Serum IgG levels to Epstein-Barr and measles viruses in patients with multiple sclerosis during natalizumab and interferon beta treatment.

Background: Patients with multiple sclerosis (MS) demonstrate higher seroprevalence of Epstein-Barr virus (EBV) and increased anti-EBV IgG levels in serum compared with healthy controls. Intrathecal antibody production to measles virus (MeV) is a common finding in patients with MS. Objective: To measure serum IgG reactivity to EBV glycoprotein 350 (gp350) and MeV nucleocapsid protein (NCORE) in patients with MS and healthy controls and to determine if reactivity changed in patients during interferon beta (IFNß) and/or natalizumab (NAT) treatment. A secondary aim was to determine the seroprevalence of EBV in patients and controls. Methods: Patients with MS (n=728) were included from the Swedish pharmacovigilance study for NAT. Paired serum samples from 714 patients drawn before and during NAT treatment and paired samples from 170 patients during prior IFNß treatment were analysed. In total, 156 patients were included in both groups. Samples from 144 matched blood donors served as controls. Indirect ELISA was applied using recombinant EBVgp350 and MeV NCORE as antigens. EBVgp350 IgG seronegative samples were also analysed using EBV nuclear antigen 1 and viral capsid antigen (VCA). Results: Patients with MS showed higher serum levels of anti-EBVgp350 and anti-MeV NCORE IgG compared with controls. During NAT treatment, the levels of anti-EBVgp350 and anti-MeV NCORE IgG declined, compared with the relatively stable levels noted during prior IFNß treatment. Ten patients failed to demonstrate anti-EBVgp350 IgG but did show detectable anti-VCA IgG, indicating EBV seropositivity. In contrast, 10/144 controls were EBV seronegative. Conclusions: Treatment with NAT, which is considered a selective immunosuppressive agent with a compartmentalised effect on the central nervous system, appeared to be associated with a moderate decrease in circulating IgG levels to EBVgp350 and MeV NCORE. All patients with MS were EBV IgG seropositive, supporting the potential role of EBV in the pathogenesis of MS.

Recombinant Epstein-Barr virus glycoprotein 350 as a serological antigen.

Epstein-Barr virus (EBV) glycoprotein 350 (gp350) is the most abundant glycoprotein expressed on the EBV envelope, the major target for neutralizing antibodies and also essential for virion attachment to B lymphocytes. Several studies have addressed EBV gp350 as a vaccine candidate, but less commonly as a potential antigen for serological assays. The aim of the current study was to develop a diagnostic tool to quantify EBV gp350-specific IgG in previously EBV-infected individuals. A construct encoding the extracellular domain of EBV gp350 (amino acid (aa) 1-860) was developed for expression in Chinese hamster ovary cells. Serum samples (n = 360) with known IgG serostatus against viral capsid antigen (VCA) and Epstein-Barr nuclear antigen 1 (EBNA1) were divided into three groups based on the differences in their serostatus: VCA + EBNA1+ (n = 120), VCA + EBNA1- (n = 120) and VCA-EBNA1- (n = 120). The samples were analyzed by indirect ELISA using recombinant EBV gp350 aa 1-860 as antigen. A clear majority, 108 of the 120 VCA + EBNA1+ samples, had detectable EBV gp350-specific IgG. Of the 120 VCA + EBNA1- samples, 79 had detectable EBV gp350-specific IgG. Only 2 of the 120 VCA-EBNA1- samples had detectable EBV gp350-specific IgG. The results reported here show that use of the EBV gp350 aa 1-860 ELISA can serve as a sensitive method for EBV-specific IgG detection in serum samples.

Intestinal MUC2 mucin supramolecular topology by packing and release resting on D3 domain assembly.

