The future is in the numbers: the power of predictive analysis in the biomedical educational environment.

Biomedical programs have a potential treasure trove of data they can mine to assist admissions committees in identification of students who are likely to do well and help educational committees in the identification of students who are likely to do poorly on standardized national exams and who may need remediation. In this article, we provide a step-by-step approach that schools can utilize to generate data that are useful when predicting the future performance of current students in any given program. We discuss the use of linear regression analysis as the means of generating that data and highlight some of the limitations. Finally, we lament on how the combination of these institution-specific data sets are not being fully utilized at the national level where these data could greatly assist programs at large.

Twelve tips for facilitating team-based learning.

BACKGROUND: Team-based learning (TBL) has become a more commonly recognized and implemented pedagogical approach in curricula of numerous disciplines. The desire to place more autonomy on the student and spend less in-class time delivering content has resulted in complete or partial adoption of this style of learning in many educational settings. AIM: Provide faculty with tools that foster a well facilitated and interactive TBL learning environment. METHODS: We examined the published literature in the area of facilitation - specifically in TBL environments, and explored learning theories associated with team learning and our own experiences to create these facilitation tips. RESULTS: We created 12 tips for TBL facilitation designed to assist faculty to achieve an effective and engaging TBL learning environment. CONCLUSIONS: Applying these twelve tips while facilitating a TBL classroom session will help to ensure maximal participation and optimal learning in a safe yet stimulating environment.

Tumor necrosis factor receptor 1 associates with CD137 ligand and mediates its reverse signaling.

Reverse signaling through CD137 ligand (CD137L) potently activates monocytes. However, the underlying mechanism is not well elucidated. This study provides evidence that tumor necrosis factor receptor 1 (TNFR1) acts as a coreceptor for CD137L and mediates CD137L signaling. CD137L colocalizes with TNFR1 on the plasma membrane and binds directly to TNFR1 via its extracellular domain. Using the human monocytic THP-1 cell line, we demonstrate that engagement of CD137L by recombinant CD137 protein promotes cell adhesion, apoptosis, expression of CD14, and production of IL-8 and tumor necrosis factor (TNF). Concomitantly, the expression of TNFR1 protein is down-regulated in response to CD137L activation, due to enhanced extracellular release and internalization of TNFR1. Activation of TNFR1 by TNF protein additively augments CD137L-induced IL-8 expression. Conversely, inhibition of TNFR1 activity by a TNFR1-neutralizing antibody inhibits CD137L-mediated cell adhesion, cell death, CD14 expression, and IL-8 production. Taken together, these data show that TNFR1 associates with CD137L and is required for CD137L reverse signaling.

Inhibition of proliferation and induction of apoptosis in multiple myeloma cell lines by CD137 ligand signaling.

BACKGROUND: Multiple myeloma (MM) is a malignancy of terminally-differentiated plasma cells, and the second most prevalent blood cancer. At present there is no cure for MM, and the average prognosis is only three to five years. Current treatments such as chemotherapy are able to prolong a patient's life but rarely prevent relapse of the disease. Even hematopoietic stem cell transplants and novel drug combinations are often not curative, underscoring the need for a continued search for novel therapeutics. CD137 and its ligand are members of the Tumor Necrosis Factor (TNF) receptor and TNF superfamilies, respectively. Since CD137 ligand cross-linking enhances proliferation and survival of healthy B cells we hypothesized that it would also act as a growth stimulus for B cell cancers. METHODOLOGY/PRINCIPAL FINDINGS: Proliferation and survival of B cell lymphoma cell lines were not affected or slightly enhanced by CD137 ligand agonists in vitro. But surprisingly, they had the opposite effects on MM cells, where CD137 ligand signals inhibited proliferation and induced cell death by apoptosis. Furthermore, secretion of the pro-inflammatory cytokines, IL-6 and IL-8 were also enhanced in MM but not in non-MM cell lines in response to CD137 ligand agonists. The secretion of these cytokines in response to CD137 ligand signaling was consistent with the observed activation of the classical NF-kappaB pathway. We hypothesize that the induction of this pathway results in activation-induced cell death, and that this is the underlying mechanism of CD137-induced MM cell death and growth arrest. CONCLUSIONS/SIGNIFICANCE: These data point to a hitherto unrecognized role of CD137 and CD137 ligand in MM cell biology. The selective inhibition of proliferation and induction of cell death in MM cells by CD137 ligand agonists may also warrant a closer evaluation of their therapeutic potential.

