Leukocyte ABCA1 gene expression is associated with fasting glucose concentration in normoglycemic men.

Adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) mediates the efflux of cholesterol to apolipoprotein A1, a process necessary for high-density lipoprotein (HDL) formation and reverse cholesterol transport. In patients with Tangier disease, mutations in ABCA1 result in low circulating HDL-cholesterol and predisposition to coronary heart disease (CHD). ABCA1 gene expression is decreased in diabetic mice. In humans, glycated hemoglobin (HbA(1c)) predicted future CHD events, even within the normal range. We hypothesised that leukocyte ABCA1 gene expression would be inversely associated with indices of glycemia in normoglycemic men. Fasting blood samples were taken from 32 healthy, nonsmoking, normoglycemic men (age 23 to 46 years). ABCA1, peroxisome proliferator-activated receptor gamma (PPARgamma), and liver X receptor alpha (LXRalpha) gene expressions in circulating leukocytes were measured using TaqMan technology. Significant inverse associations between ABCA1 gene expression and both fasting glucose concentration (r = -0.49, P =.008) and age (r = -0.39, P =.043) were found. There was no association with HbA(1c) (r = -0.23, P =.238) or HDL-cholesterol concentration (r = 0.02, P =.904). In a multiple regression model, fasting glucose remained a significant independent predictor (P =.037), whereas age did not (P =.226). Mechanisms underlying the association were explored; there were no significant associations between fasting glucose concentration and leukocyte PPARgamma gene expression, or between fasting glucose concentration and leukocyte LXRalpha gene expression. This is the first demonstration of an association between ABCA1 gene expression and fasting glucose concentration in vivo.

Functional analysis of candidate ABC transporter proteins for sitosterol transport.

Two ATP-binding cassette (ABC) proteins, ABCG5 and ABCG8, have recently been associated with the accumulation of dietary cholesterol in the sterol storage disease sitosterolemia. These two 'half-transporters' are assumed to dimerize to form the complete sitosterol transporter which reduces the absorption of sitosterol and related molecules in the intestine by pumping them back into the lumen. Although mutations altering ABCG5 and ABCG8 are found in affected patients, no functional demonstration of sitosterol transport has been achieved. In this study, we investigated whether other ABC transporters implicated in lipid movement and expressed in tissues with a role in sterol synthesis and absorption, might also be involved in sitosterol transport. Transport by the multidrug resistance P-glycoprotein (P-gp; Abcb1), the multidrug resistance-associated protein (Mrp1; Abcc1), the breast cancer resistance protein (Bcrp; Abcg2) and the bile salt export pump (Bsep; Abcb11) was assessed using several assays. Unexpectedly, none of the candidate proteins mediated significant sitosterol transport. This has implications for the pathology of sitosterolemia. In addition, the data suggest that otherwise broad-specific ABC transporters have acquired specificity to exclude sitosterol and related sterols like cholesterol presumably because the abundance of cholesterol in the membrane would interfere with their action; in consequence, specific transporters have evolved to handle these sterols.

Phosphorylation-dependent regulation of the interaction of amyloid precursor protein with Fe65 affects the production of beta-amyloid.

Neuronal Fe65 is an adapter protein that interacts with the cytoplasmic domain of the beta-amyloid precursor protein (APP). Although the interaction has been reported to occur between the second phosphotyrosine interaction domain of Fe65 and the YENPTY motif in the cytoplasmic domain of APP, the regulatory mechanism and biological function of this interaction remain unknown. We report here that (i) a single amino acid mutation at the Thr-668 residue of APP695, located 14 amino acids toward the amino-terminal end from the (682)YENPTY(687) motif, reduced the interaction between members of the Fe65 family of proteins and APP, whereas interaction of APP with the phosphotyrosine interaction domain of other APP binders such as X11-like and mammalian disabled-1 was not influenced by this mutation; (ii) the phosphorylation of APP at Thr-668 diminished the interaction of APP with Fe65 by causing a conformational change in the cytoplasmic domain that contains the Fe65-binding motif, YENPTY; and (iii) the expression of Fe65 slightly suppressed maturation of APP and decreased production of beta-amyloid (Abeta). Mutation at Thr-668 of APP abolished the effect of Fe65 on APP maturation. This mutation blocked the Fe65-dependent suppression of Abeta production and resulted in the release of increased levels of Abeta in the presence of Fe65. We previously reported that during maturation of APP in neurons, the protein is specifically phosphorylated at Thr-668 and undergoes O-glycosylation. The present results suggest that the phosphorylation of O-glycosylated mature APP at Thr-668 causes a conformational change in its cytoplasmic domain that prevents binding of Fe65 in neurons and may lead to an alteration in the production of Abeta.

pH-dependent fibrillogenesis of a VkappaIII Bence Jones protein.

