cDNA Expression Cloning Research Articles

Association of serum levels of antibodies against MMP1, CBX1, and CBX5 with transient ischemic attack and cerebral infarction.

Transient ischemic attack (TIA) is a predictor for cerebral infarction (CI), and early diagnosis of TIA is extremely important for the prevention of CI. We set out to identify novel antibody biomarkers for TIA and CI, and detected matrix metalloproteinase 1 (MMP1), chromobox homolog 1 (CBX1), and chromobox homolog 5 (CBX5) as candidate antigens using serological identification of antigens by recombinant cDNA expression cloning (SEREX) and Western blotting to confirm the presence of serum antibodies against the antigens. Amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) revealed that serum antibody levels were significantly higher in patients with TIA or acute-phase CI (aCI) compared with healthy donors (P < 0.01). Spearman’s correlation analysis and multivariate logistic regression analysis demonstrated that levels of anti-MMP1, anti-CBX1, and anti-CBX5 antibodies were associated with age, cigarette-smoking habits, and blood pressure. Thus, serum levels of antibodies against MMP1, CBX1, and CBX5 could potentially serve as useful tools for diagnosing TIA and predicting the onset of aCI.

Identification of novel serum autoantibodies against EID3 in non-functional pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (pNETs) are relatively rare heterogenous tumors, comprising only 1–2% of all pancreatic neoplasms. The majority of pNETs are non-functional tumors (NF-pNETs) that do not produce hormones, and as such, do not cause any hormone-related symptoms. As a result, these tumors are often diagnosed at an advanced stage because patients do not present with specific symptoms. Although tumor markers are used to help diagnosis and predict some types of cancers, chromogranin A, a widely used tumor marker of pNETs, has significant limitations. To identify novel NF-pNET-associated antigens, we performed serological identification of antigens by recombinant cDNA expression cloning (SEREX) and identified five tumor antigens (phosphatase and tensin homolog, EP300-interacting inhibitor of differentiation 3 [EID3], EH domain-containing protein 1, galactoside-binding soluble 9, and BRCA1-associated protein). Further analysis using the AlphaLISA® immunoassay to compare serum antibody levels revealed that antibody levels against the EID3 antigen was significantly higher in the patient group than in the healthy donor group (n = 25, both groups). In addition, higher serum anti-EID3 antibody levels in NF-pNET patients correlated with shorter disease-free survival. The AUC calculated by ROC analysis was 0.784 with moderate diagnostic accuracy. In conclusion, serum anti-EID3 antibody levels may be useful as a tumor marker for prediction of tumor recurrence in NF-pNETs.

Progression of Human Renal Cell Carcinoma via Inhibition of RhoA-ROCK Axis by PARG1

Renal cell carcinoma (RCC) is the most lethal urological malignancy with high risk of recurrence; thus, new prognostic biomarkers are needed. In this study, a new RCC antigen, PTPL1 associated RhoGAP1 (PARG1), was identified by using serological identification of recombinant cDNA expression cloning with sera from RCC patients. PARG1 protein was found to be differentially expressed in RCC cells among patients. High PARG1 expression is significantly correlated with various clinicopathological factors relating to cancer cell proliferation and invasion, including G3 percentage (P = .0046), Ki-67 score (p expression is also correlated with high recurrence of N0M0 patients (P = .0084) and poor prognosis in RCC patients (P = .0345). Multivariate analysis has revealed that high PARG1 expression is an independent factor for recurrence (P = .0149) of N0M0 RCC patients. In in vitro studies, depletion of PARG1by siRNA in human RCC cell lines inhibited their proliferation through inducing G1 cell cycle arrest via upregulation of p53 and subsequent p21Cip1/Waf1, which are mediated by increased RhoA-ROCK activities. Similarly, PARG1 depletion cells inhibited invasion ability via increasing RhoA-ROCK activities in the RCC cell lines. Conversely, overexpression of PARG1 on human embryonic kidney cell line HEK293T promotes its cell proliferation and invasion. These results indicate that PARG1 plays crucial roles in progression of human RCC in increasing cell proliferation and invasion ability via inhibition of the RhoA-ROCK axis, and PARG1 is a poor prognostic marker, particularly for high recurrence of N0M0 RCC patients.

