The synthesis and electrochemical characterization of bis(fluorosulfonyl)imide-based protic ionic liquids.

A protic ionic liquid containing the FSI anion has been synthesized for the first time and used as an electrolyte in an electrochemical storage device. This PIL-based electrolyte outperforms commonly used aprotic ionic liquids, maintaining the advantages and safety of ionic liquid-based electrolytes.

Mixtures of protic ionic liquids and propylene carbonate as advanced electrolytes for lithium-ion batteries.

In this study we investigated the chemical-physical properties of mixtures containing the protic ionic liquid (PIL) N-butyl-pyrrolidinium bis(trifluoromethanesulfonyl)imide (PYRH4TFSI), propylene carbonate (PC) and lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) in view of their use as electrolytes for lithium-ion batteries (LIBs). We showed that these electrolytic solutions might display conductivity and viscosity comparable to those of conventional electrolytes. Depending on the amount of PIL present inside the mixtures, such mixtures might also display the ability to suppress the anodic dissolution of Al. Furthermore, we showed that the coordination of lithium ions by TFSI in PIL-PC mixtures appears to be different than the one observed for mixtures of PC and aprotic ionic liquids (AILs). When used in combination with a battery electrode, e.g. lithium iron phosphate (LFP), these mixtures allow the achievement of high performance also at a very high C-rate.

Lithium coordination in protic ionic liquids.

The lithium ion-ion interactions in protic ionic liquids can be very different compared to those in aprotic ionic liquids. In this study we show that, for equal lithium ion concentration, the lithium coordination number in protic ionic liquids is lower than that in aprotic ones. This lower coordination makes lithium ions more "free" to move in protic ionic liquids and it might have an important consequence in the lithium mobility.

Identification of CD4+ and CD8+ T cell epitopes of woodchuck hepatitis virus core and surface antigens in BALB/c mice.

A therapeutic vaccine against chronic hepatitis B virus (HBV) infection requires the development of a strong and multispecific Th1 cell immune response. Woodchucks chronically infected with the woodchuck hepatitis virus (WHV) closely resemble HBV infection and represent the best animal model for this hepadnavirus-induced disease. Using the BIMAS "HLA Peptide Binding Predictions" program, we have identified and further characterized novel H-2 d-restricted CD8+ epitopes within the WHV core (peptides C#12-21, C#18-32, C#19-27, C#61-69) and surface antigens (peptides preS2#10-18, preS2#27-35, S#76-84, S#133-140 and S#257-265), respectively. These peptides bind to H-2 d with high efficiency and upon immunization of mice with peptide and Freund's adjuvant they induce the development of IFN-gamma producing T cells. More importantly, WHV core peptides C#19-27 and C#61-69 and WHV surface peptides S#133-140 and S#257-265 were also recognized by CD8+ T cells after immunization of mice with DNA/PEI nanoparticles. Direct stimulation of splenocytes obtained from such DNA-immunized mice with peptides C#18-32, S#76-84, and S#257-265 resulted in significant production of IFN-gamma. Thus, we have identified T cell determinants in mice from WHV core and surface antigens that have important value for designing and evaluating an effective vaccine against hepadnavirus infection.

Interferon-gamma-associated responses to woodchuck hepatitis virus infection in neonatal woodchucks and virus-infected hepatocytes.

Acute hepatitis and recovery from woodchuck hepatitis virus (WHV) infection involves increased intrahepatic expression of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) mRNAs. In the present study, recovery correlated with increased intrahepatic expression of mRNAs for major histocompatibility complex class 1 (MHC1), beta(2)-microglobulin, 2'5'-oligoadenylate synthetase (2'5'-OAS), and indoleamine dioxygenase (IDO). By comparison, acute WHV infection progressing to chronicity was associated with diminished expression of these IFN-gamma-associated mRNAs in liver. Transfection of WHV-infected primary hepatocytes (WPH) from WHV carriers with an IFN-gamma-expressing plasmid (pIFN-gamma) resulted in dose-dependent accumulations of MHC1, TNF-alpha, 2'5'-OAS, and IDO mRNAs within 96 h. Markers of T cells and immune-mediated cytotoxicity that accumulate in recovering liver were not apparent in WPH based on the relative lack of CD3, CD4, Fas ligand, perforin, and granzyme B mRNAs. Expression of pIFN-gamma, and TNF-alpha-expressing plasmid (pTNF-alpha), did not affect total WHV RNA, or fully double-stranded WHV DNA in WPH, but each reduced some of the replicative intermediate (RI) species of WHV DNA synthesis. WPH treated with recombinant IFN-alpha protein had a higher fold induction of 2'5'-OAS mRNA associated with partial reductions in WHV RNAs and the major RI species. Thus, IFN-gamma expression in carrier WPH induced several host responses often observed in liver of recovering woodchucks, and impaired a stage of WHV DNA synthesis by a non-cytolytic mechanism mediated by TNF-alpha. Local enhancement of IFN-gamma-associated responses in chronic WHV-infected hepatocytes may promote therapeutic antiviral effects, but additional effector mechanisms evident during recovery appear necessary for more complete clearance of WHV infection.

