Serum Autoantibodies against LRDD, STC1, and FOXA1 as Biomarkers in the Detection of Ovarian Cancer.

Purpose: This study is aimed at evaluating serum autoantibodies against four tumor-associated antigens, including LRDD, STC1, FOXA1, and EDNRB, as biomarkers in the immunodiagnosis of ovarian cancer (OC). Methods: The autoantibodies against LRDD, STC1, FOXA1, and EDNRB were measured using an enzyme-linked immunosorbent assay (ELISA) in 94 OC patients and 94 normal healthy controls (NHC) in the research group. In addition, the diagnostic values of different autoantibodies were validated in another independent validation group, which comprised 136 OC patients, 136 NHC, and 181 patients with benign ovarian diseases (BOD). Results: In the research group, autoantibodies against LRDD, STC1, and FOXA1 had higher serum titer in OC patients than NHC (P < 0.001). The area under receiver operating characteristic curves (AUCs) of these three autoantibodies were 0.910, 0.879, and 0.817, respectively. In the validation group, they showed AUCs of 0.759, 0.762, and 0.817 and sensitivities of 49.3%, 42.7%, and 48.5%, respectively, at specificity over 90% for discriminating OC patients from NHC. For discriminating OC patients from BOD, they showed AUCs of 0.718, 0.729, and 0.814 and sensitivities of 47.1%, 39.0%, and 51.5%, respectively, at specificity over 90%. The parallel analyses demonstrated that the combination of anti-LRDD and anti-FOXA1 autoantibodies achieved the optimal diagnostic performance with the sensitivity of 58.1% at 87.5% specificity and accuracy of 72.8%. The positive rate of the optimal autoantibody panel improved from 62.4% to 87.1% when combined with CA125 in detecting OC patients. Conclusion: Serum autoantibodies against LRDD, STC1, and FOXA1 have potential diagnostic values in detecting OC.

Variation at DENND1B and Asthma on the Island of Tristan da Cunha.

A high prevalence of asthma has been documented among the inhabitants of Tristan da Cunha, an isolated island in the South Atlantic. The population derives from just 28 founders. We performed lung function testing, including methacholine inhalation challenge, allergen skin prick testing, and collected DNA from essentially all of the current island population (269 individuals), and genotyped a panel of 43 single-nucleotide polymorphisms (SNPs) reported as associated with asthma and atopy. We carried out a mixed-model association analysis using the known pedigree. There were 96 individuals diagnosed as asthmatic (36%), and heritability estimates were similar to those from nonisolated population samples (multifactorial threshold model, h2 = 48%). The first component from a genetic principal components analysis using the entire SNP panel was nonlinearly associated with asthma, with the maximum risk to those intermediate to reference (Human Genome Diversity Project) European and African samples means. The single most strongly associated SNP was rs2786098 (p = 5.5 × 10-5), known to regulate the gene DENND1B. This explained approximately one-third of the trait heritability, with an allelic odds ratio for the A allele of 2.6. Among A/A carriers, 10 out of 12 individuals were asthmatic. The rs2786098*A variant was initially reported to decrease the risk of childhood (atopic) asthma in European but slightly increase the risk in African-descended populations, and does significantly alter Th2 cell function. Despite an absence of overall association with this variant in recent asthma genome wide association studies meta-analyses, an effect may exist on the particular genetic background of the Tristan da Cunha population.

Removal of syndecan-1 promotes TRAIL-induced apoptosis in myeloma cells.

Syndecan is the major transmembrane proteoglycan in cells. Of the four syndecans, syndecan-1 is the dominant form expressed in multiple myeloma and is an indicator of poor prognosis. In the current study, we observed that early TRAIL-induced apoptotic processes were accompanied by cleavage of syndecan-1 intracellular region, and explored the possibility whether removal of syndecan-1 promotes apoptotic processes. We found that syndecan-1 knockdown by specific small interfering RNA in multiple myeloma enhanced TRAIL-induced apoptosis, even though the expression of TRAIL receptors and several apoptosis-associated molecules was unaffected. The enhanced TRAIL-mediated apoptosis in syndecan-1-deficient cells was not due to a decrease in surface heparan sulfate or a reduction in TRAIL receptor endocytosis. The increase in TRAIL-induced cell death was accompanied by an elevated caspase-8 activation and an enhanced formation of death-inducing signaling complexes, which could be attributed to an increased expression of TRAIL receptor O-glycosylation enzyme in syndecan-1-deficient cells. We also found that in H9 lymphoma and Jurkat cells, knockdown of the predominant syndecan member also led to an increase in Fas ligand-induced apoptosis. Our results demonstrate that syndecan plays a negative role in death receptor-mediated cell death, suggesting potential application of syndecan downregulation in the treatment of myeloma in combination with TRAIL.

