Whole-proteome analysis of mesonephric-derived cancers describes new potential biomarkers.

Mesonephric carcinomas (MEs) and female adnexal tumors of probable Wolffian origin (FATWO) are derived from embryologic remnants of Wolffian/mesonephric ducts. Mesonephric-like carcinomas (MLCs) show identical morphology to ME of the cervix but occur in the uterus and ovary without convincing mesonephric remnants. ME, MLC, and FATWO are challenging to diagnose due to their morphologic similarities to Müllerian/paramesonephric tumors, contributing to a lack of evidence-based and tumor-specific treatments. We performed whole-proteomic analysis on 9 ME/MLC and 56 endometrial carcinomas (ECs) to identify potential diagnostic biomarkers. Although there were no convincing differences between ME and MLC, 543 proteins showed increased expression in ME/MLC relative to EC. From these proteins, euchromatic histone lysine methyltransferase 2 (EHMT2), glutathione S-transferase Mu 3 (GSTM3), eukaryotic translation elongation factor 1 alpha 2 (EEF1A2), and glycogen synthase kinase 3 beta were identified as putative biomarkers. Immunohistochemistry was performed on these candidates and GATA3 in 14 ME/MLC, 8 FATWO, 155 EC, and normal tissues. Of the candidates, only GATA3 and EHMT2 were highly expressed in mesonephric remnants and mesonephric-derived male tissues. GATA3 had the highest sensitivity and specificity for ME/MLC versus EC (93% and 99%) but was absent in FATWO. EHMT2 was 100% sensitive for ME/MLC & FATWO but was not specific (65%). Similarly, EEF1A2 was reasonably sensitive to ME/MLC (92%) and FATWO (88%) but was the least specific (38%). GSTM3 performed intermediately (sensitivity for ME/MLC and FATWO: 83% and 38%, respectively; specificity 67%). Although GATA3 remained the best diagnostic biomarker for ME/MLC, we have identified EHMT2, EEF1A2, and GSTM3 as proteins of interest in these cancers. FATWO's cell of origin is uncertain and remains an area for future research.

Expression of MHC-I and II by Microglia and Lymphocytes in the Brain of Diet-Restricted Mice.

Microglia are immunocompetent cells of the central nervous system whose function is to preserve nervous tissue homeostasis; however, under inflammatory conditions, they are associated with tissue damage. Dietary restriction is a nutritional intervention used to delay the onset of chronic disease and inflammation, in addition to improving the functions of the immune system. The aim of this study was to analyze the effect of dietary restriction on microglial expression of MHC molecules. Adult female and male BALB/c mice were fed ad libitum (controls) or kept under dietary restriction (30% reduction in food intake) for 4 wk. Purified brain mononuclear cells were analyzed by flow cytometry staining for CD45, CD11b, MHC-I and MHC-II. Our results show that female animals under dietary restriction had a significant increase in MHC-I expression in microglia (mean fluorescence intensity=7,854 (control) vs. 10,628 (diet-restricted), arbitrary units; p=0.0108), along with increased frequencies of lymphocytes compared to controls (1.39% (control) vs. 7.85% (diet-restricted); p=0.0175), whereas male animals did not show significant differences between groups. Our data suggest a differential effect for dietary restriction on female and male animals, with this nutritional regimen predominantly affecting females. Increased expression of MHC-I by diet-restricted microglia may play a role in maintaining tolerance in the absence of antigenic stimulation.

FLEXamers: A Double Tag for Universal Generation of Versatile Peptide-MHC Multimers.

