IgM to IgG Class Switching Is a Necessary Step for Pemphigus Phenotype Induction in Desmoglein 3-Specific B Cell Receptor Knock-in Mouse.

Pemphigus vulgaris is an autoimmune blistering disease caused by IgG targeting desmoglein 3 (Dsg3), an adhesion molecule of keratinocytes. Anti-Dsg3 IgG production is prevented in healthy individuals, but it is unclear how Dsg3-specific B cells are regulated. To clarify the immunological condition regulating Dsg3-specific B cells, a pathogenic anti-Dsg3 Ig (AK23) knock-in mouse was generated. AK23 knock-in B cells developed normally without undergoing deletion or acquiring an anergic phenotype in vivo. The knock-in B cells showed Ca2+ influx upon IgM cross-linking and differentiated into AK23-IgG+ B cells after LPS and IL-4 stimulation in vitro that induced a pemphigus phenotype after adoptive transfer into Rag2 -/- mice. However, the knock-in mouse itself produced AK23-IgM but little IgG without blisters in vivo. Dsg3 immunization and skin inflammation caused AK23-IgG production and a pemphigus phenotype in vivo. Furthermore, Fcgr2b deficiency or haploinsufficiency spontaneously induced AK23-IgG production and a pemphigus phenotype with poor survival rates in AK23 knock-in mice. To assess Fcgr2b involvement in Ig class-switch efficiency, postswitch transcripts of B cells were quantified and significantly higher in Fcgr2b -/- and Fcgr2b +/- mice than wild-type mice in a gene dose-dependent manner. Finally, RNA sequencing revealed reduced expression of FCGR2B and FcγRIIB-related genes in patient B cells. These results indicated that Dsg3-specific B cells do not spontaneously perform pathogenic class switching in vivo, and pemphigus phenotype induction was prevented under normal conditions. Attenuated FcγRIIB signaling is also one of the drivers for pathogenic class switching and is consistent with immunological features identified from clinical samples. This study unveiled a characteristic immune state silencing autoreactive B cells in mice.

Peripheral tolerance by Treg via constraining OX40 signal in autoreactive T cells against desmoglein 3, a target antigen in pemphigus.

Antigen-specific peripheral tolerance is crucial to prevent the development of organ-specific autoimmunity. However, its function decoupled from thymic tolerance remains unclear. We used desmoglein 3 (Dsg3), a pemphigus antigen expressed in keratinocytes, to analyze peripheral tolerance under physiological antigen-expression conditions. Dsg3-deficient thymi were transplanted into athymic mice to create a unique condition in which Dsg3 was expressed only in peripheral tissue but not in the thymus. When bone marrow transfer was conducted from high-avidity Dsg3-specific T cell receptor-transgenic mice to thymus-transplanted mice, Dsg3-specific CD4+ T cells developed in the transplanted thymus but subsequently disappeared in the periphery. Additionally, when Dsg3-specific T cells developed in Dsg3-/- mice were adoptively transferred into Dsg3-sufficient recipients, the T cells disappeared in an antigen-specific manner without inducing autoimmune dermatitis. However, Dsg3-specific T cells overcame this disappearance and thus induced autoimmune dermatitis in Treg-ablated recipients but not in Foxp3-mutant recipients with dysfunctional Tregs. The molecules involved in disappearance were sought by screening the transcriptomes of wild-type and Foxp3-mutant Tregs. OX40 of Tregs was suggested to be responsible. Consistently, when OX40 expression of Tregs was constrained, Dsg3-specific T cells did not disappear. Furthermore, Tregs obtained OX40L from dendritic cells in an OX40-dependent manner in vitro and then suppressed OX40L expression in dendritic cells and Birc5 expression in Dsg3-specific T cells in vivo. Lastly, CRISPR/Cas9-mediated knockout of OX40 signaling in Dsg3-specific T cells restored their disappearance in Treg-ablated recipients. Thus, Treg-mediated peripheral deletion of autoreactive T cells operates as an OX40-dependent regulatory mechanism to avoid undesired autoimmunity besides thymic tolerance.

Fibroblast growth factor receptor type 4 as a potential therapeutic target in clear cell renal cell carcinoma.

