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Marija Brgles, Maja Šantak, Beata Halassy, Dubravko Forcic, Jelka TomašicInstitute of Immunology, Research and Development Department, Zagreb, CroatiaBackground: Physicochemical characteristics of liposome/DNA complexes influence transfection efficiency and affect each other in a very intricate way. The result of this is discrepancies in conclusions drawn about the individual influence of each one.Methods: Aiming to elucidate the influence of liposome/DNA charge ratio and size on transfection efficiency and on each other, we used liposome/DNA complexes with charge ratio (+/-) in the range of 1–50 and extruded through membranes of 400, 200, and 100 nm. Plasmid DNA encoding green fluorescent protein was used to measure transfection efficiency by flow cytometry. Sizes of liposome/DNA complexes were measured by dynamic light scattering.Results: Liposome size was reduced after extrusion but this was mainly driven by the charge ratio and not by the size of the membrane pores. Reduction of complex size at each charge ratio positively correlated with transfection efficiency. When the size of the complexes was approximately constant, increasing the charge ratio was found to promote transfection efficiency. Cationic lipid N-(1-(2,3-dioleoyloxy)propyl)N,N,N trimethylammonium chloride was used for modulation of positive charge and a cytotoxicity test showed that increasing its amount increases cytotoxicity.Conclusion: It can be concluded that charge ratio dictates the size of the complex whereas overall size reduction and higher charge ratios promote transfection efficiency in vitro.Keywords: transfection efficiency, liposome charge, liposome size

Abstract Background The most often used mumps vaccine strains Jeryl Lynn (JL), RIT4385, Urabe-AM9, L-Zagreb and L-3 differ in immunogenicity and reactogenicity. Previous analyses showed that JL, Urabe-AM9 and L-3 are genetically heterogeneous. Results We identified the heterogeneity of L-Zagreb throughout the entire genome. Two major variants were defined: variant A being identical to the consensus sequence of viral seeds and vaccine(s) and variant B which differs from variant A in three nucleotide positions. The difference between viral variants in L-Zagreb strain is insufficient for distinct viral strains to be defined. We demonstrated that proportion of variants in L-Zagreb viral population depends on cell substrate used for viral replication in vitro and in vivo. Conclusion L-Zagreb strain should be considered as a single strain composed of at least two variant viral genomes.

The backbone of current treatment for chronic Hepatitis C virus (HCV) infection are direct-acting antivirals targeting viral nonstructural proteins (NS3, NS4A, NS5A, NS5B). To date, there are six NS5A inhibitors approved for treatment of chronic HCV infection. The presence of drug-associated resistance substitutions is mainly due to fast error-prone replication, showing differential frequency between genotypes and subtypes. The aim of this study was to determine the frequency of baseline resistance to NS5A protein inhibitors in patients with genotype 1 HCV in Croatia. Resistance-associated substitutions (RAS) were detected by Sanger sequencing of HCV NS5A region amplified from 84 patients followed by phylogenetic analysis and analysis with Geno2Pheno algorithm. The frequency of NS5A RAS was 14.3% and highly dependent on viral subtype. The overall frequency of NS5A RAS was higher in patients infected with HCV subtype 1b (24.2%) than in those infected with HCV subtype 1a (7.8%). Overall, three resistance-conferring mutations were detected (Q30R, M28T and Y93H) along with two mutations (M28V and L31I) that cause reduced susceptibility to NS5A inhibitors. Analysis of the sequences showed two distinct subtype 1a clades with RAS detected in 4.3% (1/23) clade I and 10.7% (3/28) clade II sequences. Only a few distinct NS5A RAS were detected suggesting a high degree of homogeneity of the viral population. High frequency of clinically relevant NS5A RAS in Croatia suggest that the analysis of frequency and patterns of resistance mutations in local populations and evaluation of their possible clinical impact could be beneficial., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: The families Paramyxoviridae and Pneumoviridae comprise a broad spectrum of viral pathogens that affect human health. The matrix (M) protein of these viruses has a central role in their life cycle. In line with this, molecular characteristics of the M proteins from variable viruses that circulated in Croatia were investigated., Methods: Sequences of the M proteins of human parainfluenza virus (HPIV) 1-3 within the family Paramyxoviridae, human metapneumovirus (HMPV), and human respiratory syncytial virus from the family Pneumoviridae were obtained and analyzed., Results: M proteins were very diverse among HPIVs, but highly conserved within each virus. More variability was seen in nucleotide sequences of M proteins from the Pneumoviridae family. An insertion of 8 nucleotides in the 3' untranslated region in 1 HMPV M gene sequence was discovered (HR347-12). As there are no samples with such an insertion in the database, this insertion is of interest and requires further research., Conclusion: While we have confirmed that M proteins were conserved among individual viruses, any changes that are observed should be given attention and further researched. Of special interest is inclusion of HPIV2 M proteins in this analysis, as these proteins have not been studied to the same extent as other paramyxoviruses., (© 2018 S. Karger AG, Basel.)