Peptide-MHC (pMHC) multimers have become a valuable tool for immunological research, clinical immune monitoring, and immunotherapeutic applications. Biotinylated tetramers, reversible Streptamers, or dye-conjugated pMHC multimers are distinct pMHC reagents tailored for T cell identification, traceless T cell isolation, or TCR characterization, respectively. The specific applicability of each pMHC-based reagent is made possible either through conjugation of probes or reversible multimerization in separate production processes, which is laborious, time-consuming, and prone to variability between the different types of pMHC reagents. This prohibits broad implementation of different types of pMHC reagents as a standard toolbox in routine clinical immune monitoring and immunotherapy. In this article, we describe a novel method for fast and standardized generation of any pMHC multimer reagent from a single precursor ("FLEXamer"). FLEXamers unite reversible multimerization and versatile probe conjugation through a novel double tag (Strep-tag for reversibility and Tub-tag for versatile probe conjugation). We demonstrate that FLEXamers can substitute conventional pMHC reagents in all state-of-the-art applications, considerably accelerating and standardizing production without sacrificing functional performance. Although FLEXamers significantly aid the applicability of pMHC-based reagents in routine workflows, the double tag also provides a universal tool for the investigation of transient molecular interactions in general.

Rotavirus Vaccine Take in Infants Is Associated With Secretor Status.

Rotaviruses bind to enterocytes in a genotype-specific manner via histo-blood group antigens (HBGAs), which are also detectable in saliva. We evaluated antirotavirus immunoglobulin A seroconversion ('vaccine take") among 166 Ghanaian infants after 2-3 doses of G1P[8] rotavirus vaccine during a vaccine trial, by HBGA status from saliva collected at age 4.1 years. Only secretor status was associated with seroconversion: 41% seroconversion for secretors vs 13% for nonsecretors; relative risk, 3.2 (95% confidence interval, 1.2-8.1; P = .016). Neither Lewis antigen nor salivary antigen blood type was associated with seroconversion. Likelihood of "take" for any particular rotavirus vaccine may differ across populations based on HBGAs.

Successful kidney transplantation across a positive complement-dependent cytotoxicity crossmatch by using C1q assay-directed, bortezomib-assisted desensitization: A case report.

RATIONALE: Human leukocyte antigen (HLA) is the major immunologic barrier in kidney transplantation (KT). Various desensitization protocols to overcome the HLA barrier have increased the opportunity for transplantation in sensitized patients. In addition, technological advances in solid-phase assays have permitted more comprehensive assessment of donor-specific antibodies. Although various desensitization therapies and immunologic techniques have been developed, the final transplantation decision is still based on the classic complement-dependent cytotoxicity (CDC) crossmatch (XM) technique. Some patients who fail to achieve negative XM have lost their transplant opportunities, even after receiving sufficient desensitization therapies. PATIENT CONCERNS: A 57-year-old male with end-stage renal disease secondary to chronic glomerulonephritis was scheduled to have a second transplant from his son, but CDC XM was positive. DIAGNOSES: Initial CDC XM (Initial T-AHG 1:32) and flow-cytometry XM were positive. Anti-HLA-B59 donor specific antibody was detected by Luminex single antigen assay. INTERVENTIONS: Herein, we report a successful case of KT across a positive CDC XM (T-AHG 1:8 at the time of transplantation) by using C1q assay-directed, bortezomib-assisted desensitization. After confirming a negative conversion in the C1q donor-specific antibody, we decided to perform KT accepting a positive AHG-CDC XM of 1:8 at the time of transplantation. OUTCOMES: The posttransplant course was uneventful and a protocol biopsy at 3 months showed no evidence of rejection. The patient had excellent graft function at 12 months posttransplant. LESSONS: The results of XM test and solid-phase assay should be interpreted in the context of the individual patient.

Fluorescence-based methods for screening writers and readers of histone methyl marks.

The histone methyltransferase (HMT) family of proteins consists of enzymes that methylate lysine or arginine residues on histone tails as well as other proteins. Such modifications affect chromatin structure and play a significant regulatory role in gene expression. Many HMTs have been implicated in tumorigenesis and progression of multiple malignancies and play essential roles in embryonic development and stem cell renewal. Overexpression of some HMTs has been observed and is correlated positively with various types of cancer. Here the authors report development of a continuous fluorescence-based methyltransferase assay in a 384-well format and its application in determining kinetic parameters for EHMT1, G9a, PRMT3, SETD7, and SUV39H2 as well as for screening against libraries of small molecules to identify enzyme inhibitors. They also report the development of a peptide displacement assay using fluorescence polarization in a 384-well format to assay and screen protein peptide interactions such as those of WDR5 and EED, components of MLL and EZH2 methyltransferase complexes. Using these high-throughput screening methods, the authors have identified potent inhibitors and ligands for some of these proteins.