Disorders of immunoglobulin (Ig) synthesis that occur in malignant plasma-cell proliferation may result in either granular (LCDD) or fibrillar (AL) tissue deposition of light-chain monoclonal components. The structural features that govern the transition from soluble polypeptides to either fibrillar or granular conformational states remain undefined. Among the many factors presumed to play a role in these transitions the net charge of the molecule has been associated with folding conformation changes. The majority of the proteins involved in AL amyloidosis show acidic isoelectric points (pI 3.8-5.2), whereas most L chains with basic pIs deposit in granular patterns. In our studies a 12 kD VkappaIII fragment was purified as the main component of the fibrils isolated from myocardium and adipose tissue of the pericardium obtained post-mortem from an individual with systemic AL amyloidosis. An apparently identical 12 kD VL fragment with the same N-terminal sequence constituted the BJ protein present in the urine. This urinary protein exhibited strikingly cathodic electrophoretic mobility on agarose gels and lacked retention by anionic exchange chromatography matrices, indicative of a highly basic pI (>10). When it was subjected to in vitro fibril-formation experiments, the BJ protein adopted a fibrillar conformation only at acidic pHs, remaining aggregated but not fibrillar at physiological pH. The data indicate that a specific tissue deposition pattern involves not only structural properties of the protein but rather more complex mechanisms in which acidic micro-environments may contribute to the stabilization of amyloidogenic conformations.

Isoaspartyl post-translational modification triggers autoimmune responses to self-proteins.

The normal functioning immune system is programmed to attack foreign pathogens and other foreign proteins while maintaining tolerance to self-proteins. The mechanisms by which tolerance is broken in the initiation of autoimmunity are not completely understood. In the present study, mice immunized with the murine cytochrome c peptide 90-104 showed no response by the B or T cell compartments. However, immunization with the isoaspartyl form of this peptide, where the linkage of Asp(93) to Leu(94) occurs through the beta-carboxyl group, resulted in strong B and T cell autoimmune responses. Antibodies elicited by immunization with the isoaspartyl form of self-peptide were cross-reactive in binding to both isoforms of cytochrome c peptide and to native cytochrome c self-protein. In a similar manner, immunization of mice with the isoaspartyl form of a peptide autoantigen of human systemic lupus erythematosus (SLE) resulted in strong B and T cell responses while mice maintained tolerance to the normal aspartyl form of self-antigen. Isoaspartyl linkages within proteins are enhanced in aging and stressed cells and arise under physiological conditions. These post-translationally modified peptides may serve as an early immunologic stimulus in autoimmune disease.

Chronic deletion, escape from deletion and activation of mouse mammary tumor virus superantigen-reactive T cells in C57BL/10 mice.

Though C57BL/10 mice express the mouse mammary tumor virus superantigens (sag) encoded by Mtv-8 and Mtv-9, it has been thought that these sag do not bind to the MHC class II molecule H2-Ab and consequently do not affect the T cell repertoire. However, we show that cells bearing TCR Vbeta chains specific for Mtv-8 and -9 sag are chronically deleted in C57BL/10 mice. Thymocytes and peripheral T cells escaping deletion by Mtv sag display a small reduction in the level of cell surface CD4. T cells escaping thymic deletion respond variably to endogenous Mtv sag with some, but not all, reactive populations appearing overrepresented in the activated/memory subset. The data suggest that in normal mice fine modulation of coreceptor expression levels may be a common way by which thymocytes escape elimination, that systems utilizing potentially Mtv sag-reactive TCR on a C57BL background may be inappropriate for the measurement of the affinity of TCR/MHC/peptide interactions required in thymic selection, and that detection of the activity of human sag may be aided by analysis of CD4 levels and activation markers on T cells in conjunction with studies of the frequency of cells bearing specific TCRVbeta chains.

Dual Valpha T cells.