BACKGROUND: Several clear cell renal cell carcinoma (ccRCC) cases harbour fibroblast growth factor receptor 4 (FGFR4) gene copy number (CN) gains. In this study, we investigated the functional contribution of FGFR4 CN amplification in ccRCC. METHODS: The correlation between FGFR4 CN determined via real-time PCR and protein expression evaluated using western blotting and immunohistochemistry was assessed in ccRCC cell lines (A498, A704, and 769-P), a papillary RCC cell line (ACHN), and clinical ccRCC specimens. The effect of FGFR4 inhibition on ccRCC cell proliferation and survival was assessed via either RNA interference or using the selective FGFR4 inhibitor BLU9931, followed by MTS assays, western blotting, and flow cytometry. To investigate whether FGFR4 is a potential therapeutic target, a xenograft mouse model was administered BLU9931. RESULTS: 60% of ccRCC surgical specimens harboured an FGFR4 CN amplification. FGFR4 CN was positively correlated with its protein expression. All ccRCC cell lines harboured FGFR4 CN amplifications, whereas ACHN did not. FGFR4 silencing or inhibition attenuated intracellular signal transduction pathways, resulting in apoptosis and suppressed proliferation in ccRCC cell lines. BLU9931 suppressed tumours at a tolerable dose in the mouse model. CONCLUSION: FGFR4 contributes to ccRCC cell proliferation and survival following FGFR4 amplification, making it a potential therapeutic target for ccRCC.

Anti-human leukocyte antigen and anti-ABO antibodies after SARS-CoV-2 mRNA vaccination in kidney transplant recipients.

INTRODUCTION: In this study, we evaluated whether SARS-CoV-2 mRNA vaccines induce anti-human leukocyte antigen (HLA) antibodies and anti- ABO blood type antibodies (ABOAb) in kidney transplant recipients (KTRs). METHODS: Sixty-three adult KTRs with functioning grafts who received two doses of the SARS-CoV-2 mRNA vaccine were enrolled in this cohort. Changes in anti-ABO blood type immunoglobulin IgM and IgG antibody titers, flow panel reactive antibody (PRA), de novo donor-specific anti-human leukocyte antigen antibodies (DSA), and kidney allograft function before and after vaccination were evaluated. RESULTS: Only one patient experienced conversion from negative to positive flow PRA after vaccination. However, there was no DSA in single antigen flow-bead assays. The mean fluorescence intensity (MFI) in the eight DSA-positive recipients did not significantly change before and after vaccination (p = .383), and no additional DSA was produced after vaccination in those patients. No significant elevation of ABOAb titer was observed for either IgM (p = .438) or IgG (p = .526) after vaccination. There was no significant deterioration in estimated glomerular filtration rate (eGFR) after vaccination (p = .877) or elevation of the urine protein-to-creatinine ratio (p = .209) after vaccination. One episode of AMR was observed in addition to a preexisting acute cellular rejection. CONCLUSIONS: The SARS-CoV-2 mRNA vaccine did not induce anti-HLA antibody or ABOAb production in KTRs.

Attenuation of OX40 signaling suppression by age disrupts peripheral deletion of CD4+ T cells specific for the epidermal autoantigen desmoglein 3.

Various autoimmune responses increase with age, but the underlying mechanism is not clear. In this study, we used CD4+ T cells expressing a transgenic T cell receptor specific for desmoglein 3 (Dsg3), which is the target antigen of the autoimmune bullous disease pemphigus vulgaris, to examine how peripheral immunological tolerance against pathogenic autoreactive CD4+ T cells changes with age. Dsg3-specific T cells were deleted within 14 days after adoptive transfer into young mice (8 weeks old), while they escaped deletion when transferred into older mice over 42 weeks old. Dsg3-specific T cells produced higher levels of the proinflammatory cytokine IFN-γ in aged mice than in young mice. In addition, the expression levels of both OX40 and Birc5, which are important for cell survival in T cell clonal proliferation, were higher in aged than in young mice. The dysfunction in suppressing proinflammatory cytokine secretion and Birc5 upregulation in Dsg3-specific autoreactive T cells may reflect an aspect of the preliminary steps in autoimmune disease development in the aged population. Understanding this mechanism may lead to better risk evaluation of autoimmune disease development and to onset prevention.

Immunogenicity and safety of two doses of SARS-CoV-2 mRNA vaccine in kidney transplant recipients with low-dose rituximab.

