Tissue proteomics outlines AGR2 AND LOX5 as markers for biochemical recurrence of prostate cancer.

Although many patients are cured from prostate cancer (PCa) by surgery only, there are still patients who will experience rising prostate-specific antigen (PSA) levels after surgery, a condition known as biochemical recurrence (BCR). Novel protein prognostic markers in PCa tissue might enable finding better treatment for those patients experiencing BCR with a high chance of metastasis. In this study, we aimed to identify altered proteins in prostate cancer tissue, and to evaluate their potential role as prognostic markers. We used two proteomics strategies to analyse 34 prostate tumours (PCa) and 33 normal adjacent prostate (NAP) tissues. An independent cohort of 481 samples was used to evaluate the expression of three proteins: AGR2, FASN and LOX5 as prognostic markers of the disease. Tissue microarray immunohistochemical staining indicated that a low percentage of positive tumour cells for AGR2 (HR (95% CI) = 0.61 (0.43-0.93)), and a low percentage of positive tumour cells for LOX5 expression (HR (95% CI) = 2.53 (1.23-5.22)) are predictors of BCR after RP. In contrast, FASN expression had no prognostic value for PCa.

Proteomics urine analysis of pregnant women suffering from multiple sclerosis.

Multiple sclerosis (MScl) frequently is remitted during the third trimester of pregnancy but exacerbated in the first postpartum period. In this context, we investigated protein identification, its abundance, and its change in urine related to these two periods. Using mass spectrometry (LTQ Orbitrap), we identified 1699 tryptic peptides (related to 402 proteins) in urine from 31 MScl and 8 control at these two periods. Pregnancy-related peptides were significantly elevated (p < 0.01) in MScl patients compared with controls (Analysis 1: 531 peptides in MScl and 36 peptides in controls higher abundant in the third trimester compared to postpartum). When comparing the longitudinal differences (Analysis 2), we identified 43 (related to 35 proteins) MScl disease-associated peptides (p < 0.01) with increased or decreased difference ratio in MScl compared with controls. The most discriminating peptides identified were trefoil factor 3 and lysosomal-associated membrane protein 2. Both proteins have a role in the innate immune system. Three proteins with a significant decreased ratio were plasma glutamate carboxypeptidase, Ig mu chain C region, and osteoclast associated immune like receptor. Our results indicate that the protein expression pattern in urine of MScl patients contains information about remote CNS and brain disease processes.

Mass spectrometric detection of antigen-specific immunoglobulin peptides in paraneoplastic patient sera.

Paraneoplastic neurological syndromes (PNS) are severe immune mediated effects of cancer. The presence of IgG autoantibodies against onconeural antigens in serum is a hallmark of the disease. Multiple paraneoplastic antibodies have been described, including antibodies against HuD, Yo, amphiphysin and CV2. In this study, we test the hypothesis that primary amino-acid structures of the antigen binding part of antibodies from various individuals share common sequences that are specific for each auto-antigen. We selected 60 patients with PNS, associated with antibodies against HuD, Yo, Amp or CV2. Affinity purified IgG was separated using SDS-PAGE and IgG heavy chains were excised, trypsinized and subjected to tandem mass spectrometry. We selected masses that uniquely identified a PNS autoantibody group, and used MS/MS fragmentation spectra to obtain information on peptide sequences. Out of 19,173 unique masses, 28 immunoglobulin-derived peptides were found exclusively in samples from a single autoantibody defined PNS group. Our results confirm that specific peptide structures exist in the antigen binding site of IgG that are shared between individuals harboring autoantibodies against the same onconeural antigen. Thus, the immune response in these patients followed converging paths during the rearrangement, selection and maturation of immunoglobulin sequences. The identified peptides can be applied in the diagnosis of PNS, but these data also indicate that a similar approach in a variety of other diseases involving an immune response would have an appealing outlook.

A new paraneoplastic encephalomyelitis autoantibody reactive with the axon initial segment.