Identification of Serum Anti-GADD34 Antibody as a Common Marker of Diabetes Mellitus and Parkinson Disease

Background: Growth arrest and DNA-damage-inducible gene 34 (GADD34) has been identified as an antigen by serological identification of antigens by cDNA expression cloning (SEREX) using the sera of patients with atherosclerosis. It is possible that GADD34 is associated with atherosclerosis-related diseases such as diabetes mellitus (DM), acute-phase cerebral infarction (aCI), cardiovascular disease (CVD), and chronic kidney disease (CKD) as well as endoplasmic reticulum stress-related Parkinson disease (PD). Methods: GADD34 protein was bacterially expressed and purified. Amplified luminescent proximity homogeneous assay (AlphaLISA) was used to evaluate serum antibody levels against GADD34 protein in serum samples. Results: AlphaLISA revealed significantly higher serum antibody levels against GADD34 protein in patients with DM, aCI and CVD than those in healthy donors (HDs). The difference in levels between DM and HD was more prominent than that between aCI or CVD and HDs. The anti-GADD34 antibody levels were also elevated in the sera of diabetic CKD patients; thus, the anti-GADD34 antibodies appeared to be the most associated with DM, which is also a risk factor of PD. Anti-GADD34 antibody levels were also higher in patients with PD but not in those with amyotrophic lateral sclerosis as compared with those in HDs. Conclusion: Anti-GADD34 antibody may be a useful diagnostic tool for DM and PD. GADD34 may account for the pathophysiological relationship between DM and PD.

GFP-Grb2 Translocation Assay Using High-content Imaging to Screen for Modulators of EGFR-signaling.

High-content screening is a useful tool to understand complex cellular processes and to identify genes, proteins or small molecule compounds that modulate such pathways. High-content assays monitor the function of a protein or pathway by visualizing a change in an image-based readout, such as a change in the localization of a reporter protein. Examples of this can be the translocation of a fluorescently tagged protein from the cytoplasm to the nucleus or to the plasma membrane. One protein that is known to undergo such translocation is the Growth Factor Receptor-bound protein 2 (GRB2) that is recruited to the plasma membrane upon stimulation of a growth factor receptor and subsequently undergoes internalization. We have used GFP-tagged Grb2 previously to identify genes that are involved in EGFR signaling (Petschnigg et al., 2017). Ultimately, the assay can be adapted to cDNA expression cloning (Freeman et al., 2012) and can be used in early stage drug discovery to identify compounds that modulate or inhibit EGFR signaling and internalization (Antczak and Djaballah, 2016).

Progress in studies on autoantibodies against tumor-associated antigens in hepatocellular carcinoma

The diagnosis of hepatocellular carcinoma (HCC) patients using alpha-fetoprotein (AFP) is not accurate enough because of its low specificity. Autoantibodies as a complementary tool to AFP have attracted more and more attention. Although many kinds of autoantibodies are still unknown in patients with various cancers, autoantibodies against tumor-associated antigens (TAAs) have shown high specificities in cancer diagnosis due to the abnormal production of TAAs in cancer patients. At the present, serum proteomics analyses (SERPA), serological analyses of recombinantly expressed cDNA clone (SEREX), phage display technologies and high throughput protein microarray all have been applied in the screening of autoantibodies of HCC patients. Sometimes, the diagnosis value of a single or multiple autoantibodies can be a good complement to AFP. In this review, we describe and summarize the common technologies of screening autoantibodies and the application of autoantibodies in HCC.

Immunoproteomics technologies in the discovery of autoantigens in autoimmune diseases.