OBJECTIVES: We evaluated whether the treatment history of low-dose rituximab affected safety profiles, and humoral and cellular responses induced by severe acute respiratory syndrome coronavirus 2 messenger ribonucleic acid vaccine in healthy controls and kidney transplant recipients. METHODS: We enrolled 10 healthcare workers as controls, 22 kidney transplant recipients with rituximab, and 36 kidney transplant recipients without rituximab without history of coronavirus disease 2019 who received two doses of vaccine. We assessed anti-severe acute respiratory syndrome coronavirus 2 spike antibody and the antigen-specific T cells using enzyme-linked immunospot against spike protein at baseline and after two doses of vaccine. RESULTS: All controls showed anti-severe acute respiratory syndrome coronavirus 2 antibody seroconversion and enzyme-linked immunospot positivity. Only 19/58 (33%) kidney transplant recipients experienced anti-severe acute respiratory syndrome coronavirus 2 antibody seroconversion and 31/58 (53%) kidney transplant recipients developed enzyme-linked immunospot assay positivity after vaccination. The anti-severe acute respiratory syndrome coronavirus 2 antibody seroconversion rate and enzyme-linked immunospot assay positivity rate after vaccination were not significantly different between kidney transplant recipients with or without rituximab. Multivariate regression analysis demonstrated rituximab was not associated with a lack of humoral and cellular responses to the vaccine. CONCLUSIONS: Low-dose rituximab in kidney transplant recipients did not affect humoral or cellular responses to the severe acute respiratory syndrome coronavirus 2 messenger ribonucleic acid vaccine without severe systemic adverse events including the deterioration of kidney function.

Effect of Extracellular Signal-Regulated Protein Kinase 5 Inhibition in Clear Cell Renal Cell Carcinoma.

(1) Background: Extracellular signal-regulating kinase 5 (ERK5) has been implicated in many cellular functions, including survival, proliferation, and vascularization. Our objectives were to examine the expression and effect of ERK5 in clear cell renal cell carcinoma (ccRCC). (2) Methods: The expressions of ERK5 and its regulating micro-RNA miR-143 were investigated using immunohistochemistry and quantitative reverse transcriptase PCR in surgical specimens of ccRCC patients. With invitro and in vivo studies, we used pharmacologic ERK5 inhibitor XMD8-92, RNA interference, pre-miR-143 transduction, Western blotting, MTS assay, apoptosis assay, and subcutaneous xenograft model. (3) Results: A strong ERK5 expression in surgical specimen was associated with high-grade (p = 0.01), high-recurrence free rate (p = 0.02), and high cancer-specific survival (p = 0.03). Expression levels of ERK5 and miR-143 expression level were correlated (p = 0.049). Pre-miR-143 transduction into ccRCC cell A498 suppressed ERK5 expression. ERK5 inhibition enhanced cyclin-dependent kinase inhibitor p21 expression and decreased anti-apoptotic molecules BCL2, resulting in decreased cell proliferation and survival both in ccRCC and endothelial cells. In the xenograft model, ERK5 inhibitor XMD8-92 suppressed tumor growth. (4) Conclusions: ERK5 is regulated by miR-143, and ERK5 inhibition is a promising target for ccRCC treatment.

GPI-80 Augments NF-κB Activation in Tumor Cells.

Recent studies have discovered a relationship between glycosylphosphatidylinositol (GPI)-anchored protein 80 (GPI-80)/VNN2 (80 kDa GPI-anchored protein) and malignant tumors. GPI-80 is known to regulate neutrophil adhesion; however, the action of GPI-80 on tumors is still obscure. In this study, although the expression of GPI-80 mRNA was detectable in several tumor cell lines, the levels of GPI-80 protein were significantly lower than that in neutrophils. To clarify the function of GPI-80 in tumor cells, GPI-80-expressing cells and GPI-80/VNN2 gene-deleted cells were established using PC3 prostate cancer cells. In GPI-80-expressing cells, GPI-80 was mainly detected in vesicles. Furthermore, soluble GPI-80 in the conditioned medium was associated with the exosome marker CD63 and was also detected in the plasma obtained from prostate cancer patients. Unexpectedly, cell adhesion and migration of GPI-80-expressing PC3 cells were not modulated by anti-GPI-80 antibody treatment. However, similar to the GPI-80 family molecule, VNN1, the pantetheinase activity and oxidative state were augmented in GPI-80-expressing cells. GPI-80-expressing cells facilitated non-adhesive proliferation, slow cell proliferation, NF-κB activation and IL-1ß production. These phenomena are known to be induced by physiological elevation of the oxidative state. Thus, these observations indicated that GPI-80 affects various tumor responses related to oxidation.

