Tubular Endogenous Erythropoietin Protects Renal Function against Ischemic Reperfusion Injury.

Many large-scale studies show that exogenous erythropoietin, erythropoiesis-stimulating agents, lack any renoprotective effects. We investigated the effects of endogenous erythropoietin on renal function in kidney ischemic reperfusion injury (IRI) using the prolyl hydroxylase domain (PHD) inhibitor, Roxadustat (ROX). Four h of hypoxia (7% O2) and 4 h treatment by ROX prior to IRI did not improve renal function. In contrast, 24-72 h pretreatment by ROX significantly improved the decline of renal function caused by IRI. Hypoxia and 4 h ROX increased interstitial cells-derived Epo production by 75- and 6-fold, respectively, before IRI, and worked similarly to exogenous Epo. ROX treatment for 24-72 h increased Epo production during IRI by 9-fold. Immunohistochemistry revealed that 24 h ROX treatment induced Epo production in proximal and distal tubules and worked similarly to endogenous Epo. Our data show that tubular endogenous Epo production induced by 24-72 h ROX treatment results in renoprotection but peritubular exogenous Epo production by interstitial cells induced by hypoxia and 4 h ROX treatment did not. Stimulation of tubular, but not peritubular, Epo production may link to renoprotection.

Effects of Roxadustat on Erythropoietin Production in the Rat Body.

Anemia is a major complication of chronic renal failure. To treat this anemia, prolylhydroxylase domain enzyme (PHD) inhibitors as well as erythropoiesis-stimulating agents (ESAs) have been used. Although PHD inhibitors rapidly stimulate erythropoietin (Epo) production, the precise sites of Epo production following the administration of these drugs have not been identified. We developed a novel method for the detection of the Epo protein that employs deglycosylation-coupled Western blotting. With protein deglycosylation, tissue Epo contents can be quantified over an extremely wide range. Using this method, we examined the effects of the PHD inhibitor, Roxadustat (ROX), and severe hypoxia on Epo production in various tissues in rats. We observed that ROX increased Epo mRNA expression in both the kidneys and liver. However, Epo protein was detected in the kidneys but not in the liver. Epo protein was also detected in the salivary glands, spleen, epididymis and ovaries. However, both PHD inhibitors (ROX) and severe hypoxia increased the Epo protein abundance only in the kidneys. These data show that, while Epo is produced in many tissues, PHD inhibitors as well as severe hypoxia regulate Epo production only in the kidneys.

Effects of Angiotensin II on Erythropoietin Production in the Kidney and Liver.

The kidney is a main site of erythropoietin production in the body. We developed a new method for the detection of Epo protein by deglycosylation-coupled Western blotting. Detection of deglycosylated Epo enables the examination of small changes in Epo production. Using this method, we investigated the effects of angiotensin II (ATII) on Epo production in the kidney. ATII stimulated the plasma Epo concentration; Epo, HIF2α, and PHD2 mRNA expression in nephron segments in the renal cortex and outer medulla; and Epo protein expression in the renal cortex. In situ hybridization and immunohistochemistry revealed that ATII stimulates Epo mRNA and protein expression not only in proximal tubules but also in collecting ducts, especially in intercalated cells. These data support the regulation of Epo production in the kidney by the renin-angiotensin-aldosterone system (RAS).

Development of adoptive immunotherapy with KK-LC-1-specific TCR-transduced γδT cells against lung cancer cells.