Characterization of MHC/peptide complexes refolded by a one-step ion-exchange chromatography.

The current refolding process of MHC/peptide complexes is low-yield and time-consuming, thereby limiting the wide uses of MHC/peptide multimers. Here the heavy chain protein of MHC/peptide complex (H-2K(b)/TRP2(180-188)) was immobilized onto an ion-exchange chromatography column, and the ß2m or TRP2(180-188)-fused ß2m protein, which renatured previously in refolding buffer, was able to pass through the column for the gradient refolding. This strategy refolds, concentrates and purifies MHC/peptide complexes in a single integrated step, achieving a high level of process simplification and automation. Using this on-column refolding method, MHC/peptide complexes could be prepared within 24h with a refolding yield of over 20%. Anti-H-2K(b) mAb staining and flow cytometric analyses revealed that the on-column refolded H-2K(b)/TRP2(180-188) complexes had conformational characteristics similar to the dilution refolded H-2K(b)/TRP2(180-188) complexes and the commercial ones. Furthermore, H-2K(b)/TRP2(180-188) tetramer staining and the enumeration of TRP2(180-188)-specific T cells and H-2K(b)-alloreactive T cells confirmed that the H-2K(b)/TRP2(180-188) complexes prepared by on-column refolding or dilution refolding had comparable TCR-binding ability. These data demonstrate a novel, simple and efficient refolding strategy for the generation of MHC class I/peptide complexes.

The MHC motif viewer: a visualization tool for MHC binding motifs.

In vertebrates, the onset of cellular immune reactions is controlled by presentation of peptides in complex with major histocompatibility complex (MHC) molecules to T cell receptors. In humans, MHCs are called human leukocyte antigens (HLAs). Different MHC molecules present different subsets of peptides, and knowledge of their binding specificities is important for understanding differences in the immune response between individuals. Algorithms predicting which peptides bind a given MHC molecule have recently been developed with high prediction accuracy. The utility of these algorithms is hampered by the lack of tools for browsing and comparing specificity of these molecules. We have developed a Web server, MHC Motif Viewer, which allows the display of the binding motif for MHC class I proteins for human, chimpanzee, rhesus monkey, mouse, and swine, as well as HLA-DR protein sequences. The binding motif for each MHC molecule is predicted using state-of-the-art, pan-specific peptide-MHC binding-prediction methods, and is visualized as a sequence logo, in a format that allows for a comprehensive interpretation of binding motif anchor positions and amino acid preferences.

EVI-1 interacts with histone methyltransferases SUV39H1 and G9a for transcriptional repression and bone marrow immortalization.

The ecotropic viral integration site-1 (EVI-1) is a nuclear transcription factor and has an essential function in the proliferation/maintenance of haematopoietic stem cells. Aberrant expression of EVI-1 has been frequently found in myeloid leukaemia as well as in several solid tumours, and is associated with a poor patient survival. It was recently shown that EVI-1 associates with two different histone methyltransferases (HMTs), SUV39H1 and G9a. However, the functional roles of these HMTs in EVI-1-mediated leukemogenesis remain unclear. In this study, we showed that EVI-1 physically interacts with SUV39H1 and G9a, but not with Set9. Immunofluorescence analysis revealed that EVI-1 colocalizes with these HMTs in nuclei. We also found that the catalytically inactive form of SUV39H1 abrogates the transcriptional repression mediated by EVI-1, suggesting that SUV39H1 is actively involved in EVI-1-mediated transcriptional repression. Furthermore, RNAi-based knockdown of SUV39H1 or G9a in Evi-1-expressing progenitors significantly reduced their colony-forming activity. In contrast, knockdown of these HMTs did not impair bone marrow immortalization by E2A/HLF. These results indicate that EVI-1 forms higher-order complexes with HMTs, and this association has a role in the transcription repression and bone marrow immortalization. Targeting these HMTs may be of therapeutic benefit in the treatment for EVI-1-related haematological malignancies.

