Search PubMed⌕ Search

Biomedical subjects

D J Moss

Publications and source records attributed to D J Moss.

At least 19 recordsLinked to original sources
PubMed2004

Treatment options for post-transplant lymphoproliferative disorder and other Epstein-Barr virus-associated malignancies.

Epstein-Barr virus (EBV) is associated with a range of malignancies that largely arise from a defect in EBV-specific cytotoxic T lymphocyte (CTL) immunity and function. Much work has focused on the reconstitution of CTL immunity to EBV in transplant patients, in whom immunosuppression modalities render them susceptible to post-transplant lymphoproliferative disease (PTLD). Adoptive transfer of autologous CTLs is effective at both preventing and curing PTLD in solid organ transplant recipients and can produce a long-term memory response and protection against recurring disease. In this review, the benefits and restrictions of administering EBV-specific CTLs for the treatment of PTLD are discussed and compared with emerging therapies including the generation of allogeneic human leukocyte antigen-matched CTL banks and the anti-CD20 monoclonal antibody therapy, MabThera. Furthermore, studies involving other EBV-associated disorders have described the potential benefit of adoptive transfer of EBV-specific CTLs for Hodgkin's disease, nasopharyngeal carcinoma, chronic active EBV infection, and Burkitt's lymphoma. The challenges of tailor-making therapies for individual diseases and EBV antigen expression latencies are highlighted, in addition to considering vaccination strategies for optimal treatment.

Burkitt Lymphoma↗
PubMed2003

No barrier to diffusion between cell soma and neurite membranes in sympathetic neurons for a GPI-anchored glycoprotein.

As neurons extend their axons, it is thought that newly synthesised membrane components travel in vesicles along the axon, fuse with the growth cone membrane, and diffuse back along the axonal membrane. However, it is difficult to explain how axons continue to be populated with membrane proteins as they extend in length. To investigate this problem, we have used a CEPU-green fluorescent protein (GFP) chimeric protein to study the site of insertion of new glycosyl phosphatidyl inositol (GPI)-anchored glycoproteins and their subsequent behaviour in chick dorsal root ganglia (DRG) neurons. Infection of cultures grown for 24 h revealed rapid expression of CEPU-GFP over the whole surface of the neuron, more rapidly than could be accounted for by diffusion from the growth cone, and fluorescence intensity was uniform along the length of the neurite. Photobleaching experiments of neurite membrane revealed that recovery of fluorescence was due to diffusion from adjacent membranes and there was no evidence for membrane flow in either direction. Photobleaching of membrane adjacent to the cell body also showed rapid recovery, with chimera diffusing both from cell body membrane and the distal neurite membrane into the bleached area. These results suggest there is no barrier to diffusion between the cell body and neurite membrane in DRG and sympathetic neurons cultured for 1 or 2 days in vitro. We propose that the neurite is populated by newly synthesised chimera by diffusion from both regions. This situation may also occur in neurons in the early stages of extending axons in vivo prior to polarisation and the development of the dendritic field.

Adrenergic Fibers↗
PubMed2002

High-level expression of recombinant Fc chimeric proteins in suspension cultures of stably transfected J558L cells.

Recombinant Fc chimeric proteins are useful tools for studying protein function, including the analysis of molecular interactions by techniques such as expression cloning. Here we describe a method we have used to express the IgLON family proteins, CEPU1 and OBCAM, as recombinant Fc chimeric proteins in stably transfected mouse J558L myeloma cells. The use of this cell line provided the opportunity to maximize protein production, as it secretes antibodies in large quantities and can be grown to high density in small volumes of culture medium. Isolation of recombinant OBCAMFc from the adherent COS7 cell line suggested a minimum level of expression of 0.07 mg OBCAMFc/100 mL culture medium, while the J558L cell line expressed OBCAMFc at approximately 11.4 mg/100 mL culture medium. Purification of IgLON-Fc expressed by J558L cells was simpler than purification from COS7 cells because of the lower volume of culture medium generated. Furthermore, contamination of J558L expressed IgLONFc with bovine IgG from the culture medium was negligible. The method presented, which utilizes a commercially available small-scale bioreactor, provides the nonspecialist protein expression laboratory with the means to produce recombinant proteins quickly and easily in milligram quantities.

