Split tolerance to the MHC class I molecule H-2D in animals transgenic for its soluble analog

To determine whether the function of MHC molecules in tolerance and education is related to cell surface expression, we have produced two strains of transgenic mice in the C57B1/6 background that express soluble analogs of the H-2D^d class I protein. The transgenes were stably integrated and genetically transmitted in a Mendelian fashion. Messenger RNA for the hybrid genes was detected in all tissues analyzed in a class I-like pattern of expression, with the highest levels in lymphoid tissues. All mice bearing the transgenes expressed relatively high levels (0.1 mg/ml) of the encoded protein in their serum as assessed by Western blotting and enzymelinked immunosorbent assay (ELISA). Gel filtration chromatography showed that the soluble H-2D^d protein exists as a heterodimer with β2-microglobulin and as higher order multimers in serum. Lymphoid cells from the transgenic mice showed no cell surface expression of the soluble class I protein in indirect immunofluorescence assays. Splenocytes from two independently derived transgenic lines generated primary cytotoxic and proliferative responses directed against membrane H-2D^d antigens. Mice of both strains rejected tail skin from donors that differed from the B6 background at the H-2D^d locus only, but with delayed kinetics compared to nontransgenic littermate controls. Mice expressing the transgenic protein on immunization did not produce antibodies that recognized soluble H-2D in ELISA, whereas B6 mice generated strong antibody responses to challenge with splenocytes bearing cell surface H-2D^d . Thus, transgenic mice expressing soluble H-2D were partially tolerant to stimulation by membrane-bound H-2D . As with the activation of T-cells, the induction and maintenance of immunologie tolerance apparently displayed different requirements depending upon the T-cell subpopulation involved.     

Rapid acidification and alkylation: redox analysis of the MHC class I pathway

The technique of rapid acidification and alkylation can be used to characterise the redox status of oxidoreductases, and to determine numbers of free cysteine residues within substrate proteins. We have previously used this method to analyse interacting components of the MHC class I pathway, namely ERp57 and tapasin. Here, we have applied rapid acidification/alkylation as a novel approach to analysing the redox status of MHC class I molecules. This analysis of the redox status of the MHC class I molecules HLA-A2 and HLA-B27, which is strongly associated with a group of inflammatory arthritic disorders referred to as Spondyloarthropathies, revealed structural and conformational information. We propose that this assay provides a useful tool in the study of in vivo MHC class I structure.     

Further characterization of hla homozygous typing cell lines at the LMP2 polymorphic codon 60 by an ARMS typing method

LMP2 is a subunit of the 20S proteasome within the cellular cytosolic compartment that is thought to cleave proteins into approximately 9 amino acid long oligopeptides. It is hypothesized that changes in the low molecular mass protease (LMP) gene sequence may alter the activity or specificity in which the LMP genes cleave peptides. Currently, the typing method for LMP2 involves polymerase chain reaction (PCR), restriction enzyme digestion, and gel electrophoresis. To help reduce the cost and cumbersomeness of this method, a new typing method was adapted for the LMP2 gene. To establish this new amplification refractory mutation system (ARMS) typing method, primers have been defined, amplification conditions optimized, and control cell lines sequenced to validate testing parameters. Results are listed for selected 10th and 11th International Histocompatibility Workshop homozygous cell lines.     

Effect of the format of antibodies on their specificity

The influence of alterations in the format of antibodies on their specificity has been examined. To analyze the role of Ig constant regions in recognizing antigens, a comparison was made of the specificities of full-scale murine monoclonal antibodies and scFv single-chain miniantibodies obtained from the latter with regard to a group of closely related protein antigens – Staphylococcus enterotoxins. It was found that in the scFv format the specificity and affinity of miniantibodies diminished as compared to the full-scale ones. Specificity of antibodies may be enhanced by transforming them into full-scale antibodies. Moreover it was shown that miniantibodies within a phage particle generated from combinatorial phage libraries possess greater specificity to the antigen, however during the subsequent transformation to soluble scFv antibodies their specificity diminishes.     