The assumption that T cells can only express a single receptor for antigen has in recent years been shown to be incorrect. However, the finding that a substantial number of T cells express two distinct antigen receptors at the cell surface raises a number of questions. In particular, it has been suggested that cells expressing low levels of a self-reactive T cell receptor may escape self-tolerance mechanisms and in certain situations trigger the onset of autoimmune disease. Such a hypothesis in turn raises questions central to the understanding of the nature of T cell recognition and the process of thymocyte maturation.

Selection of dual Valpha T cells.

Incomplete allelic exclusion of TCRa gene rearrangement permits the generation of dual Valpha T cells, though the issues of their frequency and whether both alphabeta pairs participate in thymic selection have not been resolved. Both questions have been investigated using lymphocytes from mice hemizygous at the TCRa locus and consequently unable to express two rearranged TCRa genes, as background controls. The data presented show that both the frequency of dual Valpha T cells and the relative expression levels of co-expressed Valpha chains are variable and are determined by thymic selection. Possession of a Valpha chain which is inefficiently positively selected appears to increase the likelihood that a second Valpha chain will be co-expressed, whilst the relative cell surface levels of a given pair of Valpha chains differ between CD4 and CD8 subsets. Further, for some but not all Valpha pairs, dual Valpha T cells appear to express elevated levels of surface TCR. Finally, contrary to previous claims, dual Valpha T cells do not appear to be relatively frequent amongst immature thymocytes.

T cell repertoire formation displays characteristics of qualitative models of thymic selection.

The use of T cell receptor elements varies between mouse strains, reflecting a balance between positive and negative selection. The presence of H-2E biases V alpha and V beta usage through major histocompatibility class II isotype preferences of V elements, and mammary tumor virus-dependent, negative selection. Quantitative models of thymic selection predict that negative selection equates to 'excess' positive selection, whereas qualitative models suggest that positive and negative selection are opposing forces. This report attempts to distinguish between the models by assessing whether, at the level of the T cell repertoire, positive and negative selection have quantitative or qualitative characteristics. The data show that the effect of bearing V alpha and V beta regions which are both preferentially (or negatively) selected in the presence of H-2E is additive or synergistic, whilst positive stimuli counteract negative ones. The data thus provide support for qualitative models of thymic selection.

Streptozotocin-induced diabetes in mice lacking alphabeta T cells.

Multiple low-dose streptozotocin (MD-STZ) is widely used for the experimental induction of diabetes, but, as non-obese diabetic (NOD)-scid/scid mice have been found to display enhanced susceptibility to MD-STZ, whether or not the model is genuinely autoimmune and T cell-mediated has been unclear. Mice bearing a targeted mutation of the T cell receptor (TCR) alpha-chain were therefore used to assess whether TCR alphabeta+ cells are involved in the diabetogenic effects of MD-STZ injections. Young NOD mice lacking TCR alphabeta cells, when given five daily injections of 40 mg/kg STZ, developed diabetes at low frequency (2/12), despite the widespread destruction of pancreatic islet cells. By comparison, most normal control mice became hyperglycaemic (12/23). We conclude that whilst much of the tissue destruction observed in this model is due to the direct toxic effect of STZ, a significant amount is also due to the action of TCR alphabeta cells tipping the balance between tolerable and clinically damaging action on islet cells.

Mice lacking alpha beta + T cells are resistant to the induction of experimental autoimmune encephalomyelitis.

T cells of the gamma delta subset have been found to localise to demyelinated lesions in both multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) and have been implicated in the pathogenesis of both diseases. We have assessed mice carrying a targeted mutation of the T cell receptor-alpha locus which consequently lack T cell receptor (TCR) alpha beta cells but have an intact gamma delta(+)-T cell population for their susceptibility to EAE. No disease was found in any of the mutant mice, nor was any infiltration of the CNS detected. These data show that, at least in the absence of TCR-alpha beta cells. TCR-gamma delta cells are not able to elicit the pathology associated with EAE.

Non-obese diabetic mice hemizygous at the T cell receptor alpha locus are susceptible to diabetes and sialitis.