Proteomics technologies are often used for the identification of protein targets of the immune system. Here, we discuss the immunoproteomics technologies used for the discovery of autoantigens in autoimmune diseases where immune system dysregulation plays a central role in disease onset and progression. These autoantigens and associated autoantibodies can be used as potential biomarkers for disease diagnostics, prognostics and predicting/monitoring drug responsiveness (theranostics). Here, we compare a variety of methods such as mass spectrometry (MS)-based [serological proteome analysis (SERPA), antibody mediated identification of antigens (AMIDA), circulating immune complexome (CIC) analysis, surface enhanced laser desorption/ionization-time of flight (SELDI-TOF)], nucleic acid based serological analysis of antigens by recombinant cDNA expression cloning (SEREX), phage immunoprecipitation sequencing (PhIP-seq) and array-based immunoscreening (proteomic microarrays), luciferase immunoprecipitation systems (LIPS), nucleic acid programmable protein array (NAPPA) methods. We also review the relevance of immunoproteomic data generated in the last 10 years, with a focus on the aforementioned MS based methods.

The Wnt7's Tale: A story of an orphan who finds her tie to a famous family.

The transformation suppressor gene RECK was isolated by cDNA expression cloning (1998), and GPR124/TEM5 was detected as a tumor endothelial marker by differential screening (2000). The importance of Wnt7a/b and Gpr124 in brain angiogenesis was demonstrated by reverse genetics in mice (2008–2010). A series of recent studies using genetically engineered mice and zebrafish as well as luciferase reporter assays in cultured cells led to the discovery of functional interactions among Reck, Gpr124, and Wnt7a/b in triggering canonical Wnt signaling with relevance to embryonic brain angiogenesis and blood–brain barrier formation.

CBM-08AUTOANTIBODIES FOR SPECIFIC GLIOMA ANTIGENS ARE CANDIDATE SERUM BIOMARKERS FOR EARLY DIAGNOSIS AND ADEQUATE THERAPEUTIC TARGETS

Symptomatic glioma is usually not curable due to its invasive nature into surrounding normal brain. Establishment of serum biomarkers for gliomas would be beneficial both for early diagnosis and adequate post-operative monitoring. We searched for novel glioma antigens by serological identification of antigens utilizing recombinant cDNA expression cloning (SEREX), and found three independent proteins including filamin C (FLNC), SH3-domain GRB2-like 1 (SH3GL1), and pyruvate kinase M2 (PKM2) as candidate proteins. We analyzed with ELISA 131 glioma patients' sera along with 77 sera from healthy volunteers, 19 sera from patients with meningioma, and 24 patients with cerebral infarction. All the levels of serum autoantibodies to these proteins were significantly higher in low-grade gliomas than in high-grade gliomas, the healthy volunteers and the patients with other benign disease. In contrast, these proteins were expressed in glioma tissues and its level got higher as tumor grade advanced. The discrepancy was caused by the immunosuppressive microenvironments due to mesenchymal cell infiltration, which was verified by immunohistochemistry and ELISA for the mesenchymal cell-specific proteins. The rat glioma models using C6 and 9L also confirmed the increase of autoantibody levels for the SEREX-identified proteins in the early stage and the suppression in the late stage. These proteins were involved in the oncogenic process of gliomas and effectively elicit autologous antibody responses in the glioma patients without immunosuppression. The immunological reaction to some glioma antigens would contribute to the establishment of a novel diagnostic and therapeutic target for gliomas.

Elevated Serum Antibody Levels against Cyclin L2 in Patients with Esophageal Squamous Cell Carcinomai

Cyclin L2 (CCNL2) (Acc. No.: NM_030937) was detected as a tumor antigen of esophageal squamous cell carcinoma (SCC) by serological identification of antigens by recombinant cDNA expression cloning (SEREX). Serum anti-CCNL2 antibodies were detected by enzyme-linked immunosorbent assay more frequent in patients with esophageal SCC than in healthy donors (32% and 15%, P<0.01). An AlphaLISA further confirmed the significant difference in serum antibody levels between the patients and healthy donors using a different set of serum specimens. The expression levels of CCNL2 mRNA detected by reverse transcription polymerase chain reaction were higher in esophageal SCC tissues than those detected in adjacent normal esophageal tissues. We then analyzed for biological function by the transient transfection of CCNL2 expression plasmids into ras-NIH3T3 mouse fibroblasts followed by analysis via luciferase assay using p53-responsive reporter plasmids. CCNL2 increased the transactivation ability of p53, which was attenuated by protein kinase C (PKC) inhibitors or a dominant negative PKCa. Thus, it is possible that CCNL2 activates p53 via PKCa activation.

