Published papers

Background Insulinoma is a tumour of insulin-producing cells of the pancreas and is known to be one of the causes of hypoglycaemia. Usually, appropriate removal of the insulinoma results in normalization of blood glucose levels. However, we found novel cases of insulinoma, in which hyperglycaemia developed soon after resection of the insulinoma. Case report We encountered two patients with repeated hypoglycaemia caused by insulinoma. Following removal of the insulinoma, unanticipated hyperglycaemia was observed in both patients. Thereafter, their blood tests revealed low levels of serum C-peptide and high titres of anti-glutamic acid decarboxylase antibody, indicating concomitant Type1 diabetes. Indeed, histological examination of the resected specimen revealed that one patient showed insulitis in non-tumorous pancreatic tissue in which β-cells had already disappeared. Moreover, inflammatory cells infiltrated the insulinoma, as if it were insulitis of Type1 diabetes, suggesting the existence of anti-islet autoimmunity. Conclusion These are first cases of insulinoma associated with underlying Type1 diabetes. Physicians should be aware of the possibility that insulinoma may mask Type1 diabetes, and measurement of anti-islet autoantibodies may be helpful to find underlying Type1 diabetes, such as in these cases. It is pathologically interesting that the immune cell infiltration into insulinoma may be suggestive of anti-islet autoimmunity. © 2012 The Authors. Diabetic Medicine © 2012 Diabetes UK.

BACKGROUND: Intestinal epithelial cells (IECs) serve as robust barriers against potentially hostile luminal antigens and commensal microbiota. Epithelial barrier dysfunction enhances intestinal permeability, leading to leaky gut syndrome (LGS) associated with autoimmune and chronic inflammatory disorders. However, a causal relationship between LGS and systemic disorders remains unclear. Ap1m2 encodes clathrin adaptor protein complex 1 subunit mu 2, which facilitates polarized protein trafficking toward the basolateral membrane and contributes to the establishment of epithelial barrier functions. METHODS: We generated IEC-specific Ap1m2-deficient (Ap1m2ΔIEC) mice with low intestinal barrier integrity as an LSG model and examined the systemic impact. FINDINGS: Ap1m2ΔIEC mice spontaneously developed IgA nephropathy (IgAN)-like