Animals↗
PubMed2002

Will a vaccine to nasopharyngeal carcinoma retain orphan status?

Epstein-Barr virus (EBV) infection is associated with infectious mononucleosis (IM) and with a number of human malignancies including nasopharyngeal carcinoma (NPC), Hodgkin's disease (HD) and immunoblastic lymphoma (IL). Their potential for immunotherapeutic treatment by cytotoxic T cells (CTL) is dependent on the degree of EBV antigen expression, with the best prospect revolving around IM where a vaccine is under development and IL of transplant patients where adoptive transfer of in vitro reactivated CTL has already been demonstrated to be effective. The opportunities for effective immunotherapy in the treatment of NPC is reduced since the available targets are limited to relatively non-immunogenic proteins. Perhaps more importantly, the development of immunotherapeutics is not considered a realistic commercial proposition. The best chance of developing an effective vaccine is to exploit the similarities in phenotype between HD and NPC since a vaccine to the former is likely to have more commercial appeal.

Antibody Formation↗
PubMed2001

Asymptomatic primary Epstein-Barr virus infection occurs in the absence of blood T-cell repertoire perturbations despite high levels of systemic viral load.

Primary infection with the human herpesvirus, Epstein-Barr virus (EBV), may result in subclinical seroconversion or may appear as infectious mononucleosis (IM), a lymphoproliferative disease of variable severity. Why primary infection manifests differently between patients is unknown, and, given the difficulties in identifying donors undergoing silent seroconversion, little information has been reported. However, a longstanding assumption has been held that IM represents an exaggerated form of the virologic and immunologic events of asymptomatic infection. T-cell receptor (TCR) repertoires of a unique cohort of subclinically infected patients undergoing silent infection were studied, and the results highlight a fundamental difference between the 2 forms of infection. In contrast to the massive T-cell expansions mobilized during the acute symptomatic phase of IM, asymptomatic donors largely maintain homeostatic T-cell control and peripheral blood repertoire diversity. This disparity cannot simply be linked to severity or spread of the infection because high levels of EBV DNA were found in the blood from both types of acute infection. The results suggest that large expansions of T cells within the blood during IM may not always be associated with the control of primary EBV infection and that they may represent an overreaction that exacerbates disease.

Antibodies, Viral↗
PubMed2001

Targeting of EBNA1 for rapid intracellular degradation overrides the inhibitory effects of the Gly-Ala repeat domain and restores CD8+ T cell recognition.

Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) includes a unique glycine-alanine repeat domain that inhibits the endogenous presentation of cytotoxic T lymphocyte (CTL) epitopes through the class I pathway by blocking proteasome-dependent degradation of this antigen. This immune evasion mechanism has been implicated in the pathogenesis of EBV-associated diseases. Here, we show that cotranslational ubiquitination combined with N-end rule targeting enhances the intracellular degradation of EBNA1, thus resulting in a dramatic reduction in the half-life of the antigen. Using DNA expression vectors encoding different forms of ubiquitinated EBNA1 for in vivo studies revealed that this rapid degradation, remarkably, leads to induction of a very strong CTL response to an EBNA1-specific CTL epitope. Furthermore, this targeting also restored the endogenous processing of HLA class I-restricted CTL epitopes within EBNA1 for immune recognition by human EBV-specific CTLs. These observations provide, for the first time, evidence that the glycine-alanine repeat-mediated proteasomal block on EBNA1 can be reversed by specifically targeting this antigen for rapid degradation resulting in enhanced CD8+ T cell-mediated recognition in vitro and in vivo.

Alanine↗
PubMed2001

Contrasting Epstein-Barr virus-specific cytotoxic T cell responses to HLA A2-restricted epitopes in humans and HLA transgenic mice: implications for vaccine design.

This study investigates the hierarchy of cytotoxic T cell (CTL) responses to twelve HLA A2-restricted epitopes from the latent, lytic and structural proteins of Epstein-Barr virus (EBV) in acute infectious mononucleosis and in healthy seropositive donors and the relative immunogenecity of these epitopes in transgenic mice. Responses to the lytic epitope were uniformly strong in all healthy seropositive individuals and acute infectious mononucleosis donors while moderate or low responses were observed to the latent and structural epitopes, respectively in both groups studied. In contrast, when HLA A2/Kb transgenic mice were immunised with these peptide epitopes, CTL responses were observed to all epitopes with a maximal response to the epitopes within the structural proteins and low to moderate responses to the latent epitopes. This hierarchy of CTL responses in mice was also reflected in an MHC stabilisation analysis. These contrasting CTL responses in humans following natural infection compared to the immunogenicity of these epitopes and their ability to stabilise MHC may need to be considered when designing an EBV vaccine.