Binding specificity of a class II-restricted hepatitis B epitope by DR molecules from responder and nonresponder vaccine recipients

A small but significant proportion of people who receive the hepatitis B vaccine do not produce anti-hepatitis B antibodies, a phenomenon associated with certain human leukocyte antigen (HLA) class II haplotypes. We were interested in determining whether natural allelic differences between two HLA-DR4 molecules associated with responder versus nonresponder subtypes differed with respect to binding of an immunodominant hepatitis B surface antigen (HBsAg) peptide as measured using a resonant mirror biosensor. In contrast to our original hypothesis, we found a ten-fold difference in the affinity in favor of the nonresponder DRB1*0401 allele, with a K_D of 6.89 × 10⁻⁸ M versus a K_D of 6.71 × 10⁻⁷ M for the responder DRB1*0404 allele. Half-times of dissociation were 1.3 min and 7.7 min, respectively, although association rate constants for both HLA class II molecules were similar (approximately 10⁴ M⁻¹s⁻¹). Of particular interest was the observation of different on-rates during the association phase, suggesting that stoichiometry of binding was not 1:1 or that different structural forms of the HLA-peptide complex exist. Our observations indicate that whereas HBsAg peptide binding to HLA class II molecules is influenced by HLA polymorphism, the nonresponse to hepatitis B vaccine associated with this HLA-DR4 subtype is not a result of failure of processed HBsAg to bind HLA class II molecules.     

Mutation in the cytoplasmic retrieval signal of porcine epidemic diarrhea virus spike (S) protein is responsible for enhanced fusion activity

Murine-adapted porcine epidemic diarrhea virus (PEDV), MK-p10, shows high neurovirulence and increased fusion activity compared with a non-adapted MK strain. MK-p10 S protein had four mutations relative to the original virus S, and one of these (H → R at position 1381, H1381R) in the cytoplasmic tail (CT) was suggested to be responsible for the increased fusion activity. To explore this, we examined fusion activity using recombinant S proteins. We expressed and compared the fusion activity of MK-p10 S, S with the H1381R mutation, S with the three other mutations that were not thought to be involved in high fusion activity, and the original S protein. The MK-p10 and MK-H1381R S proteins induced larger cell fusions than others. We also examined the distribution of these S proteins; the MK-p10 and MK-H1381R S proteins were transported onto the cell surface more efficiently than others. These findings suggest that the H1381R mutation is responsible for enhanced fusion activity, which may be attributed to the efficient transfer of S onto the cell surface. H1381 is a component of the KxHxx motif in the CT region, which is a retrieval signal of the S protein for the endoplasmic reticulum–Golgi intermediate compartment (ERGIC). Loss of this motif could allow for the efficient transfer of S proteins from ERGIC onto the cell surface and subsequent increased fusion activity.     

Human Th1 and Th2 lymphocytes are distinguished by calcium flux regulation during the first 10 min of lymphocyte activation

Preliminary data suggest different intracellular calcium handling of Th1 and Th2 lymphocytes that may contribute to distinct cytokine production patterns. In this study we explored the contribution of the main mechanisms in charge of the elevation and decrease of cytoplasmic free calcium levels, i.e., the endoplasmic calcium release, the calcium release activated calcium (CRAC) channel, the mitochondrial calcium uniporter (MCU), the sarco/endoplasmic reticulum calcium ATPase (SERCA), and the plasma membrane calcium ATPase (PMCA) during the first 10 min of activation in human Th1 and Th2 lymphocytes applying a kinetic flow cytometry approach. We isolated peripheral blood mononuclear cells from 10 healthy individuals. Cells were stained with CD4, CXCR3 and CCR4 cell surface markers to identify Th1 and Th2 cells, respectively and loaded with Fluo-3/AM calcium sensitive dye. Cells were activated with phytohemagglutinine and alterations of cytoplasmic free calcium levels were monitored for 10 min after specific inhibition of the above mechanisms. Our results revealed delicate differences in calcium flux kinetics of Th1 and Th2 lymphocytes. The lower activity of MCU, and therefore of CRAC channels, along with the higher activity of the SERCA pump account for the notion that Th2 cells go through a lower level of lymphocyte activation compared with Th1 cells upon identical activating stimuli. The observed differences in calcium flux of Th1 and Th2 cells may contribute to different calcium handling kinetics and, hence, to distinct cytokine production patterns by these subsets.     

