Charlesworth, M. Cristine

Suicide is a major public health concern; nevertheless, its neurobiology remains unknown. An area of interest in suicide research is the dorsolateral prefrontal cortex (DLPFC). We aimed to identify altered proteins and potential biological pathways in the DLPFC of individuals who died by suicide employing mass spectrometry-based untargeted proteomics. Postmortem DLPFC from age-matched male suicide mood disorder cases (n = 5) and non-suicide mood disorder cases (n = 5) were compared. The proteins that differed between groups at false discovery rate (FDR) adjusted p-values (Benjamini–Hochberg–Yekutieli) <0.3 and Log2 fold change (FC) >|0.4| were considered statistically significant and were subjected to pathway analysis by Qiagen Ingenuity software. Thirty-three of the 5162 detected proteins showed significantly altered expression levels in the suicide cases and two of them after adjustment for body mass index. The top differentially expressed protein was potassium voltage-gated channel subfamily Q member 3 (KCNQ3) (Log2FC = −0.481, p = 2.10 × 10−09, FDR = 5.93 × 10−06), which also showed a trend to downregulation in Western blot (p = 0.045, Bonferroni adjusted p = 0.090). The most notably enriched pathway was the GABA receptor signaling pathway (p < 0.001). Here, we report a reduction trend of KCNQ3 levels in the DLPFC of male suicide victims with mood disorders. Further studies with a larger sample size and equal sex representation are needed.

AbstractBackground: Patients with multiple myeloma often have Bence Jones proteins composed of free monoclonal light chains of the κ or λ type in their urine. Usually, these light chains exist as monomeric or dimeric forms, but rarely, larger molecules, such as tetramers, have been reported in the serum.Methods and Results: We report the presence of trimeric complexes of λ light chain dimers in a patient who was diagnosed with a free λ light chain multiple myeloma 2 years earlier and subsequently underwent a stem cell transplant. Recently, the patient presented with a large serum M-spike (23 g/L) by protein electrophoresis. The spike consisted of monoclonal λ light chains without a heavy chain. The urine contained only 8 mg of λ light chain in a 24-h specimen. Quantitative analysis of the serum and urinary free light chains (FLCs) indicated the probability of larger aggregates of FLCs. Size-exclusion chromatography, electrophoresis, analytical ultracentrifugation, and mass spectrometric studies of the serum revealed almost exclusively the presence of trimolecular aggregates of λ light chain dimers without other multimeric species.Conclusion: Monoclonal λ light chains may present as hexameric aggregates that cannot be cleared by renal excretion.