Circulating anti-filamin C autoantibody as a potential serum biomarker for low-grade gliomas.

BackgroundGlioma is the most common primary malignant central nervous system tumor in adult, and is usually not curable due to its invasive nature. Establishment of serum biomarkers for glioma would be beneficial both for early diagnosis and adequate therapeutic intervention. Filamins are an actin cross-linker and filamin C (FLNC), normally restricted in muscle tissues, offers many signaling molecules an essential communication fields. Recently, filamins have been considered important for tumorigenesis in cancers.MethodsWe searched for novel glioma-associated antigens by serological identification of antigens utilizing recombinant cDNA expression cloning (SEREX), and found FLNC as a candidate protein. Tissue expressions of FLNC (both in normal and tumor tissues) were examined by immunohistochemistry and quantitative RT-PCR analyses. Serum anti-FLNC autoantibody level was measured by ELISA in normal volunteers and in the patients with various grade gliomas.ResultsFLNC was expressed in glioma tissues and its level got higher as tumor grade advanced. Anti-FLNC autoantibody was also detected in the serum of glioma patients, but its levels were inversely correlated with the tissue expression. Serum anti-FLNC autoantibody level was significantly higher in low-grade glioma patients than in high-grade glioma patients or in normal volunteers, which was confirmed in an independent validation set of patients’ sera. The autoantibody levels in the patients with meningioma or cerebral infarction were at the same level of normal volunteers, and they were significantly lower than that of low-grade gliomas. Total IgG and anti-glutatione S-transferase (GST) antibody level were not altered among the patient groups, which suggest that the autoantibody response was specific for FLNC.ConclusionsThe present results suggest that serum anti-FLNC autoantibody can be a potential serum biomarker for early diagnosis of low-grade gliomas while it needs a large-scale clinical study.

Molecular cloning of propiomelanocortin cDNA and multi-tissue mRNA expression in Parasilurus asotus

We investigated the structure,evolution,and tissue specific expression of Parasilurus asotus proopiomelanocortin(POMC) cDNA using RT-PCR and RACE.The POMC cDNA was 1 099 bp in length and contained a 129 bp 5′UTR,a 322 bp 3′UTR,and a 648 bp open reading frame encoding a protein of 215 amino acids.The catfish POMC protein contained a signal peptide(SP,Met1-Ala28),an N-terminal peptide(Gln29-Asp102),adrenocorticotropic hormone(ACTH,Ser105-Met144),α-melanocyte stimulating hormone(α-MSH,Ser105-Val117),corticotropin-like intermediate lobe peptide(CLIP,Arg123–Met144),β-lipotropin(β-LPH,Glu145-Ser158),β-MSH(Asp161-Ser177),and β-Endorphin(β-EP,Tyr178-Leu215).Catfish POMC protein does not contain a γ-MSH region and the majority of the joining peptide and part of the γ-LPH were deleted.The protein shared the highest similarity(88%) with channel catfish(Parasilurus asotus) POMC.The most highly conserved active peptide was α-MSH,followed by β-MSH,β-EP,and CLIP whereas γ-MSH,joining peptide(JP),and γ-LPH were located in the most divergent region.The protein had a calculated molecular weight of 24.66 kD,a theoreti-cal isoelectric point of 7.25,was hydrophilic in most regions,and was rich in B cell antigenicity positions.Catfish POMC mRNA was primarily expressed in the pituitary and the concentration of mRNA was much higher in brain than in other tissues.