Localization of natriuretic peptides in the cardiac pacemaker of Atlantic salmon (Salmo salar L.)

This study describes the location of the primary pacemaker at the sino-atrial (SA) junction and the localization of salmon cardiac peptide (sCP) and ventricular natriuretic peptide (VNP) in Atlantic salmon (Salmo salar L.). The pacemaker tissue appeared lightly stained and composed of: (1) wavy nerve bundles with oval elongated wavy appearing nuclei with pointed ends, (2) ganglion cells (12–22 μm) with granular cytoplasm and (3) wide muscle fibers with large nuclei (modified cardiomyocytes) clearly distinguishing them from the other myocardial cells. Pacemaker tissue was further evaluated using immunohistochemical staining. Immunoreactivity of natriuretic peptides (sCP and VNP) antisera showed specific staining in pacemaker ganglion cells in addition to the cardiomyocytes. Positive staining with anti-CD3? antisera in the pacemaker ganglion cells is a novel finding in teleosts and is consistent with observations in mammals. In conclusion, the Atlantic salmon pacemaker was shown to be located at the SA node and to harbor sCP and VNP peptides, suggesting a possible neuromodulatory and/or neurotransmitter role for these cardiac hormones within the teleost heart.     

A subpopulation of serotonin1B receptors colocalize with the AMPA receptor subunit GluR2 in the hippocampal dentate gyrus

The serotonin_1B receptor (5-HT_1BR) plays a role in cognitive processes that also involve glutamatergic neurotransmission via amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptors. Accumulating experimental evidence also highlights the involvement of 5-HT_1BRs in several neurological disorders. Consequently, the 5-HT_1BR is increasingly implicated as a potential therapeutic target for intervention in cognitive dysfunction. Within the hippocampus, a brain region critical to cognitive processing, populations of pre- and post-synaptic 5-HT_1BRs have been identified. Thus, 5-HT_1BRs could have a role in the modulation of hippocampal pre- and post-synaptic conductance. Previously, we demonstrated colocalization of 5-HT_1BRs with the N-methyl-d-aspartate (NMDA) receptor subunit NR1 in a subpopulation of granule cell dendrites (Peddie et al. [53]). In this study, we have examined the cellular and subcellular distribution of 5-HT_1BRs with the AMPA receptor subunit GluR2. Of 5-HT_1BR positive profiles, 28% displayed colocalization with GluR2. Of these, 87% were dendrites, corresponding to 41% and 10% of all 5-HT_1BR labeled or GluR2 labeled dendrites, respectively. Dendritic labeling was both cytoplasmic and membranous but was not usually associated with synaptic sites. Colocalization within dendritic spines and axons was comparatively rare. These findings indicate that within the dentate gyrus molecular layer, dendritic 5-HT_1BRs are expressed predominantly on GluR2 negative granule cell processes. However, a subpopulation of 5-HT_1BRs is expressed on GluR2 positive dendrites. Here, it is suggested that activation of the 5-HT_1BR may play a role in the modulation of AMPA receptor mediated conductance, further supporting the notion that the 5-HT_1BR represents an interesting therapeutic target for modulation of cognitive function.     

Production and characterization of monoclonal antibodies against complexes of the NKT cell ligand alpha-galactosylceramide bound to mouse CD1d

The alpha-galactosylceramide (alpha-GalCer) known as KRN7000 remains the best studied ligand of the lipid-binding MHC class I-like protein CD1d. The KRN7000:CD1d complex is highly recognized by invariant natural killer T (iNKT) cells, an evolutionarily conserved subset of T lymphocytes that express an unusual semi-invariant T cell antigen receptor, and mediate a variety of proinflammatory and immunoregulatory functions. To facilitate the study of glycolipid antigen presentation to iNKT cells by CD1d, we undertook the production of mouse monoclonal antibodies (mAbs) specific for complexes of KRN7000 bound to mouse CD1d (mCD1d) proteins. Three such monoclonal antibodies were isolated that bound only to mCD1d proteins that were loaded with KRN7000 or closely-related forms of alpha-GalCer. These mAbs showed no reactivity with mCD1d proteins that were not loaded with alpha-GalCer, nor did they bind to complexes formed by loading mCD1d with the self-glycolipid and putative iNKT cell ligand isoglobotrihexosylceramide. These complex-specific monoclonal antibodies allow the direct detection and monitoring of complexes formed by the binding of KRN7000 and other alpha-GalCer analogues to mCD1d. The availability of these mAbs should facilitate a wide range of studies on the biology and potential clinical applications of CD1d-restricted iNKT cells.     