Abstract Abstract 4886 Detection of high molecular weight light chain oligomers in urinary exosomes of patients with AL amyloidosis. Background Exosomes are microvesicles that are part of the multivesicular body (MVB) pathway. They are created by the inward budding of the cell surface membrane and contain both surface bound membrane proteins and cytosolic proteins which can be used to identify the cell of origin. Immunoglobulin light chain amyloidosis (AL) occurs as the result of amyloid formation by the misfolding of monoclonal light chains (LC) and deposition of these amyloid fibrils in various soft tissues. This reaction requires the organization of the monoclonal LC's into protofibrils which are then weave into amyloid fibrils. This study was undertaken to determine whether urinary exosomes of glomerular origin contain intermediate species of amyloid formation. Method Urine samples from patients with AL, light chain deposition disease (LCDD), multiple myeloma (MM) and monoclonal clonal gammopathy of undetermined significance (MGUS) were collected. Urinary exosomes were isolated and separated into fractions by gradient centrifugation. Western blots were performed on the urinary exosome fractions using anti-kappa or anti-lambda antibodies. Glomerular fractions were identified using antibodies directed toward podocin. Results Urine samples were collected from 5 patients with AL, 2 from LCDD, 1 from MM and 1 MGUS. On the Western blot, immunoglobulin LC were seen in all exosomal fractions in patients with AL amyloidosis, LCDD, MM but not MGUS which is similar to normal controls (not shown). In patients with AL, oligomeric species were found in the highest concentrations in fraction 4 and 5 (Figure 1). Fraction 4 and 5 were also stained for podocin, a glomerular protein (not shown). The highest molecular weight species was ∼250 kd which corresponds to a LC decamer. High molecular weight species were also identified in 1 of 2 LCDD patients corresponding to a tetramer. The band was identified in fraction 10 which had polycystin-1 expression suggesting a tubular origin. No high molecular weight LC species was found in patients with MM or MGUS. Conclusion Our study found high molecular weight LC species corresponding to the intermediates involved in protofibril formation in urinary exosomes of patients with AL. Smaller (tetramer) high molecular weight LC species were also found in a patient with LCDD but not in patients with MM and MGUS. Not only were the high molecular weight LC species found exclusively in the diseases characterized by deposition of LC aggregates, they were also found in the segments of the nephron where the deposits were expected: glomerulus for AL and tubular epithelium for LCDD. This is consistent with our current understanding of the pathogenic mechanisms of these diseases. We believe urinary exosomes are a powerful tool in the study of diseases involving self-aggregation of monoclonal proteins. It has tremendous potential in both diagnostic and scientific research in this area. Disclosures Gertz: celgene: Honoraria; millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Significance Statement In approximately 70%–80% of cases of primary membranous nephropathy (MN), phospholipase A2 receptor (PLA2R)/Thrombospondin Type-1 Domain–Containing 7A (THSD7A) and anti-PLA2R/THSD7A antibodies form immune complexes along the glomerular basement membrane (GBM) that characterize the condition. In other cases of primary MN and all secondary MN, the target antigen is unknown. Using proteomics and immunohistochemistry, the authors detected two proteins, exostosin 1 (EXT1) and exostosin 2 (EXT2), in the GBM of PLA2R-negative MN. EXT1 and EXT2 were absent in all cases of PLA2R-associated MN and controls. Clinical and biopsy findings showed features of autoimmune disease, including membranous lupus nephritis, in 81% of the 26 EXT1/EXT2-associated MN cases the authors identified. These findings suggest that EXT1/EXT2-associated MN represents a distinct subtype of MN, most commonly associated with autoimmune diseases (secondary MN). Background In membranous nephropathy (MN), which is characterized by deposition of immune complexes along the glomerular basement membrane (GBM), phospholipase A2 receptor (PLA2R) and thrombospondin type 1 domain–containing 7A are target antigens in approximately 70% and 1%–5% of cases of primary MN, respectively. In other cases of primary MN and in secondary MN, the target antigens are unknown. Methods We studied 224 cases of biopsy-proven PLA2R-negative MN and 102 controls (including 47 cases of PLA2R-associated MN) in pilot and discovery cohorts. We also evaluated 48 cases of PLA2R-negative presumed primary MN and lupus MN in a validation cohort. We used laser microdissection and mass spectrometry to identify new antigens, which were localized by immunohistochemistry. Results Mass spectrometry detected exostosin 1 (EXT1) and exostosin 2 (EXT2) in 21 cases of PLA2R-negative MN, but not in PLA2R-associated MN and control cases. Immunohistochemistry staining revealed bright granular GBM staining for EXT1 and EXT2. Clinical and biopsy findings showed features of autoimmune disease, including lupus, in 80.7% of the 26 EXT1/EXT2-associated MN cases we identified. In the validation cohort, we confirmed that EXT1/EXT2 staining was detected in pure class 5 lupus nephritis (eight of 18 patients) and in presumed primary MN associated with signs of autoimmunity (three of 16 patients); only one of the 14 cases of mixed class 5 and 3/4 lupus nephritis was positive for EXT1/EXT2. Tests in seven patients with EXT1/EXT2-associated MN found no circulating anti-exostosin antibodies. Conclusions A subset of MN is associated with accumulation of EXT1 and EXT2 in the GBM. Autoimmune disease is common in this group of patients.

Abstract Tumor-associated hypoxia has been associated with malignant progression, metastasis and resistance to therapy. We studied the role of oxygen tension (pO2) in modulating the properties of cultured human prostate cancer (CaP) cells. In air, where oxygen tension (pO2) is approximately 20 kPa, cultured CaP cells expressed lower levels of transcripts associated with cancer and urologic disease; reducing pO2 to 2 kPa made these levels comparable to those in surgical CaP specimens suggesting that hypoxically cultured cells, rather than normoxically cultured cells, express genes at levels akin to those in tumor cells in situ. In human primary CaP tissue, transcripts of hypoxia-associated genes DLG7, CCNB1 and HMMR were overexpressed and associated with Gleason score and disease prognosis; this implies the potential prognostic value of these transcripts. While hypoxia affected the CaP cell proteome mostly quantitatively, it did affect expression of tumor–associated antigens and suggested the potential therapeutic relevance of the hypoxic antigenic landscape. Overall, our results suggest that hypoxia modifies cellular properties of cultured CaP cells towards a phenotype more similar to tumor cells in situ. Introduction of pO2 as a variable can constitute a tool for the identification of more effective prognostic biomarkers and development of better therapy for CaP and, possibly, other tumors. Citation Format: Christian R. Gomez, Stanimir Vuk-Pavlovic, Farhad Kosari, Jan Marie Munz, Claire Schreiber, Gaylord Knutson, Cristine Charlesworth, R. Jeffrey Karnes, John Cheville, George Vasmatzis. Prognostic value of hypoxia-associated genes in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C43.