GPI-80 as a Useful Index for Myeloid Cell Heterogeneity and a Potential Prognostic Biomarker for Metastatic Renal Cell Carcinoma.

Myeloid-derived suppressor cells (MDSCs), which include neutrophilic MDSCs and monocytic MDSCs, exhibit high immunosuppressive activity. Glycosylphosphatidylinositol-anchored 80 kD protein (GPI-80) is selectively expressed on mature neutrophils in healthy individuals. Increased GPI-80 expression on monocytes and variations in GPI-80 expression on neutrophils indicate the appearance of MDSCs in the peripheral blood of cancer patients. However, it is still unclear whether GPI-80 expression on myeloid cells, neutrophilic MDSCs and monocytic MDSCs, is correlated with the clinical outcomes of patients with cancer. In this study, we investigated the characteristics of myeloid cells expressing GPI-80 and the implication of GPI-80 expression in the clinical outcomes of patients with metastatic renal cell carcinoma (mRCC), in which primary renal cell carcinoma spreads from the kidney to other organs. The study included 20 patients with mRCC (a mean age of 66.0 years) and 16 healthy volunteers (a mean age of 47.8 years). To determine the heterogeneity of myeloid cells in peripheral blood samples, we performed the three-dimensional principal component analysis using the combination of GPI-80, CD16, and latency-associated peptide-1 (LAP), derived from the N-terminal region of transforming growth factor-ß1 precursor. The results showed that myeloid cells in mRCC patients were widely distributed and clearly distinguishable from those in the healthy volunteers. The survival analysis revealed that GPI-80 expression on neutrophils and monocytes was correlated with poor prognostic outcomes of patients with mRCC. In conclusion, the expression of GPI-80 on myeloid cells, a useful index for the heterogeneity of MDSCs, serves as a potential prognostic biomarker for mRCC.

Transferrin isoforms in cerebrospinal fluid and their relation to neurological diseases.

Iron plays many important roles in the brain, including involvement in myelination, neurotransmission and electron transfer in the respiratory chain. Transferrin (Tf), an iron transporter, is mainly biosynthesized in the liver, but can also be biosynthesized in the brain; i.e., by oligodendrocytes and the choroid plexus, a cerebrospinal fluid (CSF) producing tissue. The CSF contains two Tf isoforms, brain-type Tf and serum-type Tf, which differ in their glycan structures. Brain-type Tf is uniquely glycolsylated with biantennary asialo- and agalacto-complex type N-glycans that carry bisecting ß1,4-GlcNAc and core α1,6-Fuc. The glycans of serum-type Tf in the CSF are similar to those of Tf in serum. Biochemical analyses reveal that the apparent molecular size of brain-type Tf is smaller than that of serum-type Tf, and that hydrophobic patches are exposed on brain-type Tf as demonstrated by hydrophobic probe binding studies. We found that brain-type Tf levels were decreased in idiopathic normal pressure hydrocephalus, in which CSF production is suspected to decrease, while brain-type Tf increased in spontaneous intracranial hypotension, in which CSF production is suspected to increase. These results suggest that brain-type Tf could be a biomarker of altered CSF production.

Spontaneous intracranial hypotension is diagnosed by a combination of lipocalin-type prostaglandin D synthase and brain-type transferrin in cerebrospinal fluid.