The regulation of TNF signalling: what a tangled web we weave.

In the past 2 years there has been an explosion of information regarding molecules that regulate TNF-R1 signalling, and even reviews published in 2010 are out of date. TNF-R1 activation of NF-κB is a text book example of a signal transduction pathway regulated by ubiquitin and many of the concepts concerning the different roles of ubiquitin chains were first outlined in TNF-R1 signalling. What was once a very simple pathway with clearly defined roles for ubiquitin in regulating TNF-R1 signalling has, however, now become so complicated that we have 'an embarrassment of riches'. The less polite might claim our pathways of TNF-R1 signalling look as complicated as a web constructed by a drug-addled spider. This review will pick apart only one small strand of the web, and will address the role of ubiquitin in the activation of NF-κB by TNF with a focus on interpreting in vivo results. Nevertheless some of the concepts, for example the role of linear ubiquitin chains in regulating signalling, may be applicable to the family in general.

Changes in apoptotic gene expression in lymphocytes from rheumatoid arthritis and systemic lupus erythematosus patients compared with healthy lymphocytes.

INTRODUCTION: Systemic lupus erythematosus (SLE) and rheumatoid arthritis have complex genetic traits, but in both autoimmune diseases, dysfunctional apoptosis appears to play a part in disease pathology. This study examined the levels of in vitro apoptosis in lymphocytes from healthy, rheumatoid arthritis (RA) and SLE individuals and related observed differences to their lymphocyte apoptosis gene profiles. MATERIALS AND METHODS: Lymphocytes were assessed for cell death by nuclear pyknosis and DNA fragmentation. Control, SLE and RA apoptosis gene profiles were obtained by quantitative real-time polymerase chain reaction (QRT-PCR) analysis. RESULTS AND DISCUSSION: The mean levels of pyknosis in RA and SLE freshly isolated lymphocytes were significantly higher than in control lymphocytes. Ninety-three apoptosis genes were analysed by QRT-PCR of mRNA from RA, SLE and healthy lymphocytes. We identified significant differences (p < 0.05) in the expression of the same 11 of 93 and two of 93 apoptotic genes in individual SLE and RA patients tested as compared with controls. CONCLUSION: We propose that similarly altered expression of specific apoptotic regulatory genes (e.g., the death effector domain-containing DNA-binding protein and apoptosis-associated speck-like protein containing a CARD) occurs in the lymphocytes of individual patients with SLE or RA that may influence the extent and rate of spontaneous apoptosis in these autoimmune conditions.

Structure and regulation of cytoplasmic adapter proteins involved in innate immune signaling.

Initiation of the innate immune response requires agonist recognition by a pathogen recognition receptor. Following ligand binding, conformational rearrangement of the receptor creates a molecular scaffold from which signal transduction is propagated via complex cellular signaling pathways. This in turn leads to the induction of a pro-inflammatory immune response. A critical component of these signaling pathways is the homotypic interaction of receptor and adapter proteins via specific protein interaction domains. Within the innate immune signaling cascade, homotypic interactions between members of the death domain family and the Toll/interleukin-1 receptor domain are particularly important. Here we discuss the current understanding of the molecular basis of these homotypic receptor:adapter interactions and their role in innate immune signal transduction.

A combined approach with rituximab plus anti-TRAIL-R agonistic antibodies for the treatment of haematological malignancies.

Molecular targeted therapies have changed the landscape of cancer research. Agonistic monoclonal antibodies (MoAbs) targeting TRAIL-death receptors (TRAIL-Rs) have been developed and currently used in clinical trials. Binding of such antibodies to TRAIL-R1 and TRAIL-R2 results in death inducing signalling complex (DISC) formation and induction of apoptosis, which represents a natural mechanism of cell growth control and an ideal target for drug development. These novel fully humanized compounds have been associated with conventional chemotherapy in the treatment of advanced solid malignancies, including different types of lymphoma. Here we outline the rationale and potential of a new molecular-based strategy combining agonistic anti-TRAIL-death receptor monoclonal antibodies plus the pioneer of the new biological frontiers of cancer therapy: rituximab.

What do we know about the mechanisms of elimination of autoreactive T and B cells and what challenges remain.

