Mechanism of class I assembly with beta 2 microglobulin and loading with peptide.
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Animals↗
Biomedical subjects
T H Hansen
Publications and source records attributed to T H Hansen.
Quantitation of the cell surface level of Ld resulting in positive versus negative selection of the 2C transgenic T cell receptor in vivo.
The 2C transgenic TCR is positively selected on Kb and is alloreactive for and negatively selected on Ld. To test an avidity model for positive selection, mice were bred to express different levels of surface Ld by varying the number of gene copies encoding beta 2-microglobulin (beta 2m) or Ld heavy chain. Whereas mice expressing 35% Ld (beta 2m+/- Ld+/-) negatively selected the 2C TCR, mice expressing 2% Ld (beta 2m-/- Ld+/-) positively selected the 2C TCR. Furthermore, 2C cytotoxic T lymphocytes selected on 2% Ld showed peptide-specific cytolytic activity against Ld/p2Ca targets. These findings provide clear in vivo evidence that positive selection can occur on very low levels of the same class I antigen capable of negative selection when expressed at higher levels.
Animals↗
Class I-restricted cytotoxic T cell recognition of split peptide ligands.
We developed a novel approach to probe the molecular basis of TCR recognition of the MHC class I-peptide complex and to determine how constraints placed on peptide binding by the class I molecule influence T cell recognition. We synthesized peptide pairs derived from the N- and C-terminal regions of class I peptide ligands in which the TCR contacts and dominant binding residues were placed together or were separated. Complementary peptide pairs derived from two well-characterized Ld peptide ligands, tum- (QNHRALDL) and p2Ca (LSPFPFDL), were tested for the ability to sensitize targets for recognition by peptide-specific cytotoxic T lymphocytes (CTL). The tum-derived tetramer QNHR, containing both primary TCR contact residues (H17 and R18), is recognized only when used in combination with ALDL which contains the primary binding residues (A19, D21 and L22). This suggests that both peptides of the pair contribute to positioning of the TCR contacts. Remarkably, CTL clone P24 recognized target cells sensitized with a trimer (QNH) combined with a pentamer (RALDL), demonstrating that TCR recognition can occur when the TCR contacts are separated (placed on separate peptide subunits). For the p2Ca peptide LSPFPFDL, the C-terminal tetramer PFDL, which contains both the primary TCR contact residue (P) and the dominant binding residue (L), is sufficient for recognition. In addition, PFDL was able to bind effectively to Ld and to activate naive antigen-specific T cells. These data suggest that peptide subunits and complementary peptide pairs composed of trimeric, tetrameric or pentameric peptides can bind independently to the Ld molecule in the same register and orientation as they do when contained within the parent peptide.
Amino Acid Sequence↗
Intracytoplasmic sperm injection of in vitro-matured equine oocytes.
Intracytoplasmic sperm injection (ICSI) was performed on equine oocytes matured in vitro. The oocytes were aspirated from abattoir ovaries and matured in vitro for 36 h at 38 degrees C. ICSI was performed using frozen/thawed stallion semen after swimup in medium containing human serum albumin. Sperm-injected oocytes were either 1) cultured in vitro for 10, 20, or 72 h; 2) transferred to oviducts of pseudopregnant mice; or 3) transferred to a synchronized mare after initial in vitro culture. The transferred ova were recovered after 72 h, and all ova were subsequently fixed, stained, and processed for light and transmission electron microscopy. Single pronucleus formation was observed in 2 out of 12 presumptive zygotes 10 h postinjection, at which time abundant cortical granules were observed in the subplasmalemmal region. Twenty hours postinjection, however, 2 pronuclei were observed in 6 of 12 injected oocytes (fertilization rate 50%), and almost all cortical granules were released. The cleavage rate in vitro was 16% after 72 h in culture, and the most advanced embryo stages obtained were 6- to 8-cell embryos. The cleavage rate in vivo was very low since only 1 of 10 recovered had cleaved to the 2-cell stage. Thus, in conclusion, ICSI fertilization of equine oocytes did result in fertilization, pronucleus formation, and cortical granule release. However, the observed fertilization rate and oocyte activation was not paralleled by substantial cleavage of the zygotes.