BACKGROUND: Spontaneous intracranial hypotension (SIH) is caused by cerebrospinal fluid (CSF) leakage. Definitive diagnosis can be difficult by clinical examinations and imaging studies. METHODS: SIH was diagnosed with the following criteria: (i) evidence of CSF leakage by cranial magnetic resonance imaging (MRI) findings of intracranial hypotension and/or low CSF opening pressure; (ii) no recent history of dural puncture. We quantified CSF proteins by ELISA or Western blotting. RESULTS: Comparing with non-SIH patients, SIH patients showed significant increase of brain-derived CSF glycoproteins such as lipocalin-type prostaglandin D synthase (L-PGDS), soluble protein fragments generated from amyloid precursor protein (sAPP) and "brain-type" transferrin (Tf). Serum-derived proteins such as albumin, immunoglobulin G, and serum Tf were also increased. A combination of L-PGDS and brain-type Tf differentiated SIH from non-SIH with sensitivity 94.7% and specificity 72.6%. CONCLUSION: L-PGDS and brain-type Tf can be biomarkers for diagnosing SIH. GENERAL SIGNIFICANCE: L-PGDS and brain-type Tf biosynthesized in the brain appears to be markers for abnormal metabolism of CSF.

Lectin-Based Assay for Glycoform-Specific Detection of α2,6-sialylated Transferrin and Carcinoembryonic Antigen in Tissue and Body Fluid.

Antibodies are useful for detecting glycoprotein antigens, but a conventional antibody recognizes only a protein epitope rather than a glycan. Thus, glycan isoform detection generally requires time- and labor-consuming processes such as lectin affinity column chromatography followed by sandwich ELISA. We recently found antigen-antibody reactions that were inhibited by lectin binding to glycans on the glycoprotein antigen, leading to a convenient glycoform-specific assay. Indeed, Sambucus sieboldiana agglutinin (SSA) lectin, a binder to sialylα2,6galactose residue, inhibited antibody binding to α2,6-sialylated transferrin (Tf) (SSA inhibition). SSA inhibition was not observed with other glycoforms, such as periodate-treated, sialidase-treated and sialidase/galactosidase-treated Tf, suggesting that the assay was glycoform-specific. SSA inhibition was also applicable for visualizing localization of α2,6-sialylated-Tf in a liver section. This is the first immunohistochemical demonstration of glycoform localization in a tissue section. SSA inhibition was utilized for establishing ELISA to quantify α2,6-sialylated carcinoembryonic antigen (CEA), a marker for various cancers. In addition, α2,6-sialylated-CEA was visualized in a colonic adenocarcinoma section by SSA inhibition. The method would further be applicable to a simple and rapid estimation of other α2,6-sialylated glycoproteins and have a potential aid to histopathological diagnosis.

A unique glycan-isoform of transferrin in cerebrospinal fluid: A potential diagnostic marker for neurological diseases.

BACKGROUND: Cerebrospinal fluid (CSF) is sequestered from blood by the blood-brain barrier and directly communicates with brain parenchymal interstitial fluid, leading to contain specific biomarkers of neurological diseases. SCOPE OF REVIEW: CSF contains glycan isoforms of transferrin (Tf): one appears to be derived from the brain and the other from blood. MAJOR CONCLUSIONS: CSF contains two glycan-isoforms; brain-type Tf and serum-type Tf. Glycan analysis and immunohistochemistry suggest that serum-type Tf having α2, 6sialylated glycans is derived from blood whereas brain-type Tf having GlcNAc-terminated glycans is derived from the choroid plexus, CSF producing tissue. The ratio of serum-type/brain-type Tf differentiates Alzheimer's disease from idiopathic normal pressure hydrocephalus, which is an elderly dementia caused by abnormal metabolism of CSF. The ratios in Parkinson's disease (PD) patients were higher than those of controls and did not appear to be normally distributed. Indeed, detrended normal Quantile-Quantile plot analysis reveals the presence of an independent subgroup showing higher ratios in PD patients. The subgroup of PD shows higher levels of CSF α-synuclein than the rest, indicating that PD includes two subgroups, which differ in levels of brain-type Tf and α-synuclein. GENERAL SIGNIFICANCE: Glycosylation in central nervous system appears to be unique. The unique glycan may be a tag for glycoprotein, which is biosynthesized in the central nervous system. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.

Lectin inhibits antigen-antibody reaction in a glycoform-specific manner: Application for detecting α2,6sialylated-carcinoembryonic antigen.