Native human interferon-α (nHuIFN-α) has a stronger reductive effect on procollagen type I mRNA expression than recombinant human interferon-α (rHuIFN-α). It is partially due to the additive activity of interleukin-1β (IL-1β), which is present in small concentrations in nHuIFN-α. Here, we show that the reductive effect is also the result of the endogenous cytokines induced by the activity of nHuIFN-α. In the culture of MRC5 fibroblasts, we have further found that nHuIFN-α induces endogenous interferons in higher amounts than rHuIFN-α, measured with PCR. That is more pronounced when interferon-γ (IFN-γ) is measured. This result puts a new light on IFN-γ activity in the nHuIFN-α treatment because its role was neglected due to the loss of its activity during the nHuIFN-α preparation process. The findings lead to the conclusion that endogenous cytokines play a significant role in the nHuIFN-α -mediated reduction of procollagen type I mRNA and are therefore an important factor in potential therapeutic usage., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Measles virus (MV) strains derived from patients with subacute sclerosing panencephalitis (SSPE), SSPE strains, possess numerous mutations when compared to viruses belonging to the same genotype and circulating in similar time period. Although many SSPE strains have been extensively characterized, none of them belongs to D4 genotype which currently predominates in Europe where it has caused a number of recent outbreaks/epidemics. We sequenced an MV derived from a patient with long-term SSPE; the virus was named MVs/Zagreb.CRO/30.06[D4] (SSPE). Initial genetic analysis showed that it belongs to D4 genotype. The sequences of genes encoding matrix and fusion proteins indicate premature protein terminations. Putative hemagglutin (H) protein is lengthened for 20 amino acids, which is the longest H protein elongation so far found in SSPE viruses. Nucleotides 1421 A, 1422 G, 1507 C and 1542 C in nucleoprotein gene open reading frame seem to be specific for this D4 strain, differentiating it from other D4 non-SSPE strains. Besides, a unique mutation at position 543 of H protein was found, histidine instead of tyrosine. As persistent MV infections are initially established by "normal" wild-type MV strains, the presented comparative analyses describe alterations that could be involved in the maintenance of persistent infection, disease development and progression., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Live attenuated vaccines against mumps virus (MuV) have been traditionally produced by passaging the virus in the embryonated chicken eggs or primary chicken embryo fibroblasts (CEFs). Virus propagation on these cell substrates enables successful virus attenuation and retains it sufficiently antigenic to induce lasting protective immunity in humans. The aim of this study was to identify critical factors for MuV replication in primary CEFs grown on a small-scale level in order to explore possibilities for improvements in the virus replication and yield. The effect of differently prepared cells, culturing conditions, and infection conditions on virus yield was estimated by employing statistical design of experiments (DoE) methodology. Our results show that the preparation of primary CEFs and the way of their infection substantially impact virus yield and are critical for efficient MuV replication. These process parameters should be considered in further process optimization. We also demonstrate the applicability of DoE in optimization of virus replication as a crucial step in obtaining high virus yields.