Animals↗
Factors influencing the reliability of oral contraceptives.
BACKGROUND: The reliability of oral contraceptives (OC) is high but several known factors can potentially induce failures. The dose of estrogen (EE) has been reduced through the years. Interaction with concomitant use of other medicine is wellknown and gastroenteritis can reduce the uptake of EE. To further characterize and quantify these factors we have performed this study. METHODS: Among patients admitted for legal abortion we selected those who had taken OC (only combined preparations; gestagen-only preparations were excluded) according to the prescription but nevertheless became pregnant. Various parameters were noted. RESULTS: Among 8058 women 70 women were found. Twenty-nine used three-phased and 25 used low-dose OC while the rest used high-dose, two-phased or unknown OC (four, five and seven patients respectively). Five patients used other medicine concurrently (salbutamol, astemizol, mianserin, chlorcyclizin, paradryl, carbamazepin, lithium, chlorprotoxin and imipramin). Sixteen patients had symptoms of gastroenteritis at the time of conception. Forty-nine patients had failure without any known influencing factors. There was no significant difference between the various OC used by the patients compared to sales of them in Denmark. About two-thirds of the patients wanted to continue with OC as future prevention. CONCLUSIONS: Failure of OC is a rare event but can occur in case of concurrent gastroenteritis or use of other medicine but in many cases, however, no definite cause can be determined. The EE-content of the various OC was not found to have any influence. Although OC-failure had occurred two-thirds of the patients wanted to continue the use of OC.
Adolescent↗
The mechanisms of peptide exchange and beta 2-microglobulin exchange on cell surface Ld and Kb molecules are noncooperative.
It has previously been shown that the presence of exogenous beta 2-microglobulin (beta 2m) can dramatically enhance the binding of exogenous peptide to cell surface class I MHC. However, the mechanism by which this enhancement takes place is unknown. Two models have been proposed to explain this phenomenon. In the first model, the exchange of peptide and the exchange of beta 2m are cooperative processes. In the alternative model, beta 2m stabilizes free class I heavy chains to increase the total number of peptide binding sites available. In this report, we have examined the relationship between peptide exchange and beta 2m exchange. Comparisons of Ld and Kb complexes formed with peptides possessing widely disparate affinities revealed a reciprocal correlation between the peptide off-rate and the rate of beta 2m exchange. This result indicates that peptide exchange and beta 2m exchange are noncooperative processes that may, in fact, antagonize one another. These findings provide the first demonstration of peptide-specific influences on the rate of beta 2m exchange and suggest that exogenous beta 2m promotes peptide binding by maintaining class I heavy chains in a peptide-receptive state.
Animals↗
Localization of the mouse gene releasing sex-limited expression of Slp.
To probe genetic variation in the regulation of sexual dimorphism, we have characterized the mouse protein Slp, coded by the gene sex-limited protein (Slp). Slp expression in many strains is limited to males and is androgen-dependent. However, female expression is also observed in rare strains, due to nonlinked gene(s) termed regulator of sex-limitation (rsl). In this report we demonstrate that female expression of Slp results from homozygous recessive allele(s) at a single autosomal locus that maps to a 2.2-centimorgan interval on chromosome 13. This conclusion was supported by extensive genetic analyses including the use of polymorphic microsatellites to type numerous backcross progeny and a recombinant inbred series and to identify the congenic interval in three independently derived congenic strains. Four attractive candidate genes were identified by the localization of rsl. Interestingly, rsl was found not only to enable expression in females but to also increase expression in males. The findings suggest that the expression of Slp and perhaps other sexually dimorphic proteins is regulated by two pathways, one that is dependent upon rsl but not androgens and another that is rsl-independent but requires androgens.
Animals↗
The use of dobutamine stress echocardiography for the determination of myocardial viability.
