p.m., was associated with the IL production and IFNγ as demonstrated by abolishing the effect by specific antibodies. The data additionally support the idea that opioid-mediated responses might be to a large degree mediated by the release of cytokines.
Anion-exchange chromatography is one of the most important methods in downstream processing of plasmid DNA, both as a process and as an analytical technique. Separation of plasmid DNA on traditional ...particle-based anion-exchange supports is usually slow. Moreover, such supports have a low capacity for plasmid DNA due to the steric exclusion effects. In this work, the separation of plasmid DNA using short monolithic columns, Convective Interaction Media, will be presented. It will be demonstrated that plasmid DNA can be purified from bacterial cells using alkaline lysis followed by chromatography on a very short weak anion-exchange chromatographic columns—disks—with good purity and quality within a short time. Furthermore, the separation of plasmid DNA from cell RNA can be carried out without the need of adding RNAse. Fast and efficient method for
in-
process control of the purified plasmid will be described as well.
Virus mumpsa uzročnik je zaušnjaka, bolesti koja se može prevenirati cijepljenjem. Mumps je RNK virus koji se u kliničkim uzorcima i u supernatantima inficiranih staničnih kultura nakon izolacije RNK ...može detektirati RT-PCR testom. Prednosti RT-PCR testa su brzina, specifičnost i mogućnost detekcije malog broja kopija virusa. Genskom karakterizacijom i genotipizacijom virusa omogućeno je epidemiološko praćenje distribucije i cirkuliranja divljih tipova virusa mumpsa koji uzrokuju epidemije i u procijepljenim populacijama. Molekularna detekcija i genska karakterizacija divljih i cjepnih virusa mumpsa provodi se u Republici Hrvatskoj od kraja 90-tih godina.
In order to assure the virological safety of blood products, in addition to serological testing of individual donations and virus inactivation steps undertaken during manufacture, routine PCR testing ...for HCV RNA of starting materials (plasma, cells), intermediates or final product is necessary. The aim of this study was to determine the rate of HCV RNA positive batches of human native leukocyte interferon during large-scale production. Our findings indicate the presence of HCV RNA in 6·1% batches despite acidification of intermediates in order to inactivate Sendai virus.
Monolithic chromatography media represent a novel generation of stationary phases introduced in the last 10–15 years providing a chromatography matrix with enhanced mass transfer and hydrodynamic ...properties. These features allow for an efficient and fast separation of especially large biomolecules like e.g., DNA and viruses. In this study, the enrichment of virus RNA on short monolithic columns prior to molecular detection of viruses is described. Measles and mumps viruses were chosen as model viruses. The results show that it is possible to bind viral RNA on monoliths and concentrate viral nucleic acids from a fairly dilute sample. Consequently, a potential application of short monolithic columns is the concentration of virus RNA to improve the sensitivity and selectivity of viral detection with the possibility of isolating viral RNA from cell-free biological fluids.
Two cases of subacute sclerosing panencephalitis (SSPE), diagnosed in Croatia in 2002, were investigated. The coding regions of the matrix (M), hemagglutinin (H) and nucleoprotein (N) genes of ...measles virus were sequenced following direct RT-PCR amplification of viral RNA extracted from brain tissue. Phylogenetic analysis of the sequences of H and N genes, showed that both strains belonged to genotype D6. No vaccine strain was detected although both patients had been previously immunized. The comparison of analyzed sequences of two SSPE causative viruses with corresponding sequences of D6 genotype and with each other revealed a number of mutations in N and H gene sequences. In comparison to the Edmonston reference strain, the M gene of the SSPE viruses showed the characteristic biased hypermutation and a premature termination codon in one of the patients.
The evidence indicating the important role of natural killer (NK) cells in immune surveillance against tumours and certain infections is accumulating. Uraemic and dialysed patients are known to be at ...greater risk of infections and malignant diseases. NK cells were analysed in patients with advanced uraemia, and in patients treated with different dialysis techniques. Number of NK cells was morphologically identified as large granular lymphocytes in blood smears. NK activity was determined as mononuclear cell cytotoxicity against K562 cells. In a group of uraemic patients, large granular lymphocyte number was reduced to 39%, and NK activity to 41%-52% of control values. Large granular lymphocyte number and NK activity in patients haemodialysed on cuprophane membranes was significantly reduced, compared to corresponding values in controls and uraemic patients, declining to 17% and 8%-16% of respective control values. In a group of patients treated by CAPD, and in a group haemodialysed on polyacrylonitrile membranes, NK activity was close to values in the uraemic group, but significantly greater than those of cuprophane-haemodialysed patients. Haemodialysis on cuprophane membranes has an additional negative effect on NK cells, which are already seriously depressed by the uraemic state.