Thymidine utilization abnormality in proliferating lymphocytes and hepatocytes of the woodchuck.

Effective incorporation of tritiated thymidine ([(3)H]TdR) into proliferating lymphocytes is important because [(3)H]TdR is a standard label to study proliferate T-cell responses. We analyzed the thymidine utilization of woodchuck peripheral blood lymphocytes (PBL) since the [(3)H]TdR incorporation assay was not applicable to measure proliferative immune responses in the woodchuck, a current major virus/host model for human hepatitis B virus infection. Incorporation of [(3)H]TdR into DNA as well as the activity of the salvage pathway enzyme thymidine kinase (TK) of proliferating woodchuck PBL was low compared to human lymphocytes. Furthermore, [(3)H]TdR incorporation of proliferating woodchuck PBL remained residual regardless of the use of methotrexate, an inhibitor of the competitive deoxythymidine monophosphate de novo synthesis pathway. Using a human probe, specific for the proliferation-associated TK1, we proved the genomic presence and transcription of TK1 sequences in various species. TK1 sequences were detected in the genome of human, mouse, woodchuck, and chicken specimens. In contrast to proliferating human PBL and 3T3 mouse fibroblasts, no TK1 transcript was found in proliferating woodchuck PBL and hepatic cells. Transfection experiments with vectors containing the murine or human TK1 and selection assays demonstrated the ability of woodchuck cells to transcribe TK1 and to express functional TK1 proteins. Our study characterizes the unique failure of sufficient [(3)H]TdR incorporation into proliferating woodchuck cells and demonstrates tritiated adenine and serine as alternative labels to monitor PBL proliferation in the woodchuck.

T-Cell response to woodchuck hepatitis virus (WHV) antigens during acute self-limited WHV infection and convalescence and after viral challenge.

The infection of woodchucks with woodchuck hepatitis virus (WHV) provides an experimental model to study early immune responses during hepadnavirus infection that cannot be tested in patients. The T-cell response of experimentally WHV-infected woodchucks to WHsAg, rWHcAg, and WHcAg peptides was monitored by observing 5-bromo-2'-deoxyuridine and [2-3H]adenine incorporation. The first T-cell responses were directed against WHsAg 3 weeks after infection; these were followed by responses to rWHcAg including the immunodominant T-cell epitope of WHcAg (amino acids 97 to 110). Maximal proliferative responses were detected when the animals seroconvered to anti-WHs and anti-WHc (week 6). A decrease in the T-cell response to viral antigens coincided with clearance of viral DNA. Polyclonal rWHcAg-specific T-cell lines were established 6, 12, 18, and 24 weeks postinfection, and their responses to WHcAg peptides were assessed. Five to seven peptides including the immunodominant epitope were recognized throughout the observation period (6 months). At 12 months after infection, T-cell responses to antigens and peptides were not detected. Reactivation of T-cell responses to viral antigens and peptides occurred within 7 days after challenge of animals with WHV. These results demonstrate that a fast and vigorous T-cell response to WHsAg, rWHcAg, and amino acids 97 to 110 of the WHcAg occurs within 3 weeks after WHV infection. The peak of this response was associated with viral clearance and may be crucial for recovery from infection. One year after infection, no proliferation of T cells in response to antigens was observed; however, the WHV-specific T-cell response was reactivated after challenge of woodchucks with WHV and may be responsible for protection against WHV reinfection.

Characterization of T-cell response to woodchuck hepatitis virus core protein and protection of woodchucks from infection by immunization with peptides containing a T-cell epitope.

Specific activation of T cells appears to be a prerequisite for viral clearance during hepatitis B virus (HBV) infection. The T-cell response to HBV core protein is essential in determining an acute or chronic outcome of HBV infection, but how this immune response contributes to the course of infection remains unclear. This is due to results obtained from humans, which are restricted to phenomenological observations occurring during the clinical onset after HBV infection. Thus, a useful animal model is needed. Characterization of the T-cell response to the core protein (WHcAg) of woodchuck hepatitis virus (WHV) in woodchucks contributes to the understanding of these mechanisms. Therefore, we investigated the response of woodchuck peripheral blood mononuclear cells (PBMCs) to WHcAg and WHcAg-derived peptides, using our 5-bromo-2'-deoxyuridine assay. We demonstrated WHcAg-specific proliferation of PBMCs and nylon wool-nonadherent cells from acutely WHV-infected woodchucks. Using a cross-reacting anti-human T-cell (CD3) antiserum, we identified nonadherent cells as woodchuck T cells. T-cell epitope mapping with overlapping peptides, covering the entire WHcAg, revealed T-cell responses of acutely WHV-infected woodchucks to peptide1-20, peptide100-119, and peptide112-131. Detailed epitope analysis in the WHcAg region from amino acids 97 to 140 showed that T cells especially recognized peptide97-110. Establishment of polyclonal T-cell lines with WHcAg or peptide97-110 revealed reciprocal stimulation by peptide97-110 or WHcAg, respectively. We vaccinated woodchucks with peptide97-110 or WHcAg to prove the importance of this immunodominant T-cell epitope. All woodchucks immunized with peptide97-110 or WHcAg were protected. Our results show that the cellular immune response to WHcAg or to one T-cell epitope protects woodchucks from WHV infection.