Arsenal of pattern-recognition receptors alongside antibody production machinery make B cells vulnerable to autoimmune response if an autoantigen elicits both pathways in a self-sustained fashion. Systemic lupus erythematosus is an autoimmune disease characterized by autoantibodies to DNA, RNA and related structures. Murine studies demonstrated autoreactive B cell activation upon TLR9 stimulation with DNA-containing immune complexes. This activation could be abolished with chloroquine, a drug used in SLE treatment that also blocks TLR9 signaling. We investigated whether chloroquine modulates TLR9 expression, circulating DNA levels and B cell-related cytokines in newly discovered, untreated SLE patients. TLR9 was measured in peripheral blood B cells by flow cytometry, serum DNA by real-time PCR, and IL-10 and BAFF by ELISA before treatment, after 3weeks on corticosteroids, and 3months after introduction of chloroquine. We found that circulating DNA is higher in SLE patients than in controls in every time-point and decreases significantly after chloroquine treatment. Untreated patients had higher serum IL-10 than controls or patients on corticosteroids. Also, corticosteroids decreased and chloroquine completely abolished CpG-mediated CD86 upregulation on B cells and IL-10 secretion in PBMC culture. Providing the TLR9 pathway activation demonstrates its importance in pathogenesis of human SLE, this data supports continuation of chloroquine in SLE treatment protocol. In addition, observed modulation of cytokine and DNA levels after immunomodulatory treatment prompts for inclusion of untreated patients in studies of human immune disorders., (Copyright © 2012 Elsevier Inc. All rights reserved.)

RNA viruses display the highest replication error rate in our biosphere, leading to highly diverse viral populations termed quasispecies. The gold standard method for detection and quantification of variants in a quasispecies is cloning and sequencing, but it is expensive, laborious and time consuming. Therefore, other mutation detection approaches, including SSCP, are often used. In this study, we demonstrate development and the usage of a CE-SSCP method for quantification of two nearly identical viral variants in heterogenic population of a mumps virus strain and its comparison to RFLP-CE-fragment length analysis (RFLP-CE-FLA). Analyzed PCR fragments were of the same size (245 bp) with one difference in their nucleotide sequence. The limit of detection of both methods was at 5% of the minor variant. When PCR amplicons of the two variants were pooled, methods' results were very similar. On the contrary, the quantification results of samples in which variants were mixed prior to PCR showed substantial difference between the two methods. Our results indicate that although both methods can be used for detection and monitoring of a specific mutation within a viral population, caution should be taken when quantitative analysis of complex samples is based solely on results of one method., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The production of economically acceptable viral vaccines of high quality requires simple and efficient methods for purification and concentration of viral particles. Ion-exchange chromatography (IEC) has become one of commonly used methods for large-scale downstream purification of viruses. Viruses possess different biological and/or biochemical properties and therefore IEC conditions must be established specifically for each virus. Live attenuated rubella virus vaccines have been manufactured and successfully used widely to protect people from rubella and congenital rubella syndrome for almost 40 years. The aim of this study was to search for an efficient method for concentration and purification of rubella virus using IEC. The selected operating conditions using quaternary amine monolithic supports enabled highly efficient binding, purification and concentration of rubella virus from complex biological suspension without additional procedures. Eluted viral particles maintained their infectivity and viral recovery was almost 100%. At the same time, viral preparation was successfully depleted from host cell protein and DNA. This work indicates the possibility of using monoliths to improve the rubella virus yields in productions where high virus titers during cultivation can hardly be achieved., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Commercial preparations of native human interferon alpha (nHuIFN-alpha) contain several subtypes of interferon-alpha (IFN-alpha) and traces of other cytokines. Recently, we described its antifibrotic potential and showed nHuIFN-alpha to have a greater effect than that of recombinant human IFN-alpha (rHuIFN-alpha). We hypothesized that cooperation between different cytokines in the nHuIFN-alpha preparation is essential for this effect. Considerable concentrations of interleukin-1beta (IL-1beta) and platelet-derived growth factor AB (PDGF-AB) are present in the nHuIFN-alpha preparations., Methods: We tested the viability and the expression of procollagen type I messenger RNA (mRNA) in MRC5 fibroblasts treated with interleukin-1 beta (IL-1beta) and/or PDGF-AB, or the corresponding antibodies in combination with rHuIFN-alpha or nHuIFN-alpha., Results: We showed that neither IL-1beta nor PDGF-AB significantly affect the viability of MRC5 cells. Furthermore, cell viability was not affected when IL-1beta or PDGF-AB were applied along with rHuIFN-alpha, relative to the viability of cells treated with rHuIFN-alpha only. In contrast, both cytokines suppressed the synthesis of procollagen type I mRNA. When coadministered with rHuIFN-alpha, IL-1beta enhanced the suppression induced by rHuIFN-alpha. Conversely, PDGF-AB acted as an antagonist of rHuIFN-alpha and restored partially the synthesis of procollagen type I mRNA. Interestingly, the addition of IL-1beta to the PDGF-AB/rHuIFN-alpha mix not only abolished the antagonistic activity of PDGF-AB but also decreased the synthesis of procollagen type I mRNA beyond the level achieved by IL-1beta/rHuIFN-alpha. Therefore, IL-1beta was able to reverse the activity of PDGF-AB., Conclusion: Our study suggests that IL-1beta is an important component of nHuIFN-alpha preparations, acting directly and indirectly to modulate the action of other components. This study provides insight into these complex cytokine networks, which is necessary for better and safer antifibrotic therapy., (Copyright 2010 Mosby, Inc. All rights reserved.)