Phenotype and function of monocyte-derived dendritic cells from chinese rhesus macaques.

Dendritic cells (DCs) play a pivotal role in linking the innate immunity and acquired immunity in responses to pathogen. Non-human primates such as Chinese Rhesus Macaque (CRM) are the favorable models for preclinical study of potential therapeutic drugs, vaccines and mechanisms of human diseases. However, the phenotypical characterization of monocyte-derived dendritic cells (MDDCs) from CRM has not been elucidated. Monocytes from CRM were cultured with GM-CSF and IL-4 in RPMI-1640. Six days later, these cells were differentiated with typical dendritical morphology. CD11c and DC-SIGN were highly expressed. The immature MDDCs expressed the low levels of CD25, CD80, CD83, moderate CD40, CD86, and high MHC. After stimulation, the mature MDDCs increased expression of mature molecules CD25 and CD83, co-stimulatory molecules such as CD80, CD86 and CD40, and kept a high level of MHC. The capacity of endocytosis decreased with maturation. The mature MDDCs have strong ability of inducing allogeneic T cell proliferation and producing IL-12. In conclusion, we have characterized the phenotype and ultimate function of MDDCs from CRM for the first time.

Colorectal carcinoma rearranges cell surface protein topology and density in CD4+ T cells.

Previously, we described conserved protein clusters including MHC I and II glycoproteins, ICAM-1 adhesion molecules, and interleukin-2 and -15 receptors in lipid rafts of several human cell types. Differential protein-protein interactions can modulate function, thus influence cell fate. Therefore, we analyzed supramolecular clusters of CD4(+) T cells from draining lymph nodes and peripheral blood of colorectal carcinoma patients, and compared these to healthy controls. Superclusters of MHC I and II with IL-2/15 receptors were identified by confocal microscopy on all cell types. Flow-cytometric FRET revealed molecular associations of these proteins with each other and with ICAM-1 as well. In draining lymph nodes expression levels of all these proteins were lower, and interactions, particularly between IL-2/15 receptors and MHC molecules weakened or disappeared as compared to the control. Stimuli/local conditions can rearrange cell surface protein patterns on the same cell type in the same patient, having important implications on further function and cell fate.

Intrinsic ability of GM+IL-4 but not Flt3L-induced rat dendritic cells to promote allogeneic T cell hyporesponsiveness.

The influence of GM+IL-4 and Flt3 ligand (FL) on phenotype and function of BM-derived DC from Lewis rats was investigated. GM+IL-4-induced DC, despite expression of CD80/CD86, were less stimulatory than FL-induced DC that expressed low CD80/CD86 and were efficient stimulators of allogeneic T cells. GM+IL-4 DC were CD11b+ OX62lo, whereas FL DC were CD11blo OX62+. Following activation, GM+IL-4 DC produced IL-10 and IL-6, but no IL-12p70, and were resistant to further maturation. FL DC produced IL-12p70, IFN-alpha/beta, IL-10 and IL-6 and underwent maturation. Repeated stimulation of T cells with GM+IL-4 DC inhibited proliferation, cytokine production and induced early T cell apoptosis. FL DC-activated T cells produced large amounts of IFN-gamma/IL-10 and exhibited late T cell apoptosis/necrosis. In vivo, GM+IL-4 DC induced alloAg-specific hyporesponsiveness following T cell restimulation. These results demonstrate that GM+IL-4 DC display intrinsic regulatory properties, inducing passive-cell-death in T cells with potential for inactivation/regulation of alloreactive T cells in transplantation.

Undifferentiated murine embryonic stem cells cannot induce portal tolerance but may possess immune privilege secondary to reduced major histocompatibility complex antigen expression.

