Intravenous self‐administration studies with l ‐deprenyl (selegiline) in monkeys *

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110034/1/cptclpt1994208.pd

C4b Binding Protein Binds to CD154 Preventing CD40 Mediated Cholangiocyte Apoptosis: A Novel Link between Complement and Epithelial Cell Survival

Activation of CD40 on hepatocytes and cholangiocytes is critical for amplifying Fas-mediated apoptosis in the human liver. C4b-Binding Protein (C4BP) has been reported to act as a potential surrogate ligand for CD40, suggesting that it could be involved in modulating liver epithelial cell survival. Using surface plasmon resonance (BiaCore) analysis supported by gel filtration we have shown that C4BP does not bind CD40, but it forms stable high molecular weight complexes with soluble CD40 ligand (sCD154). These C4BP/sCD154 complexes bound efficiently to immobilised CD40, but when applied to cholangiocytes they failed to induce apoptosis or proliferation or to activate NFkB, AP-1 or STAT 3, which are activated by sCD154 alone. Thus C4BP can modulate CD40/sCD154 interactions by presenting a high molecular weight multimeric sCD154/C4BP complex that suppresses critical intracellular signalling pathways, permitting cell survival without inducing proliferation. Immunohistochemistry demonstrated co-localisation and enhanced expression of C4BP and CD40 in human liver cancers. These findings suggest a novel pathway whereby components of the complement system and TNF ligands and receptors might be involved in modulating epithelial cell survival in chronic inflammation and malignant disease

Tumor-Infiltrating T Cells Correlate with NY-ESO-1-Specific Autoantibodies in Ovarian Cancer

BACKGROUND: Tumor-infiltrating CD8+ T cells are correlated with prolonged progression-free and overall survival in epithelial ovarian cancer (EOC). A significant fraction of EOC patients mount autoantibody responses to various tumor antigens, however the relationship between autoantibodies and tumor-infiltrating T cells has not been investigated in EOC or any other human cancer. We hypothesized that autoantibody and T cell responses may be correlated in EOC and directed toward the same antigens. METHODOLOGY AND PRINCIPAL FINDINGS: We obtained matched serum and tumor tissue from 35 patients with high-grade serous ovarian cancer. Serum samples were assessed by ELISA for autoantibodies to the common tumor antigen NY-ESO-1. Tumor tissue was examined by immunohistochemistry for expression of NY-ESO-1, various T cell markers (CD3, CD4, CD8, CD25, FoxP3, TIA-1 and Granzyme B) and other immunological markers (CD20, MHC class I and MHC class II). Lymphocytic infiltrates varied widely among tumors and included cells positive for CD3, CD8, TIA-1, CD25, FoxP3 and CD4. Twenty-six percent (9/35) of patients demonstrated serum IgG autoantibodies to NY-ESO-1, which were positively correlated with expression of NY-ESO-1 antigen by tumor cells (r = 0.57, p = 0.0004). Autoantibodies to NY-ESO-1 were associated with increased tumor-infiltrating CD8+, CD4+ and FoxP3+ cells. In an individual HLA-A2+ patient with autoantibodies to NY-ESO-1, CD8+ T cells isolated from solid tumor and ascites were reactive to NY-ESO-1 by IFN-gamma ELISPOT and MHC class I pentamer staining. CONCLUSION AND SIGNIFICANCE: We demonstrate that tumor-specific autoantibodies and tumor-infiltrating T cells are correlated in human cancer and can be directed against the same target antigen. This implies that autoantibodies may collaborate with tumor-infiltrating T cells to influence clinical outcomes in EOC. Furthermore, serological screening methods may prove useful for identifying clinically relevant T cell antigens for immunotherapy

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Figure 7

<p>Immunolocalisation of C4BP in human liver tissue. This figure shows three representative sections of liver tissue stained for C4BP. Panel a shows normal liver tissue which is predominantly negative. Panel b shows PSC liver tissue showing the presence of strongly staining inflammatory cells surrounding the portal tract and within the sinusoids. Panel c shows hepatic tumour tissue taken from a hepatic resection for secondary liver cancer (colorectal hepatic metastasis) where very strong staining was observed in the tumour tissue and the inflammatory infiltrate at the tumour margin.</p

Figure 1

<p>Panel a) This histogram shows inhibition of sCD154 mediated apoptosis by C4BP but not apoptosis induced by 0.2 mM TDC. * sCD154 or **TDC induced similar levels of cholangiocyte apoptosis when experimental error was taken into account (59.7%+/−7.5 and 83.4%+/−7.7 and respectively) relative to control (p<.005)***. C4BP + sCD154 reduced apoptosis to control levels (p<0.005) whereas C4BP had no effect on TDC induced apoptosis (81.5%+/−6.7). C4BP/sCD154 also had no effect on TDC mediated apoptosis (data not shown) Panel b and c show representative cytospins stained for fragmented DNA using ISEL. Panel d) Histogram summary of the effects of sCD154 and C4BP on Cholangiocyte proliferation. Primary human cholangiocytes were cultured in 24-well culture plates and simulated with either sCD154, C4BP or a mixture of both. Following incubation for 24 hours, the cells were fixed and proliferation assessed by immunohistochemical staining for Ki-67 antigen. No significant difference was seen between the un-stimulated controls and treated samples, implying that sCD154, C4BP or the mixture had any effect on cholangiocyte proliferation. These data represent the mean of three different counted areas per well repeated for three different liver preparations.</p

Figure 5

<p>Representative Western blots showing NFkB, c-Fos and c-Jun and pSTAT 3 levels in response to CD154/C4BP stimulation. Aliquots of nuclear or cytoplasmic extracts as appropriate (40 ug protein) from cultured and stimulated primary human cholangiocytes were probed for NFkB (panel I); c-Fos and c-Jun(panel II) or pSTAT3 content. Blots were stripped and re-probed for beta-actin which allowed for normalisation of data for variations in protein loading.</p

Figure 6

<p>Densitometry Histograms showing changes in NFkB, c-Fos and c-Jun and pSTAT3 levels in response to sCD154 and C4BP stimulation determined using Western blotting (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000159#pone-0000159-g005" target="_blank">figure 5</a>). Changes in levels were assessed by densitometric analysis. These data show that the CD154/C4BP complex had no significant influence upon levels of NFkB or c-Jun, but did decrease levels of c-Fos at 24 hours compared with either unstimulated 24 hour control or the stimulated level observed at 4 hours. *p<0.05 a p<0.05These data show that stimulation of cells with CD154/C4BP complex resulted in a lower level of pSTAT3 which was sustained at 24 hours. *p<0.05 c.f. time point matched untreated control a p<0.05 c.f. treatment matched 4 hour time point.</p

Figure 8

<p>Co-localisation of C4BP and CD40 in liver tumour tissue using dual immunofluorescence. Panels a–c shows a representative section of tumour tissue from a patient with cholangiocarcinoma stained for C4BP (green - FITC) and CD40 (red - PE ). In panel a, the arrows identify an epithelial ductular structure (DS) surrounded by tumour cells (TC) and stromal tissue. Positive C4BP staining is seen within the epithelia, tumour cells, and in mononuclear infiltrate in the surrounding stromal tissue. Panel b shows the same tissue section stained for CD40 with the arrow identifying the inflammatory cells within the surrounding stroma. Panels d and e shows a sequential section from the same specimen where the primary antibodies have been substituted for non immune serum (control). Panel c shows the merged image for panels a and b. The bright yellow areas indicate regions of C4BP and CD40 co-localisation within the epithelial cells of the ductular structure, many surrounding tumour cells, and the inflammatory cells within the surrounding stromal tissue.</p