The two most commonly used methods for the determination of a virus potency are plaque assay and 50% cell culture infective dose (CCID(50)) assay, both based on cytopathic effect observation. We compared the potency estimates obtained by plaque and CCID(50) assays for nine mumps virus strains that produce different cytopathic effects in Vero cells. The ratios of CCID(50) and plaque assay quantification results differed for different strains and were in a range of 0.66-10, indicating that quantification results for some mumps virus strains are almost identical regardless of whether CCID(50) or plaque method is used, while the potency estimates of other strains strongly depend on the choice of the assay., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Human plasma is an important medical substance and a raw material for production of various therapeutics. During blood sampling, storage and processing, genomic DNA is released into plasma from nucleated blood cells that are damaged in the course of the procedure. In order to determine the concentration of contaminating DNA in plasma, we developed a method for DNA isolation by using anion-exchange chromatography on a BIA Separations CIM (convective interaction media) diethylaminoethyl column. DNA was quantified by SYBR Green based real-time polymerase chain reaction. The concentration of cell-free, non-apoptotic DNA in plasma ranged between 0.06 and 22.5 ng/ml. As substantial volumes of plasma or whole blood are administered directly into the vascular system, a recipient is exposed to high amounts of cell-free DNA, several orders of magnitude higher than the amount found in other biologicals.