Use of phage display to isolate specific human monoclonal antibody fragments against a potential target for multiple myeloma.

INTRODUCTION: Multiple myeloma (MM), a malignancy of plasma cells, accounts for 10% of all haematological malignancies and is currently incurable. Although it can be treated, the disease tends to relapse after several years and becomes increasingly resistant to conventional therapy. Investigations into using humoral therapy for MM are now underway with a view that novel therapeutic agents may provide a more targeted therapy for MM. MATERIALS AND METHODS: Here, phage display, a faster and more efficient method compared to classical hybridoma fusion technology, was used as a proof-of-concept to isolate several single-chain Fragment variables (scFv) against Ku86. RESULTS: Anti-Ku86 polyclonal scFvs biopanning was successful where third round scFvs (A(450)~1.1) showed a 1/3 increase in binding as compared to the fi rst round scFvs (A(450)~0.4) with 100 microg/mL of antigen (purified human Ku86). Subsequent selection and verification of monoclonal antibodies using third round biopanning revealed 4 good affinity binding clones ranging from A(450)~0.1 to A450~0.15 on 12.5 microg/mL of antigen as compared to low binders (A(450)~0.07) and these antibodies bind to Ku86 in a specific and dose-dependent manner. Comparative studies were also performed with commercially available murine antibodies and results suggest that 2 of the clones may bind close to the following epitopes aa506-541 and aa1-374. CONCLUSIONS: These studies using phage display provide an alternative and viable method to screen for antibodies quickly and results show that good affinity antibodies against Ku86 have been successfully isolated and they can be used for further studies on MM and form the basis for further development as anti-cancer therapeutic agents.

Association of Epstein-Barr virus with nasopharyngeal carcinoma and current status of development of cancer-derived cell lines.

It is well known that the Epstein-Barr virus (EBV) contributes directly to tumourigenesis in nasopharyngeal carcinoma (NPC), primarily in the undifferentiated form of NPC (WHO type III; UNPC or UC), which is commonly found in South East Asia. Unfortunately, research in NPC has been severely hampered by the lack of authentic EBV-positive (EBV+) human NPC cell lines for study. Since 1975, there have been more than 20 reported NPC cell lines. However, many of these NPC-derived cell lines do not express EBV transcripts in long-term culture, and therefore that finding may dispute the fundamental theory of NPC carcinogenesis. In fact, currently only one EBV+ human NPC cell line (C-666) in long-term culture has been reported. Hence, most of the NPC cell lines may not be representative of the disease itself. In order to better understand and treat NPC, there is an urgent need to develop more EBV+ human NPC cell lines. In this review, we discuss the authenticity of existing NPC cell lines and the impact of our understanding of NPC biology on the treatment of the disease and the relationship of EBV to NPC in the context of cell lines.

Use of ultraviolet-light irradiated multiple myeloma cells as immunogens to generate tumor-specific cytolytic T lymphocytes.