Acute Disease↗
PubMed2001

The immunology of Epstein-Barr virus infection.

Epstein-Barr virus is a classic example of a persistent human virus that has caught the imagination of immunologists, virologists and oncologists because of the juxtaposition of a number of important properties. First, the ability of the virus to immortalize B lymphocytes in vitro has provided an antigen presenting cell in which all the latent antigens of the virus are displayed and are available for systematic study. Second, the virus presents an ideal system for studying the immune parameters that maintain latency and the consequences of disturbing this cell-virus relationship. Third, this wealth of immunological background has provided a platform for elucidating the role of the immune system in protection from viral-associated malignancies of B cell and epithelial cell origin. Finally, attention is now being directed towards the development of vaccine formulations which might have broad application in the control of human malignancies.

Animals↗
PubMed2001

Identification and characterization of CEPU-Se-A secreted isoform of the IgLON family protein, CEPU-1.

CEPU-1/Neurotrimin is a neuronal glycoprotein thought to play a role in axon guidance and cell-cell recognition. It is a member of the IgLON family, has three C2 domains, and is attached to the plasma membrane by a GPI-anchor. We report here the characterisation of an alternatively-spliced isoform of CEPU-1 that is secreted. This isoform, termed CEPU-Se, is coexpressed with CEPU-1 in retina, cerebellum, and DRG neurons. In the cerebellum CEPU-1/CEPU-Se is expressed predominantly on granule cells and in the molecular layer. Divalent but not monovalent CEPU-Se interacts with CEPU-1 and other IgLONs, suggesting that the ability of CEPU-Se to modify the activity of the IgLON family may require an additional cofactor. CEPU-Se does not support the outgrowth of DRG neurons or the extension of established growth cones; however, neurite outgrowth on laminin is unaffected by CEPU-Se. Our data suggest that CEPU-Se may act to modulate the ability of CEPU-1, LAMP, and OBCAM to influence neurite outgrowth.

Alternative Splicing↗
PubMed2001

Immunization with tumor-associated epitopes fused to an endoplasmic reticulum translocation signal sequence affords protection against tumors with down-regulated expression of MHC and peptide transporters.

Treatment of human cancers with an inherent antigen-processing defect due to a loss of peptide transporters (TAP-1 and TAP-2) and/or MHC class I antigen expression remains a considerable challenge. There is now an increasing realization that tumor cells with down-regulated expression of TAP and/or MHC class I antigens display strong resistance to cytotoxic T lymphocyte (CTL)-mediated immune control, and often fail to respond to the conventional immunotherapeutic protocols based on active immunization with tumor-associated epitopes (TAE) or adoptive transfer of tumor-specific T cells. In the present study, we describe a novel approach based on immunization with either genetically modified tumor cells or naked DNA vectors encoding TAE fused to an endoplasmic reticulum (ER) signal sequence (ER-TAE) which affords protection against challenge by melanoma cells with down-regulated expression of TAP-1/2 and MHC class I antigens. In contrast, animals immunized with a vaccine based on TAE alone showed no protection against tumor challenge. Although MHC-peptide tetramer analysis showed a similar frequency of antigen-specific CTL in both ER-TAE- and TAE-immunized mice, functional analysis revealed that CTL activated following immunization with ER-TAE displayed significantly higher avidity for TAE when compared to animals immunized with the TAE alone. These observations provide a new strategy in anti-cancer vaccine design that allows activation of a highly effective and well-defined CTL response against tumors with down-regulated expression of TAP and MHC class I antigens.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Co-localisation, heterophilic interactions and regulated expression of IgLON family proteins in the chick nervous system.