Determination of peripheral blood mononuclear cell responses to mitogens and woodchuck hepatitis virus core antigen in woodchucks by 5-bromo-2'-deoxyuridine or 2[3H]adenine incorporation.

Characterization of cellular immune response to antigens of woodchuck hepatitis virus (WHV) can contribute to the understanding of acute resolving and chronic outcome of hepadnavirus infection. Studies were limited because peripheral blood mononuclear cells (PBMCs) of woodchucks failed to incorporate [3H]thymidine sufficiently. Therefore, we established a non-radioactive proliferation assay for woodchuck PBMCs using 5-bromo-2'-deoxyuridine (BrdU) as thymidine analogue. Mitogen- and WHV core protein-(WHcAg) induced PBMC proliferation was detected by BrdU incorporation and compared to an assay using 2[3H]adenine. After stimulation with concanavalin A (ConA) and phytohaemagglutinin (PHA) we observed significant PBMC proliferation with both assays. Mitogen-induced nucleoside uptake of PBMCs into cellular DNA was confirmed by detection of 1',2'[3H]BrdU and 2[3H]adenine in extracted DNA. PBMCs obtained during the acute phase of WHV infection could be stimulated by WHcAg, whereas no WHcAg-induced proliferation of PBMCs was found in WHV-negative animals. PBMCs of chronic WHV carriers showed only a weak response to WHcAg. The established assays will be useful in determining the kinetics of cellular immune responses to different WHV antigens in the course of WHV infection and may provide an insight into mechanisms responsible for chronic outcome of hepadnavirus infection.

Assessment of peripheral blood mononuclear cell proliferation by [2-3H]adenine uptake in the woodchuck model.

The levels of incorporated exogenous [3H]thymidine of peripheral blood mononuclear cells (PBMC) of the woodchuck were low after stimulation with mitogens concanavalin A (ConA), phytohemagglutinin (PHA), and pokeweed mitogen (PWM) when compared with other cell systems. The use of EDTA as an anticoagulant for blood sampling and AIM-V medium for culturing of PBMC improved the [3H]thymidine uptake of PBMC. A pronounced uptake is observed after use of [3H]adenine instead of [3H]thymidine for PBMC proliferation measurement. One likely explanation for the difference in [3H]adenine versus [3H]thymidine uptake is that the alternative pathway for thymidine monophosphate synthesis is important: the conversion of uridine to uridine monophosphate and, thereafter, to thymidine monophosphate. The optimal conditions for mitogen-induced proliferation of PBMC of the woodchuck were 2 micrograms/ml ConA and PHA at day 4 and 0.14 micrograms of PWM/ml at day 5. No consistent differences of [3H]adenine uptake were observed between PBMC from four woodchuck hepatitis virus-infected woodchucks and five uninfected animals.

Expression studies with the bidirectional pcbAB-pcbC promoter region from Acremonium chrysogenum using reporter gene fusions.

Two cephalosporin genes from Acremonium chrysogenum, pcbAB and pcbC encode the ACV (alpha-aminoadipyl-cysteinyl-valine) synthetase and isopenicillin N-synthetase, respectively. The two adjacent genes are orientated in opposite directions on the chromosomal DNA, separated by a 1.2-kb non-translated sequence, carrying the putative promoter sequences. Complete sequencing of this intergenic region revealed differences from homologous sequences from other strains. To assess the putative promoter strength, we constructed an expression vector carrying the beta-glucuronidase (gusA) and beta-galactosidase (lacZ) genes in opposite orientation. Fusion of the pcbAB-pcbC promoter region resulted in recombinant vector molecules, which were used for in-vivo expression studies. Using the co-transformation procedure, the reporter gene fusions were transferred into A. chrysogenum recipient strains together with vector pMW1. Individual transformants were used for protein preparations to measure specific activities of the enzymes coded by the reporter genes. The data provide in-vivo evidence that the pcbC promoter is at least five times stronger than the pcbAB promoter. Our approach should prove useful in evaluating regulatory sequences that govern gene expression in A. chrysogenum.