Determining the presence of viable myocardium has prognostic and therapeutic implications in the treatment of ischemic heart disease. Dobutamine stress echocardiography (DSE) is one possible technique to help identify both hibernating and stunned but viable myocardium. Low-dose dobutamine infusion has an increased inotropic effect, while higher doses cause both inotropic and chronotropic effects. Thus, at lower doses cardiac augmentation occurs, and at higher doses regions of ischemia may be produced in the presence of significant coronary artery disease. This is manifested echocardiographically as changes in segmental wall motion. In theory, therefore, areas of viable myocardium should show improved wall motion at low doses, and areas of irreversible myocardial damage will remain akinetic. Five studies have investigated DSE for determining viability in the setting of acute myocardial infarction, thus looking for stunned but viable myocardium. DSE was shown to compare favorably with positron emission tomography and was highly sensitive and specific for predicting functional myocardial recovery. Five additional studies examined DSE for determining the presence of hibernating myocardium. The sensitivity and specificity of DSE were found to range from 71 to 92% and from 73 to 93%, respectively. The benefits of DSE include lower cost, convenience to both patient and physician, additional ancillary information, and determination of the possible need for urgent revascularization. Limitations of DSE include occasional technical difficulty in obtaining and interpreting studies and the need for larger volumes of viable myocardium to detect changes predictive of functional recovery. Larger trials are currently underway to confirm DSE as a reliable technique for determining myocardial viability.
Dobutamine↗
Differences among various class I molecules in competition for beta2m in vivo.
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Alleles↗
TAP associates with a unique class I conformation, whereas calnexin associates with multiple class I forms in mouse and man.
To define the rules governing de novo assembly of the trimeric class I complex, we have identified the class I folding/assembly intermediates associated with calnexin or TAP, using both human and mouse cell lines. To better characterize the class I H chain structure associated with TAP, mouse mAb that distinguish open (64-3-7+) vs folded (30-5-7+) Ld heavy (H) chains were used. We report here that open forms of Ld are uniquely and specifically associated with TAP and that the conformational change in the class I H chain coincident with peptide binding induces TAP release. Chimeric Ld/Q10 displayed TAP association, demonstrating that soluble class I molecules can bind TAP. As previously reported, beta 2m was found to be required for H chain association with TAP. Interestingly, beta 2m was associated with TAP in the human class I-negative cell line LCL 721.221, suggesting that beta 2m can bind to TAP before class I H chain. In contrast to TAP, which binds a specific class I conformation, calnexin was detected in association with multiple forms of both mouse and human class I. Most significantly, we show for the first time that beta 2m-assembled forms of human as well as mouse class I molecules interact with calnexin. Based on these findings, we propose a model for the sequential assembly of class I heterotrimers and their respective interactions with TAP and calnexin.
ATP-Binding Cassette Transporters↗
Aglycosylated and phosphatidylinositol-anchored MHC class I molecules are associated with calnexin. Evidence implicating the class I-connecting peptide segment in calnexin association.
The endoplasmic reticulum resident protein calnexin interacts with several glycoproteins including class I MHC molecules. Calnexin is thought to retain free class I heavy chains and/or promote their folding and assembly with beta 2-microglobulin and peptide ligand. Whereas with other glycoproteins, Asn-linked glycans seem to be involved in calnexin association, with class I molecules the transmembrane region has been implicated. To critically define the structures on class I molecules that determine their interaction with calnexin, we have studied carbohydrate-deficient and transmembrane-variant class I molecules. Carbohydrate-deficient class I molecules were found to accumulate intracellularly in an open, non-beta 2-microglobulin-associated conformation. However, open as well as conformed class I molecules showed significant calnexin association whether they were aglycosylated or fully glycosylated. Thus, carbohydrate moieties may be necessary for efficient class I folding, but are not required for calnexin association. Calnexin was also found associated with a soluble class I molecule that has a truncated transmembrane segment, demonstrating that membrane attachment of class I is not required for interaction with calnexin. Finally, two isoforms of the class Ib molecule Q7b were compared. Unexpectedly, the glycosylphosphatidylinositol-anchored Q7b isoform was found associated with calnexin, whereas the soluble Q7b isoform was not calnexin associated. These comparisons of Q7b isoforms implicate the class I-connecting peptide segment and not the transmembrane region as a site of interaction with calnexin.
Amino Acid Sequence↗
Peptide-induced rescue of serologic epitopes on class I MHC molecules.