Serum from a patient with paraneoplastic encephalomyelitis (PEM) and small cell lung cancer (SCLC) showed high titer immunohistochemical staining of the axon initial segment (AIS) on rat and human brain sections. EM studies showed that the antigen was localized in close proximity of the microtubules in the AIS. Double labeling experiments and absence of staining at the nodes of Ranvier excluded the previously identified betaIV spectrin as autoantigen. Screening a rat hippocampal cDNA library resulted in the isolation of ubiquitin-conjugating enzyme E2E1 (UBE2E1). However, blocking and elution experiments excluded UBE2E1 as the AIS autoantigen.

B and T cell imbalances in CSF of patients with Hu-antibody associated PNS.

In paraneoplastic neurological syndromes associated with Hu-antibodies (Hu-PNS) an important role for cellular immunity is hypothesized. We characterized the cerebrospinal fluid (CSF) pleocytosis in Hu-PNS patients by assessing the major lymphocyte subsets by flow cytometry. The B cell subset in the CSF of Hu-PNS patients showed a significant absolute (approximately 20x) and relative (approximately 3x) expansion, while the numbers of CD4+ T cells, CD8+ T cells and NK cells only showed an absolute expansion (approximately 4-7x) compared to the controls. On the other hand, the NKT cell subset showed a significant relative reduction in CSF and in blood of Hu-PNS patients. The relative B cell expansion is consistent with the intrathecal synthesis of Hu-antibodies, while the increased number of T and NK cells supports an additional role for cellular immunity in the pathogenesis of Hu-PNS. In addition, the autoimmune hypothesis of Hu-PNS is supported by the relative NKT cell deficiency.

Targeting malignant gliomas with a glial fibrillary acidic protein (GFAP)-selective oncolytic adenovirus.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein abundantly expressed in malignant gliomas. We have constructed a novel oncolytic adenovirus, Ad5-gfa2(B)3-E1, for treatment of these tumors. In this construct, the E1 region is under control of the tissue-specific GFAP promoter (gfa2) with three additional copies of the glial specific 'B' enhancer. Infection of a GFAP-positive cell line with Ad5-gfa2(B)3-E1 resulted in E1A and E1B expression at 75% and 30% of the levels obtained after wtAd5 infection. Q-PCR showed that Ad5-gfa2(B)3-E1 replicated 4.5 times more efficiently in the GFAP-positive than in the GFAP-negative cell lines. Cell viability assays showed efficient elimination of GFAP-positive cells by Ad5-gfa2(B)3-E1, in some cell lines as efficiently as wtAd5, while the elimination was attenuated in GFAP-negative cell lines. When tested in human tumor xenografts in nude mice, Ad5-gfa2(B)3-E1 effectively suppressed the growth of GFAP-positive SNB-19 glial tumors but not of GFAP-negative A549 lung tumors. In Ad5-gfa2(B)3-E1, the E3 region was deleted to create space for future insertion of heterologous therapeutic genes. Experiments with dl7001, an E3-deleted variant of wtAd5, confirmed that the specificity of Ad5-gfa2(B)3-E1 replication was based on the promoter driving E1 and not on the E3 deletion. Strategies to further improve the efficacy of Ad5-gfa2(B)3-E1 for the treatment of malignant gliomas include the insertion of therapeutic genes in E3 or retargeting to receptors that are more abundantly expressed on primary glioma cells than CAR.

Identification of leptomeningeal metastasis-related proteins in cerebrospinal fluid of patients with breast cancer by a combination of MALDI-TOF, MALDI-FTICR and nanoLC-FTICR MS.

Leptomeningeal metastasis (LM) is a devastating complication occurring in 5% of breast cancer patients. However, the current 'gold standard' of diagnosis, namely microscopic examination of the cerebrospinal fluid (CSF), is false-negative in 25% of patients at the first lumbar puncture. In a previous study, we analyzed a set of 151 CSF samples (tryptic digests) by MALDI-TOF and detected peptide masses that were differentially expressed in breast cancer patients with LM. In the present study, we obtain for a limited number of samples exact masses for these peptides by MALDI-FTICR MS measurements. Identification of these peptides was performed by electrospray FTICR MS after separation by nano-scale LC. The database results were confirmed by targeted high mass accuracy measurements of the fragment ions in the FTICR cell. The combination of automated high-throughput MALDI-TOF measurements and analysis by FTICR MS leads to the identification of 17 peptides corresponding to 9 proteins. These include proteins that are operative in host-disease interaction, inflammation and immune defense (serotransferrin, alpha 1-antichymotrypsin, hemopexin, haptoglobin and transthyretin). Several of these proteins have been mentioned in the literature in relation to cancer. The identified proteins alpha1-antichymotrypsin and apolipoprotein E have been described in relation to Alzheimer's disease and brain cancer.