Tolerance to self-antigens within the adaptive immune system is safeguarded, at least in part, through deletion of autoreactive T and B lymphocytes. This deletion can occur during the development of these cells in primary lymphoid organs, the thymus or bone marrow, respectively, or at the mature stage in peripheral lymphoid tissues. Deletion of autoreactive lymphocytes is achieved to a large extent through apoptotic cell death. This review describes current understanding of the mechanisms that mediate apoptosis of autoreactive lymphocytes during their development in primary lymphoid organs and during their activation in the periphery. In particular, we discuss the roles of the proapoptotic Bcl-2 family member Bim and the small family of Nur77-related transcriptional regulators in lymphocyte negative selection. Finally, we speculate on the processes that may lead to the activation of Bim when antigen receptors are activated on autoreactive T or B cells.

Analysis of CD95 threshold signaling: triggering of CD95 (FAS/APO-1) at low concentrations primarily results in survival signaling.

Recently we generated a mathematical model (Bentele, M., Lavrik, I., Ulrich, M., Stosser, S., Heermann, D. W., Kalthoff, H., Krammer, P. H., and Eils, R. (2004) J. Cell Biol. 166, 839-851) of signaling in CD95(Fas/APO-1)-mediated apoptosis. Mathematical modeling in combination with experimental data provided new insights into CD95-mediated apoptosis and allowed us to establish a threshold mechanism of life and death. Here, we further assessed the predictability of the model experimentally by a detailed analysis of the threshold behavior of CD95 signaling. Using the model predictions for the mechanism of the threshold behavior we found that the CD95 DISC (death-inducing signaling complex) is formed at the cell membrane upon stimulation with low concentrations of agonistic anti-APO-1 monoclonal antibodies; however, activation of procaspase-8 at the DISC is blocked due to high cellular FLICE-inhibitory protein recruitment into the DISC. Given that death signaling does not occur upon CD95 stimulation at low (threshold) anti-APO-1 concentrations, we also analyzed survival signaling, focusing on mitogen-activated protein kinase activation. Interestingly, we found that mitogen-activated protein kinase activation takes place under threshold conditions. These findings show that triggering of CD95 can signal both life or death, depending on the strength of the stimulus.

Rapid up-regulation of c-FLIP expression by BCR signaling through the PI3K/Akt pathway inhibits simultaneously induced Fas-mediated apoptosis in murine B lymphocytes.

Cross-linking of BCR rapidly induces protection of B cells from Fas-mediated apoptosis, which has been assumed one of the important survival mechanisms of B cells during antigen stimulation. In the mouse B cell line A20, which is sensitive to Fas-mediated apoptosis, stimulation of BCR inhibited apoptosis induced via Fas upstream of caspase-8 activation with an associated rapid increase in the expression of both short and long forms of cellular caspase-8/FLICE-inhibitory protein (c-FLIP). The c-FLIP competitively inhibited the recruitment of caspase-8 to the death-inducing signaling complex (DISC), which took as long as 3h to form after the stimulation of Fas in A20 cells. Knockdown of c-FLIP by a short hairpin RNA-expressing method rendered BCR-stimulated A20 cells sensitive to Fas-mediated apoptosis. The BCR-induced rapid expression of c-FLIP was not affected by inactivation of NF-kappaB, but was inhibited by either treatment with a PI3K inhibitor, LY294002, or expression of a dominant negative PI3K p85 subunit, both of which suppressed phosphorylation of Akt and sensitized BCR-stimulated A20 cells to Fas-mediated apoptosis. Overexpression of constitutively active Akt was shown not only to up-regulate c-FLIP expression but also to render A20 cells resistant to Fas-mediated apoptosis. Moreover, treatment with LY294002 also suppressed BCR-induced up-regulation of c-FLIP expression in spleen B cells. Taken together, BCR-stimulation was shown to rapidly trigger a survival signal against simultaneously or ongoingly stimulated Fas-mediated apoptosis by promoting a PI3K/Akt signaling pathway-mediated up-regulation of c-FLIP expression.

The death domain superfamily in intracellular signaling of apoptosis and inflammation.

The death domain (DD) superfamily comprising the death domain (DD) subfamily, the death effector domain (DED) subfamily, the caspase recruitment domain (CARD) subfamily, and the pyrin domain (PYD) subfamily is one of the largest domain superfamilies. By mediating homotypic interactions within each domain subfamily, these proteins play important roles in the assembly and activation of apoptotic and inflammatory complexes. In this chapter, we review the molecular complexes assembled by these proteins, the structural and biochemical features of these domains, and the molecular interactions mediated by them. By analyzing the potential molecular basis for the function of these domains, we hope to provide a comprehensive understanding of the function, structure, interaction, and evolution of this important family of domains.