To test the hypothesis that T cells carrying two T cell receptor (TCR) alpha chains play a role in autoimmunity, we backcrossed the non-obese diabetic (NOD) strain with one carrying a TCR alpha gene disrupted by homologous recombination. Mice carrying one copy of the disrupted gene are incapable of generating T cells carrying two cell surface TCR alpha chains. Our early results suggested that either dual TCR alpha T cells play a role in insulin-dependent diabetes mellitus (IDDM) induction in NOD mice or that a locus co-segregating with the disrupted TCR alpha locus protected mice from diabetes induction. From the analysis both of mice in which the region co-segregating with the disrupted TCR alpha locus is minimized and of the F1 offspring of NOD mice with the 129 strain (TCR alpha hemizygous mice), the apparent protective effect of the absence of dual TCR alpha T cells is lost; thus, such cells do not appear to play a critical role in autoimmune disease in NOD mice.

Dual T cell receptor alpha chain T cells in autoimmunity.

Allelic exclusion at the T cell receptor alpha locus TCR-alpha is incomplete, as demonstrated by the presence of a number of T lymphocyte clones carrying two expressed alpha chain products. Such dual alpha chain T cells have been proposed to play a role in autoimmunity, for example, because of a second TCR-alpha beta pair having bypassed negative selection by virtue of low expression. We examined this hypothesis by generating mice of various autoimmunity-prone strains carrying a hemizygous targeted disruption of the TCR-alpha locus, therefore unable to produce dual alpha chain T cells. Normal mice have a low but significant proportion of T cells expressing two cell-surface TCR-alpha chains that could be enumerated by comparison to TCR-alpha hemizygotes, which have none. Susceptibility to various autoimmune diseases was analyzed in TCR-alpha hemizygotes that had been backcrossed to disease-prone strains for several generations. The incidence of experimental allergic encephalomyelitis and of lupus is not affected by the absence of dual TCR-alpha cells. In contrast, nonobese diabetic (NOD) TCR alpha hemizygotes are significantly protected from cyclophosphamide-accelerated insulitis and diabetes. Thus, dual alpha T cells may play an important role in some but not all autoimmune diseases. Furthermore, since protected and susceptible NOD mice both show strong spontaneous responses to glutamic acid decarboxylase, responses to this antigen, if necessary for diabetetogenesis, are not sufficient.

The T cell response of HLA-DR transgenic mice to human myelin basic protein and other antigens in the presence and absence of human CD4.

Analysis of HLA class II transgenic mice has progressed in recent years from analysis of single chain HLA class II transgenes with expression of mixed mouse/human heterodimers to double transgenic mice expressing normal human heterodimers. Previous studies have used either HLA transgenic mice in which there is a species-matched interaction with CD4 or mice which lack this interaction. Since both systems are reported to generate HLA-restricted responses, the matter of the requirement for species-matched CD4 remains unclear. We have generated triple transgenic mice expressing three human transgenes, DRA, DRB, and CD4, and compared HLA-restricted responses to peptide between human-CD4+ (Hu-CD4+) and Hu-CD4- littermates. We saw no difference between Hu-CD4+ and Hu-CD4- groups, supporting the notion that for some responses at least the requirement for species-matched CD4 may not be absolute. Evidence for positive selection of mouse T cell receptors in HLA-DR transgenic mice came both from the acquisition of new, HLA-restricted responses to various peptides and from an increased frequency of T cells using the TCR V beta 4 gene segment. An important goal with respect to the analysis of function in HLA transgenic mice is the clarification of mechanisms which underpin the recognition of self-antigens in human autoimmune disease. As a first step towards 'humanized' disease models in HLA transgenic mice, we analyzed the responses of HLA-DR transgenic mice to the human MPB 139-154 peptide which has been implicated as an epitope recognized by T cells of multiple sclerosis patients. We obtained T cell responses to this epitope in transgenic mice but not in nontransgenic controls. This study suggests that HLA transgenic mice will be valuable in the analysis of HLA-restricted T cell epitopes implicated in human disease and possibly in the design of new disease models.

Random activation of a transgene under the control of a hybrid hCD2 locus control region/Ig enhancer regulatory element.

Locus control regions such as those of human CD2 and beta-globin differ from classical enhancers in that, whereas the former confer high level, copy-dependent, position-independent expression to linked genes in transgenic mice, the latter do not, expression levels being dependent on the site of integration. We report that the position independence of the CD2 locus control region is modified by coupling it to the immunoglobulin heavy chain enhancer. Whilst in the majority of transgenic lines the Ig heavy chain enhancer has little or no effect on T cell expression of the hCD2 transgene, in others transgene expression is non-specifically extinguished in a proportion of lymphoid cells. The transgenic locus chromatin appears inaccessible to DNase I in these cells, which do not express the gene. Furthermore, mice homozygous for the hybrid hCD2-Ig heavy chain enhancer construct contain T cells with both an active and an inactive transgene. The 'decision' to express or repress the gene appears to be a random process which involves each chromosome separately, occurs at early stages in differentiation and is heritable by daughter cells. These data suggest the possibility that stochastic decisions might control a number of biological processes.