The mumps virus (MuV) molecular evolution is characterized by the co-circulation of numerous distinct strains. Standardized phylogenetic analyses based on the nucleotide sequences of the SH gene are important for mumps surveillance, but lack the information regarding antigenic properties. So far, the location of antigenic epitopes has been determined for two MuV proteins, the hemagglutinin-neuraminidase (HN) and the nucleocapsid (N) protein. We performed multiple sequence comparisons of putative HN and N protein sequences in order to describe their diversity and plasticity, and to determine the level of similarity between vaccine and wild-type strains. The results of full-length HN or N protein phylogeny showed that MuV strains form a number of differing clades which are in concordance with grouping obtained by standard MuV genotyping. When vaccine strains are compared to all wild-type strains, the highest mean percentage of amino acid differences in both HN and N protein analysis was found for Jeryl Lynn 5 and Jeryl Lynn 2 strains while the lowest value was obtained for Leningrad-3 and L-Zagreb strains. When only 3 antigenic regions of the HN protein, comprising 45 amino acids in total, were investigated, the diversity is considerably diminished: 51.5% of all putative HN proteins show identical sequences (including those of vaccine strains L-Zagreb, Leningrad-3, Hoshino and Urabe). Another 26.5% proteins (including Miyahara vaccine strain) differ in only one amino acid, while the others differ in two to five amino acids from the most common sequence. Jeryl Lynn 2 and Jeryl Lynn 5 strains differ in four amino acids each. N protein antigenic sites have been mapped within its hypervariable C-terminus. Our results indicate that there might be genotype-specific amino acids residing in this antigenic region. The results of our study present the background information for investigations of MuV heterogeneity and antigenic diversity.

Background: The most often used mumps vaccine strains Jeryl Lynn (JL), RIT4385, Urabe-AM9, L-Zagreb and L-3 differ in immunogenicity and reactogenicity. Previous analyses showed that JL, Urabe-AM9 and L-3 are genetically heterogeneous., Results: We identified the heterogeneity of L-Zagreb throughout the entire genome. Two major variants were defined: variant A being identical to the consensus sequence of viral seeds and vaccine(s) and variant B which differs from variant A in three nucleotide positions. The difference between viral variants in L-Zagreb strain is insufficient for distinct viral strains to be defined. We demonstrated that proportion of variants in L-Zagreb viral population depends on cell substrate used for viral replication in vitro and in vivo., Conclusion: L-Zagreb strain should be considered as a single strain composed of at least two variant viral genomes.

Human metapneumovirus (HMPV) is an important respiratory pathogen, especially among young children. The genetic characteristics of HMPV circulating in Croatia have not been studied so far. The aim of this study was to determine the incidence of HMPV infection in hospitalized children with acute respiratory tract infection (ARTI) in the season 2005/2006 in Croatia, as well as to perform the genotypic analysis of detected HMPV strains. From December 1 to March 31 nasopharyngeal secretions (NPSs) were collected from 402 inpatients up to 5 years of age with ARTI. NPSs were tested by real-time RT-PCR assay targeting the nucleoprotein (N) gene of HMPV. HMPV infection was detected in 33 patients (8.2%). To perform the phylogenetic study, partial nucleotide sequences were obtained for HMPV fusion (F) gene of 30 HMPV positive samples. Phylogenetic analysis showed the circulation of two main genetic lineages (A and B), with B lineages being prevalent. It also showed the existence of two sublineages within the group B (B1 and B2) and three subclusters within lineage A (A1, A2a and A2b). Further molecular analysis revealed point mutations in HMPV strains of sublineage B1.

Determination of inter- and intragenotype stability and variability are the basic tools for the molecular epidemiology and evolutionary investigation of measles virus (MV). We made a comparison between complete genome sequences of four MVs (two wt MV strains-WA.USA/17.98 and 97-45881, and two SSPE MV strains-MVs/Zagreb.CRO/47.02/and MVs/Zagreb.CRO/08.03/), all belonging to genotype D6. Results of analyses clearly confirm that MV genome continuously changes within the viruses of the same or different genotypes by accumulation of mutations in different parts of the genome. Only a small number of these accumulated mutations induce amino acid substitutions and thus possibly introduce new biological characteristics or a new genotype over a long time period. This study clearly reveals a long untranslated region between M and F genes as the most variable region of the MV genome and detects the presence of unique residues on the level of the entire genome as a new important parameter in the investigation of molecular evolution of MVs.