BACKGROUND: As the eradication of tumor cells in vivo is most efficiently performed by cytolytic T lymphocytes (CTL), various methods for priming tumor-reactive lymphocytes have been developed. In this study, a method of priming CTLs with ultraviolet (UV)-irradiated tumor cells, which results in termination of tumor cell proliferation, apoptosis, as well as upregulation of heat shock proteins (HSP) expression is described. METHODS: Peripheral blood mononuclear cells (PBMC) were primed weekly with UV-irradiated or mitomycin-treated RPMI 8226 multiple myeloma cells. Following three rounds of stimulation over 21 days, the lymphocytes from the mixed culture conditions were analyzed for anti-MM cell reactivity. RESULTS: By day 10 of cultures, PBMCs primed using UV-irradiated tumor cells demonstrated a higher percentage of activated CD8+/CD4- T lymphocytes than non-primed PBMCs or PBMCs primed using mitomycin-treated MM cells. Cytotoxicity assays revealed that primed PBMCs were markedly more effective (p < 0.01) than non-primed PBMCs in killing RPMI 8226 MM cells. Surface expression of glucose regulated protein 94 (Grp94/Gp96) and Grp78 were both found to be induced in UV-treated MM cells. CONCLUSION: Since, HSP-associated peptides are known to mediate tumor rejection; these data suggest that immune-mediated eradication of MM cells could be elicited via a UV-induced HSP process. The finding that the addition of 17-allylamide-17-demethoxygeldanamycin (17AAG, an inhibitor of HSP 90-peptide interactions) resulted in decreased CTL-induced cytotoxicity supported this hypothesis. Our study, therefore, provides the framework for the development of anti-tumor CTL cellular vaccines for treating MM using UV-irradiated tumor cells as immunogens.

Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells.

BACKGROUND: Truncated variants of Ku86 protein have previously been detected in 86% to 100% of freshly isolated patient multiple myeloma (MM) cells. Since, the Ku70/Ku86 heterodimer functions as the regulatory subunit of the DNA repair enzyme, DNA-dependent protein kinase, we have been interested in the altered expression and function of Ku86 variant (Ku86v) proteins in genome maintenance of MM. RESULTS: Although, a number of studies have suggested that truncated forms of Ku proteins could be artificially generated by proteolytic degradation in vitro in human lymphocytes, we now show using whole cell immunoblotting that the RPMI-8226 and SGH-MM5 human MM cell lines consistently express full-length Ku86 as well as a 69-kDa Ku86v; a C-terminus truncated 69-kDa variant Ku86 protein. In contrast, Ku86v proteins were not detected in the freshly isolated lymphocytes as was previously reported. Data also indicates that the Ku86v was not generated as a result of carbohydrate modification but that serine proteases may act on the full-length form of the protein. CONCLUSION: These data confirm that MM cells contain bona fide Ku86v proteins that were generated intracellularly by a post-transcriptional mechanism, which required proteolytic processing.

Detection and quantification of the abelson tyrosine kinase domains of the BCR-ABL gene translocation in chronic myeloid leukaemia using genomic quantitative real-time polymerase chain reaction.

INTRODUCTION: Since undetectable BCR-ABL mRNA transcription does not always indicate eradication of the Ph+ CML clone and since transcriptionally silent Ph+ CML cells exist, quantitation by genomic PCR of bcr-abl genes can be clinically useful. Furthermore, hotspot mutations in the Abelson tyrosine kinase (ABLK) domain of the bcr-abl gene translocation in Philadelphia chromosome-positive (Ph+) chronic myeloid leukaemia (CML) cells confer resistance on the specific kinase blocking agent, STI571. MATERIALS AND METHODS: Genomic DNA from K562, CESS and patient CML cells were amplified using rapid cycle quantitative real-time polymerase chain reaction for the gene regions spanning the mutation hotspots. In assays for ABLK exons 4 or 6, exonic or intronic PCR primers were used. RESULTS: We show that separation of cycle threshold (CT) values for log-fold amplicon quantification was 2.9 cycles for ABLK exon 4, and 3.8 cycles for exon 6 with rapid amplification times. K562 CML cells were found to have a approximately 2 log-fold ABLK gene amplification. In contrast, patient CML cells had CT differences of 2.2 for both exon, suggesting that there was no significant ABLK gene amplification. DNA sequencing confirmed that neither K562 nor patient CML cells contained ABLK hotspot mutations. Messenger RNA transcription analysis permitted the assessment of BCR-ABL transcription, which was qualitatively correlated to genomic amplification. CONCLUSIONS: This novel Q-PCR assay was found to have high fidelity and legitimacy, and potentially useful for monitoring minimal residual disease, transcriptionally silent Ph+ CML cells, and bcr-abl gene amplification.