The present study analyzed the antitumor effect of γδT cells transduced with the TCR of cancer-specific CTLs to establish forceful cancer-specific adoptive immunotherapy. We cloned the TCRαß genes from CTLs showing HLA-B15 restricted recognition of Kita-Kyushu lung cancer antigen-1 (KK-LC-1), a cancer/germline gene antigen, identified in a lung adenocarcinoma case (F1121). The TCRαß and CD8 genes were transduced into γδT cells induced from PBLs of healthy volunteers stimulated with zoledronate and IL-2. The KK-LC-1-specific TCRαß-CD8 γδT cells showed cytotoxic activity against the KK-LC-1 positive lung cancer cell line F1121L and produced IFN-γ against F1121L and KK-LC-1 peptide-pulsed F1121 EBV-B cells. These responses were blocked by HLA class I and HLA-B/C antibodies. An in vivo assay using NOD/SCID mice with xenotransplantation of human lung cancer cells was performed, and the TCRαß-CD8 transduced γδT cells (TCRαß-CD8 γδT cells) were intravenously injected. Growth inhibition of KK-LC-1+ , HLA-B15+ lung cancer cells was confirmed in mice with injection of the TCRαß-CD8 γδT cells from 1 wk after xenotransplantation of cancer cells but not in those treated 2 wk after xenotransplantation. The resected specimens of the tumor, 2 wk after xenotransplantation, highly expressed FasL but not programmed death ligand-1 (PD-L1) by immunohistochemical staining. FasL highly expressed cancer cells xenotransplanted 2 wk ago were resistant to TCRαß-CD8 γδT cells injection. These results suggested that apoptosis of Fas-positive TCRαß-CD8 γδT cells may be induced by a Fas-mediated signal after interacting with FasL-positive cancer cells.

Fludrocortisone stimulates erythropoietin production in the intercalated cells of the collecting ducts.

Erythropoietin has been thought to be secreted to plasma soon after the production because of the difficulty of Western blot analysis and immunohistochemistry. We established the new methods of Western blot analysis and immunohistochemistry. Using the new methods, we investigated the effects of aldosterone and fludrocortisone, an analogue of aldosterone on erythropoietin mRNA and protein production by the kidneys. Aldosterone stimulated Epo and HIF2α mRNA expressions in tubule suspensions and microdissected medullary thick ascending limbs and outer medullary collecting ducts. Western blot analysis showed a recombinant erythropoietin at 34-45 kDa and kidney erythropoietin at 36-40 and 42 kDa, both of which shifted to 22 kDa by deglycosylation. Erythropoietin protein expression was observed in the nephrons but not in the interstitial cells in control condition. Fludrocortisone stimulated erythropoietin mRNA and protein expressions in the distal nephrons, particularly in the intercalated cells of the collecting ducts. These data show that erythropoietin is produced by the nephrons by the regulation of renin-angiotensin-aldosterone system and not by the renal interstitial cells in control condition.

Self-Propelled Vesicles Induced by the Mixing of Two Polymeric Aqueous Solutions through a Vesicle Membrane Far from Equilibrium.

This study describes the development of self-propelled vesicles using transient interfacial energy in an aqueous two-phase system composed of polyethylene glycol (PEG), dextran (DEX), and water. The transient interfacial energy was generated at the mixing boundary between the PEG and DEX solutions when the two miscible liquids were in contact with each other far from equilibrium. Vesicles encapsulating 20 wt % DEX solution traveled spontaneously when the PEG concentration in the environmental media was >15 wt %. The motility of the vesicles varied with the permeability of the vesicle membrane. The permeability increased significantly when the concentration of PEG was >15 wt %. PEG had a profound effect not only on mass transfer through the membrane but also on the motility of the vesicles.

Expression of three isoforms of Na-K-2Cl cotransporter (NKCC2) in the kidney and regulation by dehydration.