Identification of differentially expressed genes in 72 h styles from self-incompatible Citrus reticulata

Self-incompatibility (SI) is one important factor that can result in seedless fruits of Citrus. Our previous study showed that ‘Wuzishatangju’ mandarin was gametophytic self-incompatibility (GSI) and 72h was the crucial stage for the shift from a self-compatible to a self-incompatible state. However, it is not clear what genes are involved in the process. In this study, two SSH libraries were constructed to screen differentially expressed genes using 72h styles after self-pollination of ‘Wuzishatangju’ and cross-pollination of ‘Wuzishatangju’בShatangju’. 106 and 97 differentially expressed cDNA clones were sequenced and identified from forward and reverse SSH libraries, respectively. Differentially expressed ESTs are possibly involved in the SI reaction of ‘Wuzishatangju’ through S-phase kinase-associated protein1 (SKP1)-like activity, autoinhibited Ca2+-ATPase 1, U-box domain protein, and serine/threonine kinase. Results from quantitative real-time PCR (qPCR) showed that SKP1-like and U-box were obviously up-regulated in ovaries before pollination of ‘Wuzishatangju’, and approximately 20- and 2-fold higher than that of ‘Shatangju’, respectively. The SKP1-like and U-box were obviously up-regulated in pistils at 4d after self-pollination of ‘Wuzishatangju’, and approximately 20- and 800-fold higher than that the same stage after cross-pollination of ‘Wuzishatangju’בShatangju’, respectively. The potential involvement of these genes in the SI reaction is discussed.

Dual Roles of the Transcription Factor Grainyhead-like 2 (GRHL2) in Breast Cancer

Using a retrovirus-mediated cDNA expression cloning approach, we identified the grainyhead-like 2 (GRHL2) transcription factor as novel protooncogene. Overexpression of GRHL2 in NIH3T3 cells induced striking morphological changes, an increase in cell proliferation, anchorage-independent growth, and tumor growth in vivo. By combining a microarray analysis and a phylogenetic footprinting analysis with various biochemical assays, we identified the epidermal growth factor receptor family member Erbb3 as a novel GRHL2 target gene. In breast cancer cell lines, shRNA-mediated knockdown of GRHL2 expression or functional inactivation of GRHL2 using dominant negative GRHL2 proteins induces down-regulation of ERBB3 gene expression, a striking reduction in cell proliferation, and morphological and phenotypical alterations characteristic of an epithelial-to-mesenchymal transition (EMT), thus implying contradictory roles of GRHL2 in breast carcinogenesis. Interestingly, we could further demonstrate that expression of GRHL2 is directly suppressed by the transcription factor zinc finger enhancer-binding protein 1 (ZEB1), which in turn is a direct target for repression by GRHL2, suggesting that the EMT transcription factors GRHL2 and ZEB1 form a double negative regulatory feedback loop in breast cancer cells. Finally, a comprehensive immunohistochemical analysis of GRHL2 expression in primary breast cancers showed loss of GRHL2 expression at the invasive front of primary tumors. A pathophysiological relevance of GRHL2 in breast cancer metastasis is further demonstrated by our finding of a statistically significant association between loss of GRHL2 expression in primary breast cancers and lymph node metastasis. We thus demonstrate a crucial role of GRHL2 in breast carcinogenesis.

A Carboxy-Terminally Truncated Human CPSF6 Lacking Residues Encoded by Exon 6 Inhibits HIV-1 cDNA Synthesis and Promotes Capsid Disassembly