Increased glia-specific transgene expression with glial fibrillary acidic protein promoters containing multiple enhancer elements.

The ability to direct transgene expression to astrocytes has become increasingly important as the roles for these cells continue to expand. Promoters consisting of the 5'-flanking region of the human or mouse glial fibrillary acidic protein (GFAP) gene have generally proved satisfactory. However, a more powerful promoter would be advantageous for several applications, such as expression of dominant negative RNAs or proteins, or for gene therapy. We investigated the possibility of increasing the transcriptional activity of the human GFAP promoter by inserting into it one or three additional copies of putative GFAP enhancer regions. The promoters enhanced with three additional copies gave 75-fold higher LacZ expression levels upon plasmid transfection into GFAP-expressing U251 cells than the parental gfa2 promoter. Surprisingly, in a transgenic mouse model, the enhanced promoters resulted in no or only very low expression of marker genes, probably caused by toxicity. When various cell lines were infected with replication-deficient adenoviral vectors, the enhanced promoters gave LacZ expression levels that were approximately 10-fold higher than those with the parental gfa2 promoter, while retaining specificity for GFAP-expressing cells. Injection of the adenoviral vectors carrying the enhanced promoters into nude mouse brain showed that LacZ expression was limited to GFAP-positive cells. We conclude that gfa2 enhanced promoters are useful for production of short-term, glia-specific, high expression levels of genes in an adenoviral context. Adenoviral vectors containing these enhanced promoters may be useful in glioma gene therapy.

Treatment of relapsed malignant glioma with an adenoviral vector containing the herpes simplex thymidine kinase gene followed by ganciclovir.

Between November 1998 and December 2001, we treated 14 patients with advanced recurrent high-grade gliomas with a total dose of 4.6 x 10(8), 4.6 x 10(9), 4.6 x 10(10), or 4.6 x 10(11) viral particles (VP) of a replication-incompetent adenoviral vector harboring the herpes simplex virus thymidine kinase gene driven by the adenoviral major late promoter (IG.Ad.MLPI.TK), followed by ganciclovir (GCV) treatment. The VP-to-infectious-unit ratio was 40. The vector was administered by 50 intraoperative wound-bed injections of 0.2 ml each (total volume 10 ml). The study's primary objective was to determine the safety of this treatment and establish the maximum tolerated dose (MTD). Injection of all doses of IG.Ad.MLPI.TK followed by GCV was safely tolerated and MTD was not reached. All patients had recurrence or progression of the tumor 1-24 months (median 3.5 months) after gene therapy. The overall median survival was 4 months. Four patients survived longer than 1 year following gene therapy. One patient is still alive, with histologically confirmed progression of the tumor, 29 months after treatment. Ten patients died within 8 months of treatment, all from progression of the tumor. In 5 patients residual and measurable tumor was visible on the direct (<48 h) postoperative MRI. No objective radiological response was documented on subsequent MRI. None of the patients came to autopsy. In conclusion, the administration of 4.6 x 10(11) VP of IG.Ad.MLPI.TK by 50 injections into the wound bed following resection of recurrent malignant glioma, followed by GCV treatment, was well tolerated.

Long-term potentiation of mGluR1 activity by depolarization-induced Homer1a in mouse cerebellar Purkinje neurons.