We compared complete untranslated regions (UTRs) of two subacute sclerosing panencephalitis (SSPE) measles virus (MV) strains and two wild-type (wt) MV strains, all belonging to the same genotype (D6). In comparison to wt MVs of the same genotype, base changes were identified in the two SSPE measles virus strains at 27 and 33 noncoding positions, respectively. Majority of these residues are unique for each of the SSPE virus sequences in comparison to all other reported measles virus strain sequences. The location of some of these changes indicates that they may modify cis-acting regulatory sequences including gene-end signal of the P gene, H/L gene junction and Kozak consensus element of the L gene. Further, within the long UTR between M and F genes, deletions and insertions were identified. Thus, our study could be significant for additional investigation using reverse genetics and recombinant viruses, of possible influence of mutations in UTRs on establishment and maintenance of chronic progressive CNS disease caused by MV persistence.

Interferon-alpha(IFN-alpha) inhibits fibroblast proliferation, differentiation into myofibroblasts, and extracellular matrix synthesis, which are key events during both normal wound repair and fibrotic lesion formation. Unlike recombinant human IFN-alpha (rHuIFN-alpha), a native human IFN-alpha (nHuIFN-alpha) consists of several IFN-alpha subtypes and traces of other cytokines produced by the Sendai virus-stimulated human leukocytes. This study compares the antifibrotic effect of nHuIFN-alpha and rHuIFN-alpha in normal human dermal fibroblasts (HDFs). Treatment of HDF culture with nHuIFNA-alpha markedly affects HDF viability, whereas different rHuIFN-alpha subtypes show various effects. Two of twelve rHuIFN-alpha subtypes (IFN-alpha B2 and IFN-alpha K) significantly reduce cell viability of HDFs compared with nontreated HDFs. However, nHuIFN-alpha significantly reduces HDF cell viability in comparison to both nontreated cells and cells treated with rHuIFN-alpha. The 50% inhibitory concentration (IC(50)) varied 10-fold between nHuIFN-alpha and rHuIFN-alpha (1,103 IU/mL and 10,762 IU/mL, respectively). The impact on procollagen type I mRNA synthesis level is comparable at low doses of IFN (100 and 500 IU/mL), whereas at the dose of 1,000 IU/mL, nHuIFN-alpha shows higher repression of collagen type I gene than does rHuIFN-alpha. Both, nHuIFN-alpha and rHuIFN-alpha antagonize the effect of exogenous transforming growth factor-beta (TGF-beta) and interleukin-4 (IL-4) as measured by the alpha-smooth muscle actin (alpha -SMA) and procollagen type I mRNA level, but the effect of nHuIFN-alpha is more pronounced. This study suggests that nHuIFN-alpha is a more potent suppressor of the HDF response to profibrotic stimuli than rHuIFN-alpha, probably because of the synergism between different IFN-alpha subtypes and antifibrotic cytokines and factors.

A high-performance liquid chromatography (HPLC) method for the determination of DNA entrapment efficiency in liposomes has been developed. Plasmid DNA was encapsulated into positively charged liposomes. Non-entrapped DNA was separated by ultracentrifugation from liposomes and supernatant was chromatographed on Convective Interaction Media (CIM) DEAE disk. The elution of DNA was monitored by the absorbance at 260 nm and the quantity of DNA in the tested sample was calculated from the integrated peak areas using the appropriate standard curve. This method is fast, simple, precise and does not require any kind of DNA labelling in contrast with mostly used methods for determination of DNA entrapment efficiency.

Two mumps virus strains 9218/Zg98 and Du/CRO05 were isolated in two locations in Croatia in 1998 and 2005, respectively. Genetic characterization of these temporally distinct mumps virus isolates was carried out in order to determine their genotype and putative antigenic relatedness to mumps virus vaccine strains. Sequence analysis of the small hydrophobic (SH) gene revealed that isolate 9218/Zg98 shows less than 95% of similarity to any reference strain, thus representing a potential reference strain for a new genotype. Isolate Du/CRO05 clearly belongs to genotype G with the 97% of homology to the reference strain Glouc1/UK96. When compared to each other, the two Croatian strains have extremely low level of homology of only 89% indicating no relatedness between them. Putative antigenic properties of the HN protein of these two isolates were compared to different vaccine strains. The results reveal a higher level of homology of antigenic determinants to non-A genotype vaccine strains than to A genotype vaccine strain., (Copyright 2006 Wiley-Liss, Inc.)