HLA-DR and H-2E transgenes differentially mediate TCR-specific positive selection.

The use of HLA transgenic mice in models of immunity and disease assumes that human MHC molecules are able to contribute toward the positive selection of the mouse TCR repertoire. As an initial step towards analysis of this we have compared the relative ability of DR alpha/E beta or E alpha/E beta complexes to induce T cell receptor (TCR) positive selection in H-2Ea and HLA-DRA transgenic mice lacking endogenous E alpha. The results show that, like E alpha/E beta, the hybrid DR alpha/E beta complexes are capable of mediating positive selection of V beta 2+, V beta 6+, and V beta 10+ cells. However, differences were found between the effects of the two transgenes. Thus, while V beta 6+ cells were efficiently selected in both H-2Ea and DRA transgenic mice, positive selection of V beta 10+ cells was less apparent in the DRA transgenic mice. Variation between Ea and DRA transgenic mice is consistent with the notion that this process is dependent on differential binding of endogenous peptides to the E alpha/E beta and DR alpha/E beta complexes. Furthermore, contrary to expectations, in neither set of mice was positive selection limited solely to the CD4+ subset. Thus, examples were found in which V beta-specific positive selection was confined to either the CD4+ or CD8+ subsets, and others in which both subpopulations were concomitantly increased. In the case of V beta 2 positive selection, H-2Ea transgenic mice showed expansion of these cells in both the CD4+ and CD8+ subpopulations while in DRA transgenic mice this occurred predominantly in the CD8+ subpopulation.

Mouse mammary tumor virus-mediated T-cell receptor negative selection in HLA-DRA transgenic mice.

Products of specific mouse Mtv genes expressed in association with mouse MHC class II products cause the deletion of T cells expressing particular TCR V beta gene segments. These endogenous deletion ligands have been termed superantigens due to their ability to negatively select entire T-cell families, as defined by V beta-chain usage. In most cases, deletion is preferentially effected through interaction of the Mtv ligand with H-2E products. Although human DR alpha shares only 75% identity with the E alpha chain of H-2E, it has previously been shown to substitute for the mouse homologue in its capacity to induce the deletion of V beta 11- and V beta 17a-bearing T cells. In the present study, we have undertaken a more comprehensive analysis of the interaction of mixed DR alpha/E beta pairs with various endogenous Mtv integrants in various mouse backgrounds, leading to negative selection of particular V beta families. We show in this paper that transgenic DR alpha/E beta can also efficiently interact with products of Mtv-7, causing deletion of both V beta 6+ and V beta 7+ cells. Deletion of V beta 11+ T cells in DRA transgenic mice carrying Mtv-8 and -9, however, was less efficient than in control H-2Ea transgenic mice. These data and those from other MHC transgenic mouse studies show that while the class II alpha chain can influence the interaction with superantigen, it is the identity of the beta chain that seems to be critical.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and use of an internal amino acid sequencing standard peptide.

A sequenceable internal standard has been made that can be added to samples destined for automated amino acid sequencing and that provides a means for continuously monitoring instrument performance. The 41-residue standard peptide is composed of two unnatural amino acids, norleucine and succinyl-lysine, and has the structure [norleucine-(succinyl-lysine)4]8-norleucine. The phenylthiohydantoin derivatives of the latter two amino acids are well separated via reversed-phase HPLC from the phenylthiohydantoin derivatives of the 20 naturally occurring amino acids. In addition, the standard peptide can be readily synthesized and it has excellent solubility, stability, and sequencing characteristics. The placement of norleucine at every fifth residue in this peptide permits accurate repetitive yield and carryover calculations following runs that are as short as six cycles. Use of this internal standard permits the early detection of suboptimal instrument performance and is especially helpful in differentiating between two common reasons (blocked NH2-terminus versus an instrument problem) for the failure of an automated protein sequencer to provide an NH2-terminal sequence.