Since HIV-1 replication is modulated at multiple stages by host cell factors, identification and characterization of those host cell factors are expected to contribute to the development of novel anti-HIV therapeutics. Previous studies showed that a C-terminally truncated cytosolic form of cleavage and polyadenylation-specific factor 6 (CPSF6-358) inhibits HIV-1 infection through interference with HIV-1 trafficking to the nucleus. Here we identified and characterized a different configuration of C-terminally truncated human CPSF6 (hCPSF6-375) through cDNA expression cloning coupled with ganciclovir-mediated lethal selection. Notably, hCPSF6-375, but not mouse CPSF6-358 (mCPSF6-358) as previously reported, remarkably interfered with viral cDNA synthesis after HIV-1 infection. Moreover, we found that hCPSF6-375 aberrantly accelerated the disassembly of the viral capsid in target cells, while CPSF6-358 did not. Sequence comparison of CPSF6-375 and CPSF6-358 cDNAs showed a lack of exon 6 and additional coding sequence for 54 amino acid residues in the C terminus of hCPSF6-375. Mutational analyses revealed that the residues encoded by exon 6, but not the C-terminal 54 residues in hCPSF6-375, is responsible for impaired viral cDNA synthesis by hCPSF6-375. This is the first report demonstrating a novel mode of HIV-1 inhibition by truncated forms of CPSF6 that involves rapid capsid disassembly and inhibition of viral cDNA synthesis. These findings could facilitate an increased understanding of viral cDNA synthesis in light of the viral capsid disassembly.

Autoantibody against WD repeat domain 1 is a novel serological biomarker for screening of thyroid neoplasia

Thyroid nodules are common among adults, and accurate diagnosis is critical in for management decisions. Ultrasound and fine needle aspiration cytology are the most common methods to evaluate nodules, but they are not practical for screening large numbers of patients because of cost and time considerations. The aim of this study was to isolate an autoantibody to tumour antigen, WD repeat domain 1 (WDR1), and evaluate its diagnostic sensitivity and specificity for thyroid neoplasms. We investigated serological biomarkers in patients with thyroid carcinoma who had a poor prognosis. Using a serological analysis of recombinant cDNA expression cloning (SEREX) strategy, we isolated WDR1 and its specific autoantibody in the sera of patients with undifferentiated thyroid carcinoma (UTC). We examined using indirect ELISA, the titre of the anti-WDR1 antibody (AWA) in 54 study patients: 10 with UTC, 20 with papillary thyroid carcinoma (PTC), 17 with benign thyroid nodule (BTN), 7 with autoimmune thyroid disease (AITD), as well as 38 controls (N). WDR1 was ubiquitously expressed in various types of thyroid tissues. However, the titre of AWA in UTC and PTC was significantly higher than that in BTN, AITD and N (P<0·001). No significant correlation was observed between thyroid function, serum thyroglobulin and tumour diameter. The cut-off value estimated using ROC to differentiate malignancies from others was 0·95 (sensitivity 96·7%, specificity 91·9%, AUC 0·969, P<0·001). Anti-WDR1 antibody could be a novel approach for serological screening of PTC and UTC, and could be an efficient and inexpensive biomarker.

Identifying differentially expressed genes in pollen from self-incompatible "Wuzishatangju" and self-compatible "Shatangju" mandarins.

Self-incompatibility (SI) is one of the important factors that can result in seedless fruit in Citrus. However, the molecular mechanism of SI in Citrus is not yet clear. In this study, two suppression subtractive hybridization (SSH) libraries (forward, F and reverse, R) were constructed to isolate differentially expressed genes in pollen from “Wuzishatangju” (SI) and “Shatangju” (self-compatibility, SC) mandarins. Four hundred and sixty-eight differentially expressed cDNA clones from 2077 positive clones were sequenced and identified. Differentially expressed ESTs are possibly involved in the SI reaction of “Wuzishatangju” by regulating pollen development, kinase activity, ubiquitin pathway, pollen-pistil interaction, and calcium ion binding. Twenty five SI candidate genes were obtained, six of which displayed specific expression patterns in various organs and stages after self- and cross-pollination. The expression level of the F-box gene (H304) and S1 (F78) in the pollen of “Wuzishatangju” was 5-fold higher than that in “Shatangju” pollen. The F-box gene, S1, UBE2, UBE3, RNaseHII, and PCP were obviously up-regulated in pistils at 3 d after self-pollination of “Wuzishatangju”, approximately 3-, 2-, 10-, 5-, 5-, and 2-fold higher, respectively than that at the same stage after cross-pollination of “Wuzishatangju” × “Shatangju” pistils. The potential involvement of these genes in the pollen SI reaction of “Wuzishatangju” is discussed.