The chick glycoprotein GP55 has been shown to inhibit the growth and adhesion of DRG and forebrain neurons. GP55 consists of several members of the IgLON family, a group of glycoproteins including LAMP, OBCAM, CEPU-1 (chick)/neurotrimin (rat) and neurotractin (chick)/kilon (rat) thought to play a role in the guidance of growing axons. IgLONs belong to the Ig superfamily and have three C2 domains and a glycosyl phosphatidylinositol anchor which tethers them to the neuronal plasma membrane. We have now completed the deduced amino acid sequence for two isoforms of chicken OBCAM and used recombinant LAMP, OBCAM and CEPU-1 to raise antisera specific to these three IgLONs. LAMP and CEPU-1 are co-expressed on DRG and sympathetic neurons, while both overlapping and distinct expression patterns for LAMP, OBCAM and CEPU-1 are observed in retina. Analysis of IgLON mRNA expression reveals that alternatively spliced forms of LAMP and CEPU-1 are developmentally regulated. In an attempt to understand how the IgLONs function, we have begun to characterise their molecular interactions. LAMP and CEPU-1 have already been shown to interact homophilically. We now confirm that OBCAM will bind homophilically and also that LAMP, OBCAM and CEPU-1 will interact heterophilically with each other. We propose that IgLON activity will depend on the complement of IgLONs expressed by each neuron.

Amino Acid Sequence↗
PubMed2000

Differential splicing of antigen-encoding RNA reduces endogenous epitope presentation that regulates the expansion and cytotoxicity of T cells.

The activation of CTLs is dependent on the recognition of MHC-bound peptide present on the surface of APCs. We give evidence in this study that differential splicing of Ag-encoding RNA can decrease the antigenic dose in APCs and regulate the recall of human memory CTLs. Differential splicing of RNA that encoded an immunodominant HLA-B8-restricted CTL epitope of EBV reduced the functional presentation of this epitope, and consequently the in vitro expansion and activity of CTLs, as measured by MHC/peptide-tetramer staining and cytotoxicity assays. The reduced activity of the stimulated CTLs was not only due to lower numbers of Ag-specific CTLs but, surprisingly, was also characterized by decreased cytotoxicity of the CTLs to target cells presenting limiting amounts of the peptide epitope. As indicated by TCR repertoire analysis, the reduction in CTL activity was not caused by stimulation of distinct populations of TCR clonotypes. This study demonstrates how a common eukaryotic posttranscriptional mechanism of gene regulation can modulate the endogenous presentation of Ag and ultimately contribute to the fine tuning of immunological memory cells, which are important in the fight against pathogens and tumors and in autoimmunity.

Alternative Splicing↗
PubMed1999

Activation and adoptive transfer of Epstein-Barr virus-specific cytotoxic T cells in solid organ transplant patients with posttransplant lymphoproliferative disease.

The treatment of Epstein-Barr virus (EBV)-associated lymphoproliferative disease (PTLD) in EBV seronegative solid organ transplant recipients who acquire their EBV infection after engraftment poses a considerable challenge because of underlying immunosuppression that inhibits the virus-specific cytotoxic T cell (CTL) response in vivo. We have developed a protocol for activating autologous EBV-specific CTL lines from these patients and show their potential use for immunotherapy against PTLD in solid organ transplant patients. Peripheral blood mononuclear cells from a panel of solid organ transplant recipients with and without active PTLD were used to assess EBV-specific memory CTL responses. The activation protocol involved cocultivation of peripheral blood mononuclear cells with an autologous lymphoblastoid cell line under conditions that favored expansion of virus-specific CTL and hindered the proliferation of allospecific T cells. These CTL consistently showed (i) strong EBV-specificity, including reactivity through defined epitopes in spite of concurrent immunosuppressive therapy, and (ii) no alloreactivity toward donor alloantigens. More importantly, adoptive transfer of these autologous CTLs into a single patient with active PTLD was coincident with a very significant regression of the PTLD. These results demonstrate that a potent EBV-specific memory response can be expanded from solid organ recipients who have acquired their primary EBV infection under high levels of immunosuppressive therapy and that these T cells may have therapeutic potential against PTLD.

Adoptive Transfer↗
PubMed1999

Developing immunotherapeutic strategies for the control of Epstein-Barr virus-associated malignancies.