Sodium reabsorption via Na-K-2Cl cotransporter 2 (NKCC2) in the thick ascending limbs has a major role for medullary osmotic gradient and subsequent water reabsorption in the collecting ducts. We investigated intrarenal localization of three isoforms of NKCC2 mRNA expressions and the effects of dehydration on them in rats. To further examine the mechanisms of dehydration, the effects of hyperosmolality on NKCC2 mRNA expression in microdissected renal tubules was studied. RT-PCR and RT-competitive PCR were employed. The expressions of NKCC2a and b mRNA were observed in the cortical thick ascending limbs (CAL) and the distal convoluted tubules (DCT) but not in the medullary thick ascending limbs (MAL), whereas NKCC2f mRNA expression was seen in MAL and CAL. Two-day dehydration did not affect these mRNA expressions. In contrast, hyperosmolality increased NKCC2 mRNA expression in MAL in vitro. Bradykinin dose-dependently decreased NKCC2 mRNA expression in MAL. However, dehydration did not change NKCC2 protein expression in membrane fraction from cortex and outer medulla and in microdissected MAL. These data show that NKCC2a/b and f types are mainly present in CAL and MAL, respectively. Although NKCC2 mRNA expression was stimulated by hyperosmolality in vitro, NKCC2 mRNA and protein expressions were not stimulated by dehydration in vivo. These data suggest the presence of the inhibitory factors for NKCC2 expression in dehydration. Considering the role of NKCC2 for the countercurrent multiplier system, NKCC2f expressed in MAL might be more important than NKCC2a/b.

Reevaluation of erythropoietin production by the nephron.

Erythropoietin production has been reported to occur in the peritubular interstitial fibroblasts in the kidney. Since the erythropoietin production in the nephron is controversial, we reevaluated the erythropoietin production in the kidney. We examined mRNA expressions of erythropoietin and HIF PHD2 using high-sensitive in situ hybridization system (ISH) and protein expression of HIF PHD2 using immunohistochemistry in the kidney. We further investigated the mechanism of erythropoietin production by hypoxia in vitro using human liver hepatocell (HepG2) and rat intercalated cell line (IN-IC cells). ISH in mice showed mRNA expression of erythropoietin in proximal convoluted tubules (PCTs), distal convoluted tubules (DCTs) and cortical collecting ducts (CCDs) but not in the peritubular cells under normal conditions. Hypoxia induced mRNA expression of erythropoietin largely in peritubular cells and slightly in PCTs, DCTs, and CCDs. Double staining with AQP3 or AE1 indicated that erythropoietin mRNA expresses mainly in ß-intercalated or non α/non ß-intercalated cells of the collecting ducts. Immunohistochemistry in rat showed the expression of HIF PHD2 in the collecting ducts and peritubular cells and its increase by anemia in peritubular cells. In IN-IC cells, hypoxia increased mRNA expression of erythropoietin, erythropoietin concentration in the medium and protein expression of HIF PHD2. These data suggest that erythropoietin is produced by the cortical nephrons mainly in the intercalated cells, but not in the peritubular cells, in normal hematopoietic condition and by mainly peritubular cells in hypoxia, suggesting the different regulation mechanism between the nephrons and peritubular cells.

Ten-Gram-Scale Total Synthesis of the Anticancer Drug Candidate E7130 to Supply Clinical Trials.

E7130 is a novel drug candidate with an exceedingly complex chemical structure of the halichondrin class, discovered by a total synthesis approach through joint research between the Kishi group at Harvard University and Eisai. Only 18 months after completion of the initial milligram-scale synthesis, ten-gram-scale synthesis of E7130 was achieved, providing the first good manufacturing practice (GMP) batch to supply clinical trials. This paper highlights the challenges in developing ten-gram-scale synthesis from the milligram-scale synthesis.

Single-cell sequencing on CD8+ TILs revealed the nature of exhausted T cells recognizing neoantigen and cancer/testis antigen in non-small cell lung cancer.