Carcinoembryonic antigen (CEA) is a glycoprotein marker, which is widely used for diagnosing various cancers, especially colon adenocarcinoma. In addition, CEA mediates homotypic adhesion of colon adenocarcinoma cells, which appears to favor hematogenous metastasis. CEA carries α2,6sialyl residues on its N-glycans whereas a normal counterpart, normal fecal antigen-2, does α2,3sialyl residues, suggesting that cancer-specific  α2,6sialylation on CEA may play a role for cell invasion and metastasis. A simple and rapid estimation of α2,6sialyled CEA in detergent extracts from formalin-fixed colon adenocarcinoma by "lectin inhibition" is reported. In the lectin inhibition method, Sambucus sieboldiana Agglutinin (SSA) lectin, an α2,6sialic acid binder, was used as a glycoform-specific inhibitor for antigen-antibody reaction in ELISA. Detergent extracts from colon adenocarcinoma showed a fair amount of ELISA signal in the absence of SSA whereas the signal was markedly reduced (45≈74%) in the presence of SSA, suggesting that the extracts contains α2,6sialyled CEA. The presence of α2,6sialyled CEA in the extracts was confirmed by lectin microarray, in which SSA, Sambucus nigra agglutinin, and Trichosanthes japonica agglutinin I lectins were used as α2,6sialyl binders. Thus lectin inhibition is a simple and rapid method for detecting α2,6sialyled CEA even in crude detergent extracts from formalin-fixed adenocarcinoma tissue.

GSK-3 directly regulates phospho-4EBP1 in renal cell carcinoma cell-line: an intrinsic subcellular mechanism for resistance to mTORC1 inhibition.

BACKGROUND: The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin 1 (mTORC1) signaling pathway is aberrantly activated in renal cell carcinoma (RCC). We previously demonstrated glycogen synthase kinase-3ß (GSK-3ß) positively regulated RCC proliferation. The aim of this study was to evaluate the role of GSK-3 in the PI3K/Akt/mTORC1 pathway and regulation of the downstream substrates, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), ribosomal protein S6 kinase (S6K), and ribosomal protein S6 (S6RP). METHODS: We used human RCC cell lines (ACHN, Caki1, and A498) and, as normal controls, human renal proximal tubular epithelial cell (HRPTEpC) and non-tumorous kidney tissues that were obtained surgically for treatment of RCC patients. Rapamycin-resistant ACHN (ACHN/RR) cells were generated with chronic exposure of ACHN to rapamycin ranging from 1nM finally to 1 µM. Cell viability, cell cycling and direct interaction between GSK-3ß and 4EBP1 were evaluated with MTS assay, flowcytometry and in vitro kinase assay with recombinant GSK-3ß and 4EBP1products, respectively. Protein expression and phosphorylation of molecules associated with the PI3K/Akt/mTORC1 pathway were examined by immunoblotting. Effects of drug combination were determined as the combination index with CompuSyn software. RESULTS: Overexpression and phosphorylation of 4EBP1 and S6RP together with GSK-3 activation were observed in RCC cell lines, but not in human normal kidney cells and tissues. Cell proliferation, p4EBP1 and pS6RP were strongly suppressed by GSK-3 inhibition. Rapamycin and LY294002 sufficiently decreased pS6RP, but only moderately p4EBP1. In vitro kinase assays showed that recombinant GSK-3ß phosphorylated recombinant 4EBP1, and the effect was blocked by GSK-3 inhibitors. Different from rapamycin, AR- A014418 remarkably inhibited cell proliferation, and rapidly suppressed p4EBP1 and pS6RP in ACHN and ACHN/RR (in 30 min to 1 h). AR- A014418 and rapamycin combination showed additivity at lower concentrations, but antagonism at higher concentrations. CONCLUSIONS: GSK-3ß could directly phosphorylate 4EBP1 and activate the mTORC1 downstream signaling cascades to enhance protein biosynthesis and cell proliferation in RCC cell lines independent of rapamycin sensitivity. The direct GSK-3ß/4EBP1 pathway might be an important subcellular mechanism as an inherent equipment for RCC cells to acquire clinical chemoresistance to mTORC1 inhibitors.

A diagnostic marker for superficial urothelial bladder carcinoma: lack of nuclear ATBF1 (ZFHX3) by immunohistochemistry suggests malignant progression.