The biology of Ku and its potential oncogenic role in cancer.

Ku is a heterodimeric protein made up of two subunits, Ku70 and Ku80. It was originally identified as an autoantigen recognized by the sera of patients with autoimmune diseases. It is a highly versatile regulatory protein that has been implicated in multiple nuclear processes, e.g., DNA repair, telomere maintenance and apoptosis. Accordingly, Ku is thought to play a crucial role in maintenance of chromosomal integrity and cell survival. Recent reports suggest that there is a positive relationship between Ku and the development of cancer, making Ku an important candidate target for anticancer drug development. Specifically, prior studies suggest that a delicate balance exists in Ku expression, as overexpression of Ku proteins promotes oncogenic phenotypes, including hyperproliferation and resistance to apoptosis; whereas deficient or low expression of Ku leads to genomic instability and tumorigenesis. Such observations through various experimental models indicate that Ku may act as either a tumor suppressor or an oncoprotein. Hence, understanding the link between the various functions of Ku and the development of cancer in different cell systems may help in the development of novel anticancer therapeutic agents that target Ku. These studies may also increase our understanding of how Ku autoantibodies are generated in autoimmune diseases.

Defective B cell responses in the absence of SH2D1A.

More than half of patients with X-linked lympho-proliferative disease, which is caused by a defect in the intracellular adapter protein SH2D1A, suffer from an extreme susceptibility to Epstein-Barr virus. One-third of these patients, however, develop dysgammaglobulenemia without an episode of severe mononucleosis. Here we show that in SH2D1A(-/-) mice, both primary and secondary responses of all Ig subclasses are severely impaired in response to specific antigens. Because germinal centers were absent in SH2D1A(-/-) mice upon primary immunization, and because SH2D1A was detectable in wt germinal center B cells, we examined whether SH2D1A(-/-) B cell functions were impaired. Using the adoptive cotransfer of B lymphocytes from hapten-primed SH2D1A(-/-) mice with CD4(+) T cells from primed wt mice into irradiated wt mice provided evidence that signal transduction events controlled by SH2D1A are essential for B cell activities resulting in antigen specific IgG production. Defects in naive SH2D1A(-/-) B cells became evident upon cotransfer with non-primed wt CD4(+) cells into Rag2(-/-) recipients. Thus, both defective T and B cells exist in the absence of SH2D1A, which may explain the progressive dysgammaglobulinemia in a subset of X-linked lympho-proliferative disease patients without involvement of Epstein-Barr virus.

Expression of the SH2D1A gene is regulated by a combination of transcriptional and post-transcriptional mechanisms.

The SH2D1A gene, which is altered or deleted in patients with X-linked lymphoproliferative disease, encodes the small protein SAP (for SLAM-associated protein) that is expressed in T and NK cells. A 22-bp fragment in close proximity to an initiator-like site was defined as the basal promoter of mouse SH2D1A, and a highly homologous 33-bp segment was defined as the human basal promoter. When an Ets consensus site was mutated, no reporter activity was detectable. Gel mobility supershift assays revealed that the two transcription factors Ets-1 and Ets-2 bind to the human and mouse sequences. The involvement of Ets-1 and Ets-2 in expression of SH2D1A was functionally confirmed by overexpression studies of their dominant-negative forms. We also found that SH2D1A mRNA decays very rapidly in mouse T cells, and its 3' untranslated region (UTR) has RNA-destabilizing activity in transfection studies with reporter/3' UTR constructs. As judged by RNA-gel mobility shift assays, this rapid degradation of SH2D1A mRNA was due to a balance in binding of the factors AUF1 and HuR to its 3' UTR. Although the SH2D1A mRNA level decreased upon triggering of the T cell receptor (TCR), the RNA degradation rate itself was not altered by TCR engagement.