Metabotropic glutamate receptor 1 (mGluR1) plays a crucial role in synaptic plasticity and motor learning in the cerebellum. We have studied activity-dependent changes in mGluR1 function in mouse cultured Purkinje neurons. Depolarizing stimulation potentiated Ca2+ and current responses to an mGluR1 agonist for several hours in the cultured Purkinje neurons. It also blocked internalization of mGluR1 and increased the number of mGluR1s on the cell membrane. We found that depolarization simultaneously increased transcription of Homer1a in Purkinje neurons. Homer1a inhibited internalization and increased cell-surface expression of mGluR1 when coexpressed in human embryonic kidney (HEK)-293 cells. Depolarization-induced Homer1a expression in Purkinje neurons was blocked by a mitogen-activated protein kinase (MAPK) inhibitor. Changes in internalization and mGluR1-mediated Ca2+ response were also blocked by inhibition of MAPK activity, suggesting that localization and activity of mGluR1 were regulated in the same signalling pathway as Homer1a expression. It is thus suggested that depolarization of the Purkinje neuron leads to the increment in mGluR1 responsiveness through MAPK activity and induction of Homer1a expression, which increases active mGluR1 on the cell surface by blocking internalization of mGluR1.

Imaging expression of adenoviral HSV1-tk suicide gene transfer using the nucleoside analogue FIRU.

Substrates for monitoring HSV1-tk gene expression include uracil and acycloguanosine derivatives. The most commonly used uracil derivative to monitor HSV1-tk gene transfer is 1-(2-fluoro-2-deoxy--D-arabinofuranosyl)-5-[*I]iodouracil (fialuridine; I*-FIAU), where the asterisk denotes any of the radioactive iodine isotopes that can be used. We have previously studied other nucleosides with imaging properties as good as or better than FIAU, including 1-(2-fluoro-2-deoxy--D-ribofuranosyl)-5-[*I]iodouracil (FIRU). The first aim of this study was to extend the biodistribution data of 123I-labelled FIRU. Secondly, we assessed the feasibility of detecting differences in HSV1-tk gene expression levels following adenoviral gene transfer in vivo with 123I-FIRU. 9L rat gliosarcoma cells were stably transfected with the HSV1-tk gene (9L-tk+). 123I-FIRU was prepared by radioiodination of 1-(2-fluoro-2-deoxy--D-ribofuranosyl)-5-tributylstannyl uracil (FTMRSU; precursor compound) and purified using an activated Sep-Pak column. Incubation of 9L-tk+ cells and the parental 9L cells with 123I-FIRU resulted in a 100-fold higher accumulation of radioactivity in the 9L-tk+ cells after an optimum incubation time of 4 h. NIH-bg-nu-xid mice were then inoculated subcutaneously with HSV1-tk (-) 9L cells or HSV1-tk (+) 9L-tk+ cells into both flanks. Biodistribution studies and gamma camera imaging were performed at 15 min and 1, 2, 4 and 24 h p.i. At 15 min, the tumour/muscle, tumour/blood and tumour/brain ratios were 5.2, 1.0 and 30.3 respectively. Rapid renal clearance of the tracer from the body resulted in increasing tumour/muscle, tumour/blood and tumour/brain ratios, reaching values of 32.2, 12.5 and 171.6 at 4 h p.i. A maximum specific activity of 22%ID/g tissue was reached in the 9L-tk+ tumours 4 h after 123I-FIRU injection. Two Ad5-based adenoviral vectors containing the HSV1-tk gene were constructed: a replication-incompetent vector with the transgene in the former E1 region, driven by a modified CMV promoter, and a novel replication-competent vector with the HSV1-tk gene in E3 driven by the natural E3 promoter. The human glioma cell lines U87MG and T98G were infected with a multiplicity of infection (m.o.i.) of 10. Forty-eight hours later the cells were incubated with 123I-FIRU and radioactivity was measured in a gamma counter. We found significantly higher levels of radioactivity in both cell lines following infection with the replication-competent vector (P<0.001). NIH-bg-nu-xid mice were then inoculated subcutaneously with U87MG cells. Tumours (approximately 1,000 mm3) were injected with 108 and 109 Infectious Units (I.U.) of either vector. After 48 h, the tracer was injected, followed by gamma camera imaging and direct measurement of radioactivity in the tumours at 4 h p.i. Images and direct measurements indicated increased uptake of tracer with higher I.U. and also demonstrated increased accumulation of tracer in the tumours treated with the replication-competent adenoviral vector (P=0.03). These results demonstrate that 123I-FIRU in combination with HSV1-tk is a valuable tracer for in vivo monitoring of adenoviral gene transfer.