BACKGROUND: Pathological stage and grade have limited ability to predict the outcomes of superficial urothelial bladder carcinoma at initial transurethral resection (TUR). AT-motif binding factor 1 (ATBF1) is a tumor suppressive transcription factor that is normally localized to the nucleus but has been detected in the cytoplasm in several cancers. Here, we examined the diagnostic value of the intracellular localization of ATBF1 as a marker for the identification of high risk urothelial bladder carcinoma. METHODS: Seven anti-ATBF1 antibodies were generated to cover the entire ATBF1 sequence. Four human influenza hemagglutinin-derived amino acid sequence-tagged expression vectors with truncated ATBF1 cDNA were constructed to map the functional domains of nuclear localization signals (NLSs) with the consensus sequence KR[X10-12]K. A total of 117 samples from initial TUR of human bladder carcinomas were analyzed. None of the patients had received chemotherapy or radiotherapy before pathological evaluation. RESULTS: ATBF1 nuclear localization was regulated synergistically by three NLSs on ATBF1. The cytoplasmic fragments of ATBF1 lacked NLSs. Patients were divided into two groups according to positive nuclear staining of ATBF1, and significant differences in overall survival (P = 0.021) and intravesical recurrence-free survival (P = 0.013) were detected between ATBF1+ (n = 110) and ATBF1- (n = 7) cases. Multivariate analysis revealed that ATBF1 staining was an independent prognostic factor for intravesical recurrence-free survival after adjusting for cellular grading and pathological staging (P = 0.008). CONCLUSIONS: Cleavage of ATBF1 leads to the cytoplasmic localization of ATBF1 fragments and downregulates nuclear ATBF1. Alterations in the subcellular localization of ATBF1 due to fragmentation of the protein are related to the malignant character of urothelial carcinoma. Pathological evaluation using anti-ATBF1 antibodies enabled the identification of highly malignant cases that had been overlooked at initial TUR. Nuclear localization of ATBF1 indicates better prognosis of urothelial carcinoma.

Comparison of analytical methods for profiling N- and O-linked glycans from cultured cell lines : HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study.

The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.

[Asynchronous Bilateral Testicular Infarction with Suspected Polyarteritis Nodosa : A Case Report].

This report documents a case of asynchronous bilateral testicular infarction. The patient was a 42- year-old man who presented with left testicular pain and swelling. He had a past history of right idiopathic testicular infarction and underwent a right orchiectomy 6 years ago. He also had received treatment for 5 years for suspected polyarteritis nodosa (PAN). The left scrotal pain persisted for a week and left orchiectomy was performed. Pathological evaluations demonstrated a benign testis with testicular hemorrhage and chronic vasculopathy. There was no fibrinoid necrosis of medium-size vessel walls which characterizes PAN. In this report, we review the pathogenesis, risk of contralateral testicular infarction, and management of testicular infarction.

Increased SPHK2 Transcription of Human Colon Cancer Cells in Serum-Depleted Culture: The Involvement of CREB Transcription Factor.

Sphingosine kinases (SPHK) are important to determine cells' fate by producing sphingosine 1-phosphate. Reportedly, exogenous SPHK2 overexpression induces cell cycle arrest or cell death. However, the regulatory mechanism of SPHK2 expression has not been fully elucidated. Here, we analyzed this issue using human colon cancer cell lines under various stress conditions. Serum depletion (FCS(-)) but not hypoxia and glucose depletion increased mRNA, protein and enzyme activity of SPHK2 but not SPHK1. In HCT116 cells mostly used, SPHK2 activity was predominant over SPHK1, and serum depletion increased both nuclear and cytoplasmic SPHK2 activity. Based on previous reports analyzing cellular response after serum depletion, the temporal changes of intracellular signaling molecules and candidate transcription factors for SPHK2 were examined using serum-depleted HCT116 cells, and performed transfection experiments with siRNA or cDNA of candidate transcription factors. Results showed that the rapid and transient JNK activation followed by CREB activation was the major regulator of increased SPHK2 transcription in FCS(-) culture. EMSA and ChIP assay confirmed the direct binding of activated CREB to the CREB binding site of 5' SPHK2 promoter region. Colon cancer cells examined continued to grow in FCS(-) culture, although mildly, while hypoxia and glucose depletion suppressed cell proliferation or induced cell death, suggesting the different role of SPHK2 in different stress conditions. Because of the unique relationship observed after serum depletion, we examined effects of siRNA for SPHK2, and found the role of SPHK2 as a growth or survival factor but not a cell proliferation inhibitor in FCS(-) culture.