PurposeAutoantibodies are implicated in the pathogenesis of cardiovascular diseases and cardiac arrhythmias. In this pilot study, we tested the hypothesis that autoantibodies are present in patients with postural orthostatic tachycardia syndrome (POTS).Experimental designSeven control subjects (6 F:1 M, average age 36.1 years) and ten patients with the diagnosis of POTS (7 F: 3 M, average age 35.1 years) provided informed consent and 30 mL of venous blood. Human heart membrane proteins were resolved by 2DE and immunoblotted against purified IgGs from controls and patients.ResultsEighteen protein spots immunoreactive specifically against patient IgGs were detected and they were excised from gels, trypsin‐digested, and analyzed by nanoLC‐electrospray MS/MS. Forty unique proteins were identified and these include proteins that are associated with cardiac hypertrophy (mimecan, myozenin), cardiac remodeling (periostin), cardiomyopathy (desmin, desmoplakin), cell survival (laminin), structural integrity (filamin), chaperone proteins (crystalline, HSP70), mitochondrial enzymes, and channel proteins. Ingenuity Pathway Analysis showed multiple pathways were involved including those that regulate energy metabolism, redox, fibrosis, cardiac hypertrophy, and degeneration.Conclusions and clinical relevanceAutoantibodies are present in patients with POTS. These autoantibodies cross‐react with a wide range of cardiac proteins and may induce alterations in cardiac function. Autoimmune pathogenetic mechanisms should be further explored in these patients.

Abstract In early clinical trials, vaccination with allogeneic cancer cells enhanced the overall survival in some patients with malignancies. Because the administered vaccine cells were cultured at pO2=20 kPa, it is unclear whether they provided an adequate antigen match to tumor cells in situ where pO2 is generally much lower. Thus, we postulate that hypoxically grown vaccine cells will provide a better antigen match to tumors in situ. We are testing this hypothesis by studying the effects of hypoxia on prostate cancer (CaP) cells. LnCaP and VCaP cells were cultured at pO2=2 kPa or 20 kPa and analyzed by 2-D gel electrophoresis. The difference in pO2 affected the proteome mostly quantitatively (i.e., by change in spot intensity). Using a threshold of fivefold change, we found that hypoxia decreased the levels of thirteen proteins and increased of four in VCaP cells. These results are in line with the reports showing that hypoxia affects expression only of a small fraction of total cellular protein and that the content of total protein is not altered significantly. To determine if the hypoxia-dependent changes affected immune reactivity of the cells, we took advantage of the spontaneous autoantibodies against tumor-associated antigens (TAAs). Hence, we compared the binding to lysates of LnCaP and VCaP cells of antibodies pooled from CaP patients (n=25), healthy controls (n=25), rheumatoid arthritis (n=17), colorectal cancer (n=10) and lung cancer (n=10). CaP patient sera plasma reacted with numerous spots, some even in control groups and thus considered nonspecific. CaP sera plasma specifically bound to seven spots; four were hypoxia-specific. All selected spots were excised from the gel, trypsin-digested, and identified by MALDI-TOF mass spectrometry as heat shock 70 kDa protein 4; protein disulfide isomerase A3; heterogeneous nuclear ribonucleoprotein L and leucine-rich repeat-containing protein 47. With the exception of the latter, the proteins have been identified or validated as TAAs before, but apparently none in CaP. CaP patient antibodies bound to lysates of LnCaP cells grown at pO2=2 kPa, but less than to such VCaP cell lysates. Our results confirmed the findings that hypoxia affects the proteome mostly quantitatively. Lysates of hypoxic CaP cells revealed novel potential TAAs suggesting the potential immunologic relevance of antigens in the hypoxic proteome. Thus, pO2 control can provide a tool for the development of more effective immunotherapy for CaP and possibly other cancers. Support: DOD PC094680 (CRG), PCF Creativity Award (CRG), Mayo Clinic Prostate SPORE 5P50CA091956 (SV-P); Mrs. Adelyn L. Luther, Singer Island, Florida and Mayo Clinic Cancer Center (SV-P). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C223.