Epstein-Barr virus (EBV) infection is associated with various physical human malignancies. The potential for immunotherapeutic treatment by cytotoxic T cells (CTL) depends on the degree of EBV-antigen expression, with the best prospect revolving around the immunoblastic lymphomas of organ transplant patients in which adoptive transfer of in vitro reactivated CTL has already been demonstrated to be effective. Opportunities for effective immunotherapy in the treatment of nasopharyngeal carcinoma (NPC) is reduced because the available targets are limited to relatively nonimmunogenic proteins. However, analysis of NPC cells has revealed normal expression of the major histocompatibility complex (MHC)-encoded peptide transporters TAP-1 and TAP-2, together with high levels of human leukocyte antigen (HLA) class I alleles on the cell surface. Burkitt's lymphoma (BL) displays downregulated expression of MHC class I and TAP-1 and TAP-2 proteins, whereas viral antigen expression is limited to a protein incapable of processing class I CTL epitopes. It therefore seems likely that effective treatment of BL will revolve around protocols designed to reverse its undifferentiated phenotype.

AIDS-Related Opportunistic Infections↗
PubMed1999

cDNA cloning of the CEPUS, a secreted type of neural glycoprotein belonging to the immunoglobulin-like opioid binding cell adhesion molecule (OBCAM) subfamily.

GP55 is a family of glycoproteins distributed predominantly in the nervous system, and its previously characterized members, including the GP55A (EMBL Y08170) and E19S (EMBL Y08171) reveal a typical glycosyl phosphatidyl inositol (GPI)-anchored pattern for membrane proteins. CEPUS identified in this study appeared to represent the third member of GP55. This 3.2 kb long complete cDNA clone from the chicken brain exhibited 3 Ig-like domains. The open reading frame of CEPUS contains 313 amino acids, which can encode a 31.7 kDa core protein (pI 5.75) for the mature form. The signal peptide cleavage site was predicted at Gln25. The structural features of the CEPUS cDNA sequence represented a soluble counterpart to the recently identified cerebellar Purkinje cell specific antigen, CEPU-1. The sequence difference between CEPU-1 and CEPUS was only found in the C-terminus in which the CEPUS lacked the GPI-anchored binding site. It displays significant sequence homology to GP55-related molecules, including OBCAM, GP55A, E19S/LAMP, neurotrimin, and CEPU-1, which are all membrane attached types. The absence of the hydrophobic tail sequence in CEPUS may, therefore, suggest that CEPUS would represent the first identified secreted member in this group of genes. We defined that this molecule forms the opioid-binding cell adhesion molecule (OBCAM) subfamily in the molecular phylogeny. Structurally, these molecules represent acidic proteins (pI 5.47-6.09). Six cysteins, as well as 5 Asn-linked potential glycosylation sites were evolutionary-conserved, suggesting that this OBCAM subfamily resembles immunoglobulin-like and highly glycosylated molecules. The presence of CEPUS would probably suggest to us that the spatial/local expression of the CEPU gene may provide a favorable route for migrating CEPU-positive population of neurons to generate a neuron-specific guidance in developing neurons in vivo.

Amino Acid Sequence↗
PubMed1999

Direct alloreactivity by human cytotoxic T lymphocytes can Be inhibited by altered peptide ligand antagonism.

Alloreactive T lymphocytes that respond directly to foreign major histocompatibility complex (MHC) molecules and bound peptide are known to be central mediators of graft-versus-host disease (GVHD) and allograft rejection. We have recently identified a peptide from the human protein, cytochrome P450 (isotypes IIC9, 10, or 18), that is recognized in association with human leukocyte antigen (HLA) B*3501 by alloreactive cytotoxic T lymphocytes (CTLs). These CTLs with this specificity were isolated from several unrelated individuals and were found to express a common T-cell receptor (TCR). Synthetic analogs of the cytochrome P450 peptide were generated by introducing single amino acid substitutions at putative TCR contact positions. Four altered peptide ligands were powerful competitive antagonists of these CTL clones, reducing lysis levels of target cells expressing the alloantigen HLA B*3501 by over 80%. This first demonstration that it is possible to suppress CTL alloreactivity with structural variants of allodeterminants raises the prospect that such TCR antagonists could be exploited within the clinical arena to specifically modulate GVHD and allograft rejection.

Amino Acid Substitution↗