Induction of donor-specific tolerance using embryonic stem (ES) cells followed by transplantation of ES cell-derived tissues from the same allogeneic strain could theoretically engender successful transplantation without immunosuppression. We sought to induce tolerance using bona fide murine ES cells in immunocompetent mice. ES cells were evaluated for the expression of markers restricted to undifferentiated cells [stage-specific embryonic antigen-1 (SSEA-1) and OCT-4] and the ability to form teratomas in immunodeficient mice. BALB/cByJ mice underwent intraportal inoculation with YC5-EYFP ES cells (129 strain; R1-derived) or saline followed by transplantation with 129X1/SvJ, CBA/J, or BALB/cByJ nonvascularized, neonatal cardiac grafts. Mice were sacrificed at graft failure and underwent histologic evaluation of transplanted grafts and lymphoid organs. ES cells and early differentiated progeny underwent real time (RT)-PCR and fluorescence-activated cell sorting (FACS) analysis to detect major histocompatibility complex (MHC) gene transcription and antigen expression. ES cells expressed markers restricted to undifferentiated cells while maintaining the ability to form teratomas in immunodeficient mice. No prolongation of allograft survival or evidence of lymphoid chimerism was observed in immunocompetent recipient mice despite hepatic teratoma formation. MHC class I, class II, and nonclassical antigens were undetectable on ES cells and early differentiated progeny despite the presence of mRNA transcripts. Class I expression was strongly upregulated upon exposure to gamma-interferon. Intraportal inoculation with murine ES cells does not produce lymphoid chimerism or induce donor-specific unresponsiveness to neonatal cardiac grafts in unmanipulated immunocompetent hosts. However, specific differentiated cell types such as ES cellderived dendritic cells, or alternate routes of ES cell administration, may be effective. ES cells appear to have immune privilege, allowing them to form teratomas in immunocompetent mice.

Which complement assays and typings are necessary for the diagnosis of complement deficiency in patients with lupus erythematosus? A study of 25 patients.

INTRODUCTION: Deficiencies in components of the classical pathway of complement activation are strong risk factors for lupus erythematosus (LE).Yet, it has not been addressed whether the conventional measurements of the serum hemolytic CH50 activity and antigenic concentrations of C3 and C4 are sufficient to asses a deficiency in C4A, C4B or C2 components, the most common deficiencies associated with LE. PATIENTS AND METHODS: In a retrospective series, we performed complement analyses in 35 patients with LE who were systematically screened for a complement deficiency. The majority of patients had cutaneous LE with mild systemic involvement and no complement consumption. Of 25 patients (72%) with complement deficiency we found 13 with a partial C4A deficiency, 2 with a complete C4A deficiency, 6 with a partial C4B deficiency, 2 with a complete C4B deficiency and 2 with a combined partial C2 and C4A deficiency. RESULTS: The total complement activity (CH50) was decreased in only one out of two patients with complete C4B deficiency. CH50 level was found to be low-normal (35-38 U/ml(-1)) in one patient with partial C4B deficiency, one patient with complete C4B deficiency and both patients with combined partial C4A and C2 deficiency. Total C4 levels were normal in 9 out of 13 the patients with a partial C4A deficiency and in 2 out of 6 patients with a complete C4B deficiency. The antigenic concentration of C3 was low in only 1 patients with a complete C4B deficiency and within the normal range in all the others patients. Overall, 50% of the patients had normal or elevated C3, C4, and CH50 levels. DISCUSSION: This study emphasizes that the usual measurements of CH50, C3 and C4 levels are not adequate to detect a C4 and/or C2 deficiency in patients with LE. In epidemiologic or investigative studies addressing the prevalence of complement deficiency, more elaborated diagnostic tests, such as C4 protein allotyping, C2 level measurement and genetic screening for type I C2 deficiency should also be performed.

Human NK cells can lyse porcine endothelial cells independent of their expression of Galalpha(1,3)-Gal and killing is enhanced by activation of either effector or target cells.