Regions recognized on the light chain of botulinum neurotoxin type A by T lymphocytes of SJL and BALB/c mice primed with inactivated toxin

Lymph node cells (LNC) from SJL (H-2^s) and BALB/c (H-2^d) mice primed once with inactivated botulinum neurotoxin type A (BoNT/A) were examined for their T-cell responses to each of 32 synthetic overlapping peptides (19 residues each, L1–L32) that encompass the entire L chain (residues 1–448) of BoNT/A. LNC of SJL gave strong responses to 6 regions on, L2 (residues 15–23), L10/11/12 (127–173), L19 (253–271) and L21 (281–299), and moderate to weak responses to L9 (113–131), L14/15 (183–215) and L27 (365–383). In BALB/c, LNC gave a substantial T-cell response only against peptide L12 (residues 155–173), and responded very weakly to 9 other peptides. The results were compared with the recognition profiles determined previously in these two strains after multiple BoNT/A injections. Overall responses to the L-chain peptides of T cells in later profiles were found to be somewhat weakened in SJL and stayed essentially at a similar level in BALB/c, although responses to BoNT/A increased. In SJL, response to L10 (127–145) remained the highest in the later profile. Strong responses against L12 (155–173) observed in both strains at early stage were reduced to an insignificant level. Cross-reactivity to tetanus neurotoxin by BoNT/A-specific T cells was observed in SJL but not in BALB/c. Design of an effective synthetic peptide vaccine will require incorporation of both T cell- and Ab-recognition elements of the BoNT molecule. Significance and possible implications of these results on BoNT/A-specific T-cell responses of BoNT-treated patients are discussed.     

The ubiquitin proteasome system in neurodegenerative diseases: Culprit,accomplice or victim?

A shared hallmark for many neurodegenerative disorders is the accumulation of toxic protein species which is assumed to be the cause for these diseases. Since the ubiquitin proteasome system (UPS) is the most important pathway for selective protein degradation it is likely that it is involved in the aetiology neurodegenerative disorders. Indeed, impairment of the UPS has been reported to occur during neurodegeneration. Although accumulation of toxic protein species (amyloid β) are in turn known to impair the UPS the relationship is not necessarily causal. We provide an overview of the most recent insights in the roles the UPS plays in protein degradation and other processes. Additionally, we discuss the role of the UPS in clearance of the toxic proteins known to accumulate in the hallmarks of neurodegenerative diseases. The present paper will focus on critically reviewing the involvement of the UPS in specific neurodegenerative diseases and will discuss if UPS impairment is a cause, a consequence or both of the disease.     

The generation of rat dorsal root ganglion cell lines to identify the target of KW-7158, a novel treatment for overactive bladder

KW-7158 is a drug candidate for the treatment of overactive bladder. Although pharmacological studies have suggested that it suppresses afferent nerve conduction, its molecular target is unknown. We herein report the establishment of dorsal root ganglion (DRG) cell lines useful for identification of the target of this compound. First, we confirmed that the target exists in rat primary DRG by [³H]KW-7158 binding. To establish DRG cell lines, we used DRG from transgenic rats harboring the temperature-sensitive large T-antigen. The immortalized cells were initially screened for their expression of neuronal markers, and 72 positive clones were obtained (designated as TRD cells). Next, in order to select TRD cells expressing the target of KW-7158, we measured the binding affinity and amount of the binding sites present in each clone. Most clones expressed two binding sites, one with low affinity and one with high affinity. Differential binding of KW-7158 derivatives to each site revealed that the high affinity site is pharmacologically relevant. Therefore, we successfully identified “TRD-10” which express the largest amount of the high affinity site. These cell lines will therefore be useful tools to identify the target of KW-7158.