Restriction enzymes have proven to be among the most valuable tools in molecular biology. In this work, we demonstrate that the cleavage of fluorescently labeled, PCR-amplified DNA can be used as a simple and highly sensitive technique for detection of sequences present in a percentage as low as 0.6% in a DNA pool. Due to the fact that fluorescent labeling of DNA fragments enables such sensitive detection and quantification of restriction enzyme cleavage, the method was further exploited in monitoring of the enzymatic digestion completeness and in determination of factors that influence restriction enzyme effectiveness. We analyzed the activity of six restriction endonucleases; the percentage of uncleaved DNA fragments predominantly ranged between 2.0 and 2.5 and the highest value was 8.00%. We conclude that, since the enzymatic digestion completeness may not always be assured, each assay based on restriction enzyme cleavage that is intended to be used in investigations of heterogeneity in a DNA pool should be constructed so that the presence of cleaved sequences is the indication of pool nonuniformity. When the presence of uncleaved sequences indicates pool heterogeneity, the results could be misleading due to possible incompleteness of enzymatic cleavage.

CBF1 is a cellular highly conserved DNA binding factor that is ubiquitously expressed in all tissues and acts as a repressor of cellular genes. In Epstein-Barr virus growth-transformed B-cell lines, CBF1 serves as a central DNA adaptor molecule for several viral proteins, including the viral transactivator Epstein-Barr virus nuclear antigen 2 (EBNA-2). EBNA-2 binds to CBF1 and thereby gains access to regulatory regions of target genes and activates transcription. We have inactivated the CBF1 gene by homologous recombination in the human B-cell line DG75 and characterized changes in cellular gene expression patterns upon loss of CBF1 and activation of EBNA-2. CBF1-negative DG75 cells were viable and proliferated at wild-type rates. Loss of CBF1 was not sufficient to release repression of the previously described EBNA-2 target genes CD21 or CCR7, whereas induction of both target genes by EBNA-2 required CBF1. In contrast, repression of immunoglobulin M by EBNA-2 was mainly CBF1 independent. CBF1-negative DG75 B cells thus provide an excellent tool to dissect CBF1-dependent and -independent functions exerted by the EBNA-2 protein in future studies.

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., (Copyright 2001 The International Association for Biologicals.)

Peptidoglycan monomer, GlcNAc-MurNAc-L-Ala-D-isoGln-mesoDAP(omega NH2)-D-Ala-D-Ala (PGM) originating from Brevibacterium divaricatum and synthetic adamantyltripeptides, diastereoisomers of D,L-(adamant-2-yl)-Gly-L-Ala-D-isoGln (AdTP1 and AdTP2) exhibit immunomodulating activity. An experimental model in the mouse has been established with suboptimal amounts of ovalbumin (OVA) as the immunogen, and parallel testing of adjuvant activity of these three immunomodulators was carried out in Balb/c, C57B16 or CBA mice. Tested compounds (100 or 200 micrograms/mouse) mixed with OVA in saline (50 micrograms/mouse) were administered s.c. Anti-OVA was assayed by ELISA in the sera of mice taken 7 days after the boosters (given on days 14 and 28). The treatment with PGM and one of the diastereoisomers, AdTP2, resulted in significantly higher increase in anti-OVA IgG levels (stimulation index up to 46) with respect to controls and groups treated with AdTP1. The effect of AdTP2 treatment was comparable to that of PGM in most experiments after the first booster, but after the second booster PGM exhibited markedly better effect. PGM and AdTP2 also induced markedly higher levels of IgG1 and IgG2 anti-OVA subclasses than detected in controls and AdTP1 treated mice, indicating that these two immunomodulators might upregulate both Th1-like and Th2-like immune responses.