To monitor conformational changes in MHC class I structure induced by interaction with peptide or beta 2-microglobulin (beta 2-m), we have taken a serologic approach. Previous studies by us and others have defined circumstances wherein specific peptides can decrease serologic recognition of class I molecules. However, such blocking of serologic epitopes has often been interpreted as steric hindrance by peptide side chains. In this paper, we describe peptide-induced gains in recognition by mAbs 30-5-7, 34-1-2, and B22/249. In experiments with mAb 30-5-7, impaired reactivity, which resulted from an Ld loop mutation, was specifically rescued by the binding of a beta-galactosidase-derived peptide to the Ld mutant. In studies with mAb 34-1-2, poor Ld detection was enhanced by mutations in Ld at beta 2-m interaction sites or by changes within the peptide-binding groove. To evaluate whether known peptides in the Ld groove could influence 34-1-2 recognition, we tested six peptide ligands, four of which increased the reactivity of 34-1-2 with the Ld-expressing cell to various degrees (up to 14-fold). It is of interest that Ld mutations at position 9 and 95/97 made significant differences in the ranking of the peptides in regard to their ability to increase recognition by 34-1-2 and B22/249. This finding suggests that mutations in the binding groove can alter peptide conformation and result in secondary changes in class I structure. On the basis of the cumulative serologic data, we propose that the class I molecule displays considerable fluidity, and is structurally influenced by both beta 2-m and peptide.
Amino Acid Sequence↗
Induction of peptide-specific CD8+ CTL clones in beta 2-microglobulin-deficient mice.
We have examined the ability of beta 2-m- mice to produce CD4-8+ T cells by generating CD8+ CTLs to a defined ligand. We report here the first demonstration of peptide-specific, self-class I MHC-restricted CTLs from beta 2-m-deficient mice. We have used the KOD mouse, an H-2d beta 2-m- strain, to generate CTLs that recognize the class I MHC molecule Ld in association with one of two Ld-binding immunogenic peptides. Testing of these CTLs on a panel of Ld-binding peptides reveals a high degree of peptide specificity. Peptide-specific CTL bulk cultures from KOD mice differ from those generated in beta 2-m+ mice in that they possess altered affinities for their peptide ligands. In addition, we show that CTLs generated from beta 2-m- mice in the presence of beta 2-m+ stimulator cells and exogenous peptide are specific either for the exogenous peptide or for endogenous peptides that are present in association with Ld on the surface of beta 2-m+ cells, but are not present at detectable levels on beta 2-m- cells. These results demonstrate that positive selection of CD8+ CTLs can occur in vivo on the very low levels of class I MHC found in the KOD mouse. Furthermore, CTLs from the KOD mouse maintain a high degree of peptide specificity despite reduced levels of class I MHC.
Amino Acid Sequence↗
Beta 2-microglobulin with an endoplasmic reticulum retention signal increases the surface expression of folded class I major histocompatibility complex molecules.
With beta 2-microglobulin- (beta 2m-) cell lines such as R1E/Db, the surface expression of class I major histocompatibility complex molecules is greatly impaired, and class I molecules that are on the surface are generally misfolded. To determine whether beta 2m must be continually present with the class I heavy chain for the class I molecule to reach the surface in a folded conformation, a sequence encoding an endoplasmic reticulum (ER) retention signal (KDEL) was attached onto the 3' end of a beta 2m cDNA. After this chimeric cDNA was transfected into R1E/Db cells, beta 2m-KDEL protein was detectable by an anti-beta 2m serum within the cells but not at the cell surface. Interestingly, R1E/Db cells transfected with beta 2m-KDEL were found to express a high level of conformationally correct Db molecules at the cell surface. This observation implies that beta 2m has a critical and temporal role in the de novo folding of the class I heavy chain. We propose that the critical time for beta 2m association is when the class I molecule is docked with the transporter associated with antigen processing (TAP) and first interacts with peptide.
Amino Acid Sequence↗
Mouse H2 congenic intervals: analysis and use for mapping.
In this study we exploit the unique genetic resource of inbred mouse major histocompatibility complex (H2) congenic and recombinant strains to construct a high-resolution map of microsatellite loci in and around the H2 region, as well as an independent genetic map of other loci on mouse Chromosome (Chr) 17. Microsatellite loci were analyzed in 11 C57BL/10 (B10) strains to determine the size of the congenic interval in each. The length of the congenic interval found in each strain varied widely. Interestingly, the intervals were generally smaller than statistical expectations. However, the observed congenic intervals were still sufficiently long that these strains and probably wild-derived H2 congenics are an important source of genetic variability. The staggered ends of the various congenic intervals and the recombinants were used to construct the map. This map will be useful for physical cloning and to help localize novel genes. As evidence of the mapping application of congenic strains, locational information was derived about Trp53-ps and Stl.