Identification of autoantibodies expressed in acquired aplastic anaemia

Acquired aplastic anaemia (aAA) is recognized as an autoimmune disorder; however, the autoantigens and target cells involved remain elusive. Expression of autoantibodies and their target cells were examined using the haematopoietic cell line K562 and bone marrow stromal cell line hTS-5; 43·5% and 21·7% of aAA expressed autoantibody against K562 and hTS-5 cells, respectively. The autoantigens were identified by serological identification of antigens through recombinant cDNA expression cloning. This study indicates that haematopoietic cells are the targets of immune abnormality in aAA. These autoantibodies may be utilized to distinguish patients associated with immune abnormality from bone marrow failure syndrome.

Autologous antibody to src-homology 3-domain GRB2-like 1 specifically increases in the sera of patients with low-grade gliomas

BackgroundGlioma is the most common primary malignant central nervous system tumor in adult, and is usually not curable in spite of various therapeutic approaches. Clarification of the oncogenic process in its early stage is important for the diagnosis and effective therapy.MethodsIn the present study, we used the serological identification of antigens by recombinant cDNA expression cloning (SEREX) to explore the subtle changes of the protein expression in low-grade glioma. The levels of serum autoantibodies to the SEREX-identified glioma-related antigens were analyzed by ELISA, and the epitope site was identified using deletion mutants and overlap peptide array. Changes in the serum autoantibody levels were examined in the rat glioma model using C6 and 9 L glioma cell lines.ResultsWe identified 31 glioma-related antigens by SEREX. Among them, the serum level of autoantibody to src-homology 3-domain GRB2-like 1 (SH3GL1) was significantly higher in patients with low-grade glioma than healthy volunteers or high-grade gliomas. The 10 amino-acids at the C-terminal were identified as the epitope site by the overlap peptide array and the ELISA using deletion mutants. The tissue expression of SH3GL1 protein increased in proportion to glioma progression. The rat glioma models confirmed the increase of anti-SH3GL1 autoantibody level in the early stage and the suppression in the late stage.ConclusionSH3GL1 may be involved in the oncogenic process of gliomas and effectively elicit an autologous antibody response in low-grade gliomas. The immunological reaction to SH3GL1 would contribute to the establishment of a novel diagnostic and therapeutic target for gliomas.

Molecular cloning reveals nearly half of patients with Crohn's disease have an antibody to peroxiredoxin 6‐like protein

Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) of unknown etiology. We aimed to identify the etiological agent of CD using a molecular cloning strategy that was particularly focused on identifying agents causing immune abnormalities and infectious agents. We constructed a cDNA library derived from the inflamed intestinal tissue of a CD patient, and screened 1.5 million clones in this library with the serum from another typical CD patient. The expressed cDNA clones that positively reacted with the serum were then expressed as fusion proteins with glutathione S-transferase, and western blotting was performed using the sera of 22 CD, 13 ulcerative colitis (UC), and 16 non-IBD patients. We identified nine positive clones that did not contain any viral or bacterial genomic DNA. Of these, we selected one clone (clone 50) with which the typical CD patient's serum most strongly reacted. Clone 50 is highly homologous to the antioxidant protein peroxiredoxin 6. In western blotting, the sera of 47.6% CD patients (small intestine type 80%, large and small intestine type 43%, large intestine type 0%) showed strong reactivity to clone 50, none of the UC patients were reactive to clone 50, and 18.8% of non-IBD patients were very weakly reactive to it. We also found that the expression of peroxiredoxin 6 was significantly increased in inflamed intestinal epithelia of CD. The present study first showed that some CD patients have an antibody against peroxiredoxin 6-like protein, which may be involved in the pathogenesis of CD.