MUC2 is the major gel-forming mucin of the colon forming a protective gel barrier organized into an inner stratified and an outer loose layer. The MUC2 N-terminus (D1-D2-D'D3 domains) has a dual function in building a net-like structure by disulfide-bonded trimerization and packing the MUC2 polymer into an N-terminal concatenated polygonal platform with the C-termini extending perpendicularly by pH- and calcium-dependent interactions. We studied the N-terminal D'D3 domain by producing three recombinant variants, with or without Myc tag and GFP (green fluorescent protein), and analyzed these by gel filtration, electron microscopy and single particle image processing. The three variants were all trimers when analyzed upon denaturing conditions but eluted as hexamers upon gel filtration under native conditions. Studies by electron microscopy and three-dimensional maps revealed cage-like structures with 2- and 3-fold symmetries. The structure of the MUC2 D3 domain confirms that the MUC2 mucin forms branched net-like structures. This suggests that the MUC2 mucin is stored with two N-terminal concatenated ring platforms turned by 180° against each other, implicating that every second unfolded MUC2 net in mature mucus is turned upside down.

Increased understanding of the biochemistry and biosynthesis of MUC2 and other gel-forming mucins through the recombinant expression of their protein domains.

The gel-forming mucins are large and heavily O-glycosylated proteins which build up mucus gels. The recombinant production of full-length gel-forming mucins has not been possible to date. In order to study mucin biosynthesis and biochemistry, we and others have taken the alternative approach of constructing different recombinant proteins consisting of one or several domains of these large proteins and expressing them separately in different cell lines. Using this approach, we have determined that MUC2, the intestinal gel-forming mucin, dimerizes via its C-terminal cysteine-knot domain and also trimerizes via one of the N-terminal von Willebrand D domains. Both of these interactions are disulfide bond mediated. Via this assembly, a molecular network is built by which the mucus gel is formed. Here we discuss not only the functional understanding obtained from studies of the recombinant proteins, but also highlight the difficulties encountered when these proteins were produced recombinantly. We often found an accumulation of the proteins in the ER and consequently no secretion. This was especially apparent when the cysteine-rich domains of the N- and C-terminal parts of the mucins were expressed. Other proteins that we constructed were either not secreted or not expressed at all. Despite these problems, the knowledge of mucin biosynthesis and assembly has advanced considerably through the studies of these recombinant proteins.

Varicella-zoster virus (VZV) glycoprotein E is a serological antigen for detection of intrathecal antibodies to VZV in central nervous system infections, without cross-reaction to herpes simplex virus 1.

Herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) cause serious central nervous system (CNS) diseases that are diagnosed with PCR using samples of cerebrospinal fluid (CSF) and, during later stages of such infections, with assays of intrathecal IgG antibody production. However, serological diagnoses have been hampered by cross-reactions between HSV-1 and VZV IgG antibodies and are commonly reported in patients with herpes simplex encephalitis (HSE). In this study we have evaluated VZV glycoprotein E (gE) as a new antigen for serological diagnosis of VZV-induced CNS infections. Paired samples of CSF and serum from 29 patients with clinical diagnosis of VZV CNS infection (n = 15) or HSE (n = 14), all confirmed by PCR, were analyzed. VZV gE and whole VZV were compared as antigens in enzyme-linked immunosorbent assays (ELISAs) for serological assays in which the CSF/serum sample pairs were diluted to identical IgG concentrations. With the gE antigen, none of the HSE patients showed intrathecal IgG antibodies against VZV, compared to those shown by 11/14 patients using whole-VZV antigen (P < 0.001). In the patients with VZV infections, significantly higher CSF/serum optical density (OD) ratios were found in the VZV patients using the VZV gE antigen compared to those found using the whole-VZV antigen (P = 0.001). These results show that gE is a sensitive antigen for serological diagnosis of VZV infections in the CNS and that this antigen was devoid of cross-reactivity to HSV-1 IgG in patients with HSE. We therefore propose that VZV gE can be used for serological discrimination of CNS infections caused by VZV and HSV-1.

Recombinant glycoprotein E produced in mammalian cells in large-scale as an antigen for varicella-zoster-virus serology.