BACKGROUND: Xenotransplantation of pig organs may provide an approach to alleviate the severe shortage of human organs. Natural antibodies against Galalpha(1,3)-Gal (alphaGal) epitopes cause hyperacute rejection of pig organs in primates. However, evidence for the role of alphaGal in the natural killer (NK) cell-mediated xenoresponse has been contradictory. METHODS: We investigated the recognition of alphaGal by human NK cells using endo-beta-galactosidase C, an enzyme that cleaves alphaGal, and endothelial cells (EC) from alpha1,3-galactosyltransferase null pigs that do not synthesize alphaGal. Endo-beta-galactosidase C treatment variably reduced the susceptibility of porcine EC to lysis by fresh human NK cells. RESULTS: Removal of alphaGal from porcine EC using endo-beta-galactosidase C, produced variable results, i.e. cytotoxicity was decreased in half of the human NK cell donors tested. The two EC strains from alphaGal-/- pigs were marginally, and not significantly, less susceptible to lysis by naïve human NK cells compared with alphaGal-expressing cells obtained from animals from the same herd, but these differences were not statistically significant (P > 0.10). Treatment of porcine EC with recombinant human tumor necrosis factor (TNF)-alpha, which is known to activate porcine EC, enhanced the susceptibility of all target cells to lysis by fresh human NK cells. Surface expression of MHC or adhesion molecules on alphaGal-/- cells, compared with wild type cells, showed no consistent difference in either MHC or adhesion molecules CD106 (VCAM-1), CD31 (PECAM) or CD62E (E-selectin), either with or without TNF-alpha stimulation, that could explain the differential susceptibility to lysis. Strikingly, all alphaGal-/- and wild type EC exhibited similar susceptibility to human NK cells that had been cultured for 5 days with or without interleukin-2. CONCLUSIONS: These findings demonstrate that human NK cells can kill porcine targets in the absence of alphaGal, and donor variability plays a major role in whether alphaGal has a role in determining susceptibility of porcine EC to lysis. Moreover, susceptibility to lysis of alphaGal null EC is enhanced to the level of wild type EC by activation of either effector or target cells. Elimination of alphaGal alone from source pigs will be insufficient to circumvent the NK cell mediated destruction of porcine EC.

[HLA typing, analysis methods, and clinical applications]. / Typage HLA, méthodes d'analyses et applications cliniques.

The polygenic HLA system is a well known region of the human genome. Its main function is to present antigenic peptides to the immune system and thereby regulate induction of immune responses. Extensive genetic polymorphisms characterize some HLA genes. Initially, genetic variations were analyzed by a serological typing technique (microlymphocytotoxicity). The introduction of polymerase chain reaction (PCR) in the mid-1980s led the development of a variety of methods that use molecular biology. An international nomenclature, regularly updated, governs the names of HLA antigens defined by serology as well as of HLA alleles. Knowledge of the specific polymorphisms of individuals is essential in organ and stem cell transplantation and highly useful in disease association studies.

Thiol redox and immune regulation in corneal transplantation.

Penetrating keratoplasty (PKP) is the most common type of clinical grafting performed in humans. Although PKP has emerged as the most successful form of transplantation, PKP in "high-risk" eyes shows high incidence of allograft rejection. The incidence of epithelial rejection after limbal transplantation (LT) is extremely higher and swifter than PKP rejection, and even intensive systemic immunosuppressive therapy is often of no avail. Because failure of corneal grafts is an important cause of blindness, developing new strategies for suppressing graft rejection is a worthy goal for research. Corneal allograft rejection is mainly mediated by the TH1-type immune response, which leads to a delayed-type hypersensitivity reaction. Because the TH2-type immune response regulates the TH1-type immune response, we have successfully elicited allograft survival after both PKP and LT by inducing systemic TH2-type immune responses. Because intracellular thiol redox status of antigen-presenting cells (APC) reportedly regulates TH1/TH2 balance via distinctive cytokine production by APC, we also investigated the effect of modulating macrophage intracellular thiol redox status on corneal allograft survival. These strategies are quite effective in major histocompatibility complex (MHC) matching in mice, although it is believed that MHC matching has no effect on corneal allograft survival according to many rodent studies. Recently, many laboratories are reconsidering HLA matching for allograft survival in human corneal transplantation. It may be possible that MHC matching improves corneal allograft survival in the context of TH1 suppression. We propose that the suppression of the TH1-type immune response and MHC matching together may promote allograft survival in humans.