Animals↗
Chronic pancreatitis and extrapancreatic cancer: a retrospective study among 181 patients with chronic pancreatitis.
The relationship between chronic pancreatitis (CP) and extrapancreatic cancer has been debated in the recent years. In prospective studies, it has been found that pancreatic cancer develops in 0-5% of patients with chronic pancreatitis. Many papers describe an increased relative risk for developing extrapancreatic cancer in patients suffering from chronic pancreatitis. In this study including 181 patients with CP, we found 14 patients with extrapancreatic cancer (three of these had two different types of cancer). No patient had pancreatic cancer. It was found that the respiratory airways and upper gastrointestinal tract were the dominating locations (five and four cases, respectively), but also genital and hemolymphopoietic cancers were represented (four and two cases, respectively). Two patients had metastatic cancer with unknown primary tumor. The patients with cancer tended to be older than those without cancer. The patients with CP had a 2.43 times greater risk of developing cancer than the general Danish population (age and sex standardized comparison). The relatively large number of cancers in the upper gastrointestinal tract and respiratory airways suggest that tobacco and alcohol may be responsible, as these organs have the highest exposure to these compounds, which are well known carcinogens.
Adult↗
Conformational changes induced in the MHC class I molecule by peptide and beta 2-microglobulin.
Assembly of the class I MHC molecule is inextricably linked to the antigen presentation function of the class I molecule. Association of the class I MHC molecule with beta 2-microglobulin (beta 2m) is a prerequisite for association with the heterodimeric protein TAP, and once peptide is acquired, the class I molecule folds and begins its sojourn to the cell surface. To maintain its folded conformation, class I MHC requires peptide but not beta 2m, and the sequence of the peptide bound exercises a subtle influence on the structure of the class I molecule that is likely to be a factor in T cell receptor discrimination of MHC/peptide complexes.
Animals↗
Characterization of class I MHC folding intermediates and their disparate interactions with peptide and beta 2-microglobulin.
Newly synthesized class I heavy chains achieve domain structure using disulfide bonds, assemble with beta-2 microglobulin (beta 2m), and bind peptide ligand to complete the trimeric complex. Although each of these initial events is thought to be critical for class I folding, their sequential order and effect on class I structure are unknown. Using mAb specific for distinct conformations of H-2Ld and Lq, we have defined folding intermediates of class I molecules. We show here that non-peptide-associated forms of Ld or Lq, detected by mAb 64-3-7 and designated L alt, lack numerous conformational epitopes surrounding their ligand binding sites. These results support the notion that L alt molecules have an open conformation. Interestingly, a significant proportion of L alt molecules were detected in association with beta 2m and these L alt/beta 2m heterodimers were preferentially folded by peptide in cell lysates. These findings indicate that class I heavy chain/beta 2m association can precede ligand binding and that peptide is probably the limiting factor for completion of the Ld/beta 2m/peptide trimeric complex in vivo. The characteristics of L alt molecules were investigated further by ascertaining the disulfide bond status of these molecules and their association with beta 2m and peptide. Treatment of cells with dithiothreitol (DTT), a membrane-permeable reducing agent, demonstrated that L alt molecules constitute a heterogeneous population including reduced, partially reduced and native class I molecules. Furthermore, partially reduced Ld alt molecules, in a cell line expressing a mutant Ld molecule lacking the alpha 2 domain disulfide bond, accumulated intracellularly, were not beta 2m-associated and displayed marginal peptide-induced folding in vitro. In accordance with this latter finding, peptide was found to preferentially convert fully disulfide-bonded forms of Ld alt to conformed Ld. Thus, we propose that intrachain disulfide bond formation precedes the association of class I heavy chain with beta 2m and peptide, and that disulfide bond formation is required for efficient assembly, ligand binding and folding of the class I heavy chain.
Animals↗