A recombinant glycoprotein E (gE) from varicella-zoster virus (VZV) was generated and produced in Chinese Hamster Ovary (CHO) cells, in the development of a specific antigen for analysis of IgG antibodies to VZV. Several stable gE-secreting clones were established and one clone was adapted to growth in serum-free suspension culture. When the cells were cultured in a perfusion bioreactor, gE was secreted into the medium, from where it could be easily purified. The recombinant gE was then evaluated as a serological antigen in ELISA. When compared to a conventional whole virus antigen, the VZV gE showed similar results in ELISA-based seroprevalence studies of 854 samples derived from blood donors, students, ischemic stroke patients and their controls, including samples with border-line results in previous analyses. Eight samples (0.9%) were discordant, all being IgG-negative by the VZV gE ELISA and positive by the whole virus ELISA. The sensitivity and specificity of the VZV gE ELISA were 99.9% and 100%, respectively, compared to 100% and 88.9% for the VZV whole virus ELISA. The elderly subjects showed similar reactivities to both antigens, while VZV gE gave lower signals in the younger cohorts, suggesting that antibodies to gE may increase with age. It was concluded that the recombinant VZV gE from CHO cells was suitable as a serological antigen for the detection of IgG antibodies specific for VZV.

Function of the CysD domain of the gel-forming MUC2 mucin.

The colonic human MUC2 mucin forms a polymeric gel by covalent disulfide bonds in its N- and C-termini. The middle part of MUC2 is largely composed of two highly O-glycosylated mucin domains that are interrupted by a CysD domain of unknown function. We studied its function as recombinant proteins fused to a removable immunoglobulin Fc domain. Analysis of affinity-purified fusion proteins by native gel electrophoresis and gel filtration showed that they formed oligomeric complexes. Analysis of the individual isolated CysD parts showed that they formed dimers both when flanked by two MUC2 tandem repeats and without these. Cleavages of the two non-reduced CysD fusion proteins and analysis by MS revealed the localization of all five CysD disulfide bonds and that the predicted C-mannosylated site was not glycosylated. All disulfide bonds were within individual peptides showing that the domain was stabilized by intramolecular disulfide bonds and that CysD dimers were of non-covalent nature. These observations suggest that CysD domains act as non-covalent cross-links in the MUC2 gel, thereby determining the pore sizes of the mucus.

Cervical mucins carry alpha(1,2)fucosylated glycans that partly protect from experimental vaginal candidiasis.

Cervical mucins are glycosylated proteins that form a protective cervical mucus. To understand the role of mucin glycans in Candida albicans infection, oligosaccharides from mouse cervical mucins were analyzed by liquid chromatography-mass spectrometry. Cervical mucins carry multiple alpha(1-2)fucosylated glycans, but alpha(1,2)fucosyltransferase Fut2-null mice are devoid of these epitopes. Epithelial cells in vaginal lavages from Fut2-null mice lacked Ulex europaeus agglutinin-1 (UEA-I) staining for alpha(1-2)fucosylated glycans. Hysterectomy to remove cervical mucus eliminated UEA-I and acid mucin staining in vaginal epithelial cells from wild type mice indicating the cervix as the source of UEA-I positive epithelial cells. To assess binding of alpha(1-2) fucosylated glycans on C. albicans infection, an in vitro adhesion assay was performed with vaginal epithelial cells from wild type and Fut2-null mice. Vaginal epithelial cells from Fut2-null mice were found to bind increased numbers of C. albicans compared to vaginal epithelial cells obtained from wild type mice. Hysterectomy lessened the difference between Fut2-null and wild type mice in binding of C. ablicans in vitro and susceptibility to experimental C. albicans vaginitis in vivo. We generated a recombinant fucosylated MUC1 glycanpolymer to test whether the relative protection of wild type mice compared to Fut2-null mice could be mimicked with exogenous mucin. While a small portion of the recombinant MUC1 epitopes displayed alpha(1-2)fucosylated glycans, the predominant epitopes were sialylated due to endogenous sialyltransferases in the cultured cells. Intravaginal instillation of recombinant MUC1 glycanpolymer partially reduced experimental yeast vaginitis suggesting that a large glycanpolymer, with different glycan epitopes, may affect fungal burden.

The effect of lactic acid on anaerobic carbon or nitrogen limited chemostat cultures of Saccharomyces cerevisiae.