Computer program for analyzing donor photobleaching FRET image series.

BACKGROUND: The photobleaching fluorescence resonance energy transfer (pbFRET) technique is a spectroscopic method to measure proximity relations between fluorescently labeled macromolecules using digital imaging microscopy. To calculate the energy transfer values one has to determine the bleaching time constants in pixel-by-pixel fashion from the image series recorded on the donor-only and donor and acceptor double-labeled samples. Because of the large number of pixels and the time-consuming calculations, this procedure should be assisted by powerful image data processing software. There is no commercially available software that is able to fulfill these requirements. METHODS: New evaluation software was developed to analyze pbFRET data for Windows platform in National Instrument LabVIEW 6.1. This development environment contains a mathematical virtual instrument package, in which the Levenberg-Marquardt routine is also included. As a reference experiment, FRET efficiency between the two chains (beta2-microglobulin and heavy chain) of major histocompatibility complex (MHC) class I glycoproteins and FRET between MHC I and MHC II molecules were determined in the plasma membrane of JY, human B lymphoma cells. RESULTS: The bleaching time constants calculated on pixel-by-pixel basis can be displayed as a color-coded map or as a histogram from raw image format. CONCLUSION: In this report we introduce a new version of pbFRET analysis and data processing software that is able to generate a full analysis pattern of donor photobleaching image series under various conditions. .

Novel antibody tags from the rat lysosomal protein RT1.DM for immunodetection of recombinant proteins.

Previously, two mouse monoclonal antibodies (12B8 and 6D4) were raised against the alpha- and beta-subunits of the rat non-classical MHC class-II heterodimer RT1.DM. Here, I describe the epitope mapping of mAb 12B8 to amino acids alpha3-14 (EASPQAWWDESQ) and mAb 6D4 to amino acids beta35-44 (WDPEEGQIVP). Epitope mapping was conducted by preparing fusion proteins between the alpha and beta chain of RT1.DM for Western detection with mAb 12B8 and 6D4. By mutating non-conserved amino acids of the human orthologue of RT1.DM, the rat epitopes were introduced, thereby making the alpha and beta polypeptides sensitive for mAb 12B8 and 6D4 detection. The epitopes, designated as 12B8 and 6D4, were tested for protein tagging. They were appended to the N- or C-terminus of four human proteins, the tumour suppressor protein VHL (von Hippel-Lindau), SUMO4, MHC class-II DQbeta and -DPbeta for expression in mammalian cells. Western detection, immunoprecipitation and localisation of the tagged proteins were successfully demonstrated. Thus, the 12B8 and 6D4 epitope tag can be universally used for the immunodetection of recombinant proteins and to study protein-protein interactions.

Digital HLA allelotyping.

Stem cell transplantation is a potentially curative therapy for a number of neoplastic disorders. Successful transplantation of bone marrow, and other sources of stem cells, depends upon the extent to which human leukocyte antigen (HLA) alleles in donor and recipient are matched. HLA incompatibility can result in cytotoxic T-cell mediated graft failure or graft versus host disease. It has been shown that accurate matching between donor and recipient minimizes both of these outcomes and results in improved patient survival. Therefore, methods for high resolution HLA analysis have substantial clinical impact. Heterozygosity at the HLA loci interferes with current methods of DNA sequence-based HLA typing. We present a novel method for HLA analysis, which we term Digital HLA Allelotyping, or dig-HLA. This method employs a strategy in which the expressed HLA alleles are randomly sorted and individually identified.