BACKGROUND: CD8+tumor infiltrating lymphocytes (TILs) are often observed in non-small cell lung cancers (NSCLC). However, the characteristics of CD8+ TILs, especially T-cell populations specific for tumor antigens, remain poorly understood. METHODS: High throughput single-cell RNA sequencing and single-cell T-cell receptor (TCR) sequencing were performed on CD8+ TILs from three surgically-resected lung cancer specimens. Dimensional reduction for clustering was performed using Uniform Manifold Approximation and Projection. CD8+ TIL TCR specific for the cancer/testis antigen KK-LC-1 and for predicted neoantigens were investigated. Differentially-expressed gene analysis, Gene Set Enrichment Analysis (GSEA) and single sample GSEA was performed to characterize antigen-specific T cells. RESULTS: A total of 6998 CD8+ T cells was analyzed, divided into 10 clusters according to their gene expression profile. An exhausted T-cell (exhausted T (Tex)) cluster characterized by the expression of ENTPD1 (CD39), TOX, PDCD1 (PD1), HAVCR2 (TIM3) and other genes, and by T-cell oligoclonality, was identified. The Tex TCR repertoire (Tex-TCRs) contained nine different TCR clonotypes recognizing five tumor antigens including a KK-LC-1 antigen and four neoantigens. By re-clustering the tumor antigen-specific T cells (n=140), it could be seen that the individual T-cell clonotypes were present on cells at different stages of differentiation and functional states even within the same Tex cluster. Stimulating these T cells with predicted cognate peptide indicated that TCR signal strength and subsequent T-cell proliferation and cytokine production was variable but always higher for neoantigens than KK-LC-1. CONCLUSIONS: Our approach focusing on T cells with an exhausted phenotype among CD8+ TILs may facilitate the identification of tumor antigens and clarify the nature of the antigen-specific T cells to specify the promising immunotherapeutic targets in patients with NSCLC.

Helicobacter pylori, a carcinogen, induces the expression of melanoma antigen-encoding gene (Mage)-A3, a cancer/testis antigen.

Cancer/testis antigens (CTAs) are known to be expressed in various cancer types but are minimally or not expressed in normal tissues except for germline tissues. CTAs are attractive targets for cancer immunotherapy and diagnosis because of their restricted expression. The mechanisms of CTAs expression are unclear because the inducers of CTAs expression remain to be elucidated. We hypothesized that carcinogens may induce the cellular expression of CTAs. To prove this, we attempted to inoculate Helicobacter pylori, a known carcinogen, in Meth-A cells, normal gastric cells, and normal splenocytes and induce the expression of a CTA. Melanoma antigen-encoding gene (Mage)-A3, one of the CTAs, was not expressed in both normal cells but in Meth-A cells inoculated with H. pylori. Furthermore, we performed limiting dilution using Meth-A cells inoculated with H. pylori and established derivative clone from Meth-A designated as Meth-A/pylori/3C3 which permanently express Mage-A3 after excluding H. pylori. We herein report the first successful induction of a CTA in a cell line via exposure to a carcinogenic agent. Furthermore, the establishment of Meth-A/pylori/3C3, which is Meth-A expressing Mage-A3, is considered to contribute to the resolution of the mechanism of CTAs expression.

Immune profile analysis of peripheral blood and tumors of lung cancer patients treated with immune checkpoint inhibitors.

Background: Immune checkpoint inhibitors (ICIs) have become central to lung cancer drug therapy, and establishing biomarkers that can predict effects and adverse events (AEs) is awaited. We prospectively analyzed the association between the immune-related molecular expression in peripheral blood mononuclear cells (PBMCs) and lung cancer tissues, and the effects of ICI monotherapy. Methods: Twenty-one patients with advanced non-small cell lung cancer (NSCLC) who received ICI monotherapy were included. Changes in the expression of immune-related molecules in PBMCs before and after the administration of ICI were analyzed by flow cytometry. The major histocompatibility complex (MHC) class I and programmed cell death-ligand 1 (PD-L1) expression of cancer cells, and the PD-L1, CD8 and CD103 expression of tumor infiltrating immune cells in lung cancer tissue before the administration of ICI were confirmed by immunohistochemistry (IHC). Results: Twenty-one patients were investigated, including 11 adenocarcinoma and 10 squamous cell carcinoma cases. Anti-programmed cell death protein-1 (PD-1) antibody (n=18) and anti-PD-L1 antibody (n=3) were administered. The clinical responses were graded as follows: complete response (CR) (n=1), partial response (PR) (n=7), stable disease (SD) (n=10) and progressive disease (PD) (n=3). Among immune-related molecules expressed in PBMCs, the CD103+ CD39+ CD8+ T cell change after administration closely correlated with the clinical response. In the univariate analyses of the factors associated with progression-free survival (PFS), CD103+ CD39+ CD8+ cell change after administration was identified as a significant prognostic factor, while the CD103+ CD39+ CD8+ cell change after administration and Brinkman index were independent prognostic factors in a multivariate analysis of the factors associated with PFS. Conclusions: The CD103+ CD39+ CD8+ cell change after administration may predict the efficacy of ICIs.

Detection of Kita-Kyushu Lung Cancer Antigen-1, a Cancer/Testis Antigen, in the Stomach Close to a Cancerous Condition.

Background: Kita-Kyushu lung cancer antigen-1 (KK-LC-1), encoded by CT83, is a cancer/testis antigen (CTA) and an attractive target for immunotherapy. Our previous study demonstrated frequent CT83 expression in gastric cancers (GCs) and non-tumor sites of the stomach with tumors. Additionally, there was a correlation with Helicobacter pylori (Hp) infection. Since it currently remains unclear whether KK-LC-1 is expressed in the stomach without GC, this study investigated KK-LC-1 expression in non-GC stomach. Methods: We investigated differences in CT83 gene expression at non-tumor sites of stomachs with or without tumors in 118 GC patients and 115 non-GC patients. Fisher's exact test was used for statistical analyses. Results: CT83 expression was detected in 77% of non-tumor sites in stomachs with tumors, which was significantly higher than in stomachs without tumors (7%, p < 0.0001). All patients with CT83 expression at non-tumor sites of their stomachs without tumors carried Hp. Conclusion: CT83 appears to be rarely expressed in the atrophic stomach, and furthermore, a part of patients positive for its expression will develop GC in the future, suggesting that CT83 expression is a useful marker for predicting GC.

An analysis of the immunological tumor microenvironment of primary tumors and regional lymph nodes in squamous cell lung cancer.

BACKGROUND: Various immune cells that play a central role in antitumor immunity accumulate in primary tumors and regional lymph nodes. Such cellular accumulation and the molecular expression were analyzed to elucidate the immunological tumor microenvironment. METHODS: Fifty squamous cell lung cancer patients with complete resection were included. Resected specimens from primary lung tumors and regional lymph nodes were immunostained for immune-related molecules, such as CD8, CD103, major histocompatibility complex (MHC) class I, and programmed cell death protein ligand-1 (PD-L1), and the relationship between the prognosis and clinicopathological factors was retrospectively analyzed. RESULTS: Tumor-infiltrating lymphocytes and CD8+ lymphocytes, intratumoral and intrastromal CD103+ lymphocytes, tumor diameter, pathological T and N factors, and pathological stage were significant prognostic factors for the disease-specific survival (DSS) in a univariate analysis. In a multivariate analysis, intratumoral and intrastromal CD103+ lymphocytes and pathological T and N factors were independent prognostic factors of the DSS. Significant concordance was found between the PD-L1 expression of primary tumors and metastatic lymph nodes as well as among tumor-infiltrating lymphocytes, CD8+ lymphocytes and CD103+ lymphocytes. Infiltration of CD103+ lymphocytes into the tumor was significantly correlated with an increased PD-L1 expression of cancer cells in both primary tumors and reginal lymph node metastases. Both the intratumoral infiltration of CD103+ lymphocytes and PD-L1 expression of cancer cells were significantly higher in lymph node metastases than in primary tumors. CONCLUSIONS: CD103+ lymphocyte infiltration in the primary tumor was shown to be strongly involved in the prognosis.

Relationship between Kita-Kyushu Lung Cancer antigen-1 expression and prognosis of cases with lung squamous cell carcinoma.

BACKGROUND: Previously, we identified the highly immunogenic cancer testicular antigen named Kita-Kyushu Lung Cancer antigen-1 (KK-LC-1). In this study, we analyzed the effect of KK-LC-1 expression on the prognosis of patients with resected squamous cell lung cancer. METHODS: Fifty squamous cell lung cancer patients, who received complete resection, were enrolled in this study. The expressions of KK-LC-1, CD8, human leukocyte antigen (HLA) class I, and programmed cell death protein ligand-1 (PD-L1) were assessed via immunohistochemistry staining using the specimens obtained from the participants. The association between the expression of the abovementioned molecules and patient prognosis was investigated. RESULTS: KK-LC-1 expression was observed in 21 of 50 recruited cases (42%). However, no significant correlation was found between KK-LC-1 expression and patient prognosis. The prognosis was significantly better in lung cancer cases with KK-LC-1 expression in which CD8+ T cells infiltrated the tumor. Regardless of the HLA class I expression or the PD-L1 expression, the KK-LC-1 expression in squamous cell lung cancer could not be detected as a significant prognostic factor. Furthermore, considering the polarity of the cancer tissue as epithelium, staining of KK-LC-1 tended to be strong in the area corresponding to the basal side of the tumor tissue. The Ki-67 expression was frequently observed in cancer cells on the basal side, which was consistent with the KK-LC-1 expression in representative four cases with KK-LC-1-positive squamous cell lung cancer. CONCLUSIONS: Our results indicated that lung squamous cell cancer patients with KK-LC-1 expression and the tumor infiltrating CD8+ T cells might exhibit better prognosis. KK-LC-1 might be highly expressed in cancer cells with high proliferative capacity. Larger cohort analysis is still required for further elucidation and validation of the results of this study.

Should Lung-Sparing Surgery Be the Standard Procedure for Malignant Pleural Mesothelioma?

BACKGROUND: Surgical procedures for malignant pleural mesothelioma (MPM) include extrapleural pneumonectomy (EPP), extended pleurectomy/decortication (P/D) and P/D. EPP has been applied to MPM for a long time, but the postoperative status is extremely poor due to the loss of one whole lung. We compared the mortality, morbidity and median survival time (MST) of lung-sparing surgery (extended P/D or P/D) and lung-sacrificing surgery (EPP) for MPM by performing a systematic review. METHODS: We extracted the number of events and patients from the literature identified in electronic databases. Ultimately, 15 reports were selected, and 2674 MPM patients, including 1434 patients undergoing EPP and 1240 patients undergoing extended P/D or P/D, were analyzed. RESULTS: Our systematic review showed that lung-sparing surgery was significantly superior to lung-sacrificing surgery in both the surgical-related mortality (extended P/D vs. EPP: 3.19% vs. 7.65%, p < 0.01; P/D vs. EPP: 1.85% vs. 7.34%, p < 0.01) and morbidity (extended P/D vs. EPP: 35.7% vs. 60.0%, p < 0.01; P/D vs. EPP: 9.52% vs. 20.89%, p < 0.01). Lung-sparing surgery was not inferior to EPP in terms of MST. CONCLUSION: Although no prospective randomized controlled trial has been conducted, it may be time to change the standard surgical method for MPM from lung-sacrificing surgery to lung-sparing surgery.

Cancer/testis antigen, Kita-Kyushu lung cancer antigen-1 and ABCD stratification for diagnosing gastric cancers.

BACKGROUND: The ABCD stratification [(combination of serum pepsinogen (PG) levels and titers of antibody (immunoglobulin G, IgG) against Helicobacter pylori (H. pylori)] is effective for the classification of individuals at risk of developing gastric cancer (GC). The Kita-Kyushu lung cancer antigen-1 (KK-LC-1) is a Cancer/Testis antigen frequently expressed in GC. AIM: To evaluate the effectiveness of KK-LC-1 and ABCD stratification in the diagnosis of GC. METHODS: We analyzed the gene expression of KK-LC-1 in surgical specimens obtained from GC tumors. The levels of serum PG I/PG II and IgG against H. pylori were measured. According to their serological status, the patients were classified into the four groups of the ABCD stratification. RESULTS: Of the 77 examined patients, 63 (81.8%) expressed KK-LC-1. The IgG titers of H. pylori and PG II were significantly higher in patients expressing KK-LC-1 than those measured in patients not expressing KK-LC-1 (P = 0.0289 and P = 0.0041, respectively). The expression of KK-LC-1 in group C [PG method (+)/H. pylori infection (+)] was as high as 93.9% high. KK-LC-1 was also detected in group A [-/-]. CONCLUSION: The KK-LC-1 expression in GC was associated with H. pylori infection and atrophic status, so that, KK-LC-1 may be a useful marker for the diagnosis of GC.

Differentiation of endogenous erythropoietin and exogenous ESAs by Western blotting.

Doping tests for the illegal use of erythropoiesis-stimulating agents (ESAs) have been developed. We developed a new Western blotting method to detect and distinguish endogenous erythropoietin (Epo, 35-38 kDa) and exogenous ESAs (epoetin α and ß, 38-42 kDa; darbepoetin α, 47-50 kDa; epoetin ß pegol, 93-110 kDa). Epo and ESAs are glycoproteins and deglycosylation using peptide-N-glycosidase F shifted all Epo and ESA bands except epoetin ß pegol to 22 kDa. We cut the bands of Epo and ESAs from SDS-PAGE gels and analyzed them by Liquid Chromatography/Mass Spectrometry (LC/MS). LC/MS detected all endogenous Epo and exogenous ESAs as deglycosylated 22 kDa Epo, indicating that LC/MS analysis could confirm the presence of Epo or ESA, but could not distinguish between endogenous Epo and exogenous ESAs. We propose the following Epo doping tests: 1) detect Epo or ESAs by Western blotting of the glycosylated form; 2) increase the reliability by the band shift following deglycosylation; and 3) complete confirmation of Epo or ESA by LC/MS analysis using cut gels. One of the advantages of our method is that pre-purification of samples for Epo is not required in our Western blotting.

Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation.

The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG-bound epoetin ß pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Severe hypoxia (7% O2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney but not the liver is the main site of Epo production in control and severe hypoxia. Our method will make the tests for Epo doping and detection easy.

Antitumor effect of antibody against a SEREX-defined antigen (UOEH-LC-1) on lung cancer xenotransplanted into severe combined immunodeficiency mice.

We previously reported the humoral immune response of tumor-infiltrating B lymphocytes in a lung cancer patient and 22 genes coding tumor-associated antigens identified using the serological identification of antigens by recombinant expression cloning method. In this study, we investigated one of these genes, designated University of Occupational and Environmental Health-Lung cancer antigen-1 (UOEH-LC-1), which has an extracellular domain. Quantitative reverse transcription-PCR revealed that UOEH-LC-1 was expressed ubiquitously in the normal tissues tested. However, it was overexpressed in 5 of 11 (45.5%) lung cancer cell lines and also in 9 of 15 (60%) lung cancer tissues compared with the paired normal lung tissues. A sequence analysis revealed that UOEH-LC-1 has a transmembrane domain. Flow cytometry analysis using a polyclonal antibody against UOEH-LC-1 revealed positive staining on lung cancer cell lines that were positive for expression of mRNA of UOEH-LC-1. Phage plaque assay showed the specific reactivity of anti-UOEH-LC-1 antibody against UOEH-LC-1 protein derived from the antigen encoding phage. By immunohistochemical staining with the anti-UOEH-LC-1 antibody, 7 of 28 (25.0%) lung cancer specimens showed positive staining on the cell surface. The administration of anti-UOEH-LC-1 antibody inhibited the growth of the UOEH-LC-1-positive tumors that were xenotransplanted into severe combined immunodeficiency mice. Complement-dependent cytotoxicity was one of the mechanisms to suppress the tumor growth. These results suggest that the antibody against UOEH-LC-1 therefore seems to have a promising therapeutic potential as a treatment for lung cancer.