Heat shock proteins: to present or not, that is the question.

The contribution of major histocompatibility complex (MHC) I and II to the adaptive immune response has been well documented. In 1996, Peter Doherty and Rolf Zinkernagel were awarded the Nobel Prize, for their fundamental observations concerning the genetic elements involved in specific antigen (Ag) recognition. These elements encode molecules that present self and non-self peptide fragments to both CD4+ and CD8+ cytolytic T lymphocytes (CTL). The recognition by Srivastava and coworkers that heat shock proteins (HSPs) might also present Ag in chemically induced sarcomas brought about many new questions concerning the central dogma of Ag processing and presentation. HSPs, in particular glucose-regulated peptide 94 (GRP94), HSP70 and to a lesser extent HSP90, bind peptides that are immunogenic in vitro and in vivo. There is mounting evidence that these HSP-peptide complexes provide alternative Ag-specific recognition in many systems. Whether a separate genetic program evolved in addition to MHC that increases the antigenic repertoire of the cell or if this newly observed function of HSP is predominantly a laboratory-based phenomena and/or a normal chaperone function of this family of proteins remains to be answered. Nevertheless, there are clinical therapeutic strategies that involve HSP-derived peptides isolated from various tumors that look extremely promising.

The cell surface receptor SLAM controls T cell and macrophage functions.

Signaling lymphocyte activation molecule (SLAM), a glycoprotein expressed on activated lymphocytes and antigen-presenting cells, has been shown to be a coregulator of antigen-driven T cell responses and is one of the two receptors for measles virus. Here we show that T cell receptor-induced interleukin (IL)-4 secretion by SLAM(-/-) CD4(+) cells is down-regulated, whereas interferon gamma production by CD4(+) T cells is only slightly up-regulated. Although SLAM controls production of IL-12, tumor necrosis factor, and nitric oxide in response to lipopolysaccharide (LPS) by macrophages, SLAM does not regulate phagocytosis and responses to peptidoglycan or CpG. Thus, SLAM acts as a coreceptor that regulates signals transduced by the major LPS receptor Toll-like receptor 4 on the surface of mouse macrophages. A defective macrophage function resulted in an inability of SLAM(-/-) C57Bl/6 mice to remove the parasite Leishmania major. We conclude that the coreceptor SLAM plays a central role at the interface of acquired and innate immune responses.

The role of SAP in murine CD150 (SLAM)-mediated T-cell proliferation and interferon gamma production.

CD150 (signaling lymphocyte activation molecule [SLAM]) is a self-ligand cell surface glycoprotein expressed on T cells, B cells, macrophages, and dendritic cells. To further explore the role of CD150 signaling in costimulation and T(H)1 priming we have generated a panel of rat antimouse CD150 monoclonal antibodies. CD150 cell surface expression is up-regulated with rapid kinetics in activated T cells and lipopolysaccharide/interferon gamma (IFN-gamma)-activated macrophages. Anti-CD150 triggering induces strong costimulation of T cells triggered through CD3. DNA synthesis of murine T cells induced by anti-CD150 is not dependent on SLAM-associated protein (SAP, SH2D1A), because anti-CD150 induces similar levels of DNA synthesis in SAP(-/-) T cells. Antibodies to CD150 also enhance IFN-gamma production both in wild-type and SAP(-/-) T cells during primary stimulation. The level of IFN-gamma production is higher in SAP(-/-) T cells than in wild-type T cells. Anti-CD150 antibodies also synergize with interleukin 12 (IL-12) treatment in up-regulation of IL-12 receptor beta(2) mRNA during T(H)1 priming, and inhibit primary T(H)2 polarization in an IFN-gamma-dependent fashion. Cross-linking CD150 on CD4 T cells induces rapid serine phosphorylation of Akt/PKB. We speculate that this is an important pathway contributing to CD150-mediated T-cell proliferation.