Weak organic acids are well-known metabolic effectors in yeast and other micro-organisms. High concentrations of lactic acid due to infection of lactic acid bacteria often occurs in combination with growth under nutrient-limiting conditions in industrial yeast fermentations. The effects of lactic acid on growth and product formation of Saccharomyces cerevisiae were studied, with cells growing under carbon- or nitrogen-limiting conditions in anaerobic chemostat cultures (D=0.1 h(-1)) at pH values 3.25 and 5. It was shown that lactic acid in industrially relevant concentrations had a rather limited effect on the metabolism of S. cerevisiae. However, there was an effect on the energetic status of the cells, i.e. lactic acid addition provoked a reduction in the adenosine triphosphate (ATP) content of the cells. The decrease in ATP was not accompanied by a significant increase in the adenosine monophosphate levels.

Rapamycin pre-treatment preserves viability, ATP level and catabolic capacity during carbon starvation of Saccharomyces cerevisiae.

Saccharomyces cerevisiae growing exponentially in anaerobic batch cultures that are suddenly exposed to carbon starvation will rapidly lose almost all ATP. This will cause an energy deficiency and adaptation to starvation conditions is prohibited. As a result, viability and fermentative capacity will be drastically reduced during prolonged starvation. However, if the cells are incubated in the presence of rapamycin (which will inactivate the TOR pathway) before carbon starvation ATP levels, viability and fermentative capacity will be preserved to a much larger extent compared to untreated cells. The beneficial effect of rapamycin cannot be explained by induction of a stationary phase phenotype. In fact, under these anaerobic well-controlled growth conditions, rapamycin-treated cells were still metabolically active and continued to grow, albeit not exponentially and with a reduced protein content. It is hypothesized that the loss of ATP during carbon starvation occurs because protein synthesis does not make an immediate arrest at the onset of starvation. Since there are no external or internal energy sources, this will rapidly deplete the cells of ATP. Rapamycin-treated cells, on the other hand, have already downregulated the protein-synthesizing machinery and are thus better suited to cope with a sudden carbon starvation condition. This hypothesis is strengthened by the fact that treating the cells with the protein synthesis inhibitor cycloheximide also improves the carbon starvation tolerance, although not to the same extent as rapamycin. The even better effect of rapamycin is explained by accumulation of storage carbohydrates, which is not observed for cycloheximide-treated cells.

Starvation response of Saccharomyces cerevisiae grown in anaerobic nitrogen- or carbon-limited chemostat cultures.

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h(-1) at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population.

Carbon starvation can induce energy deprivation and loss of fermentative capacity in Saccharomyces cerevisiae.

Seven different strains of Saccharomyces cerevisiae were tested for the ability to maintain their fermentative capacity during 24 h of carbon or nitrogen starvation. Starvation was imposed by transferring cells, exponentially growing in anaerobic batch cultures, to a defined growth medium lacking either a carbon or a nitrogen source. After 24 h of starvation, fermentative capacity was determined by addition of glucose and measurement of the resulting ethanol production rate. The results showed that 24 h of nitrogen starvation reduced the fermentative capacity by 70 to 95%, depending on the strain. Carbon starvation, on the other hand, provoked an almost complete loss of fermentative capacity in all of the strains tested. The absence of ethanol production following carbon starvation occurred even though the cells possessed a substantial glucose transport capacity. In fact, similar uptake capacities were recorded irrespective of whether the cells had been subjected to carbon or nitrogen starvation. Instead, the loss of fermentative capacity observed in carbon-starved cells was almost surely a result of energy deprivation. Carbon starvation drastically reduced the ATP content of the cells to values well below 0.1 micro mol/g, while nitrogen-starved cells still contained approximately 6 micro mol/g after 24 h of treatment. Addition of a small amount of glucose (0.1 g/liter at a cell density of 1.0 g/liter) at the initiation of starvation or use of stationary-phase instead of log-phase cells enabled the cells to preserve their fermentative capacity also during carbon starvation. The prerequisites for successful adaptation to starvation conditions are probably gradual nutrient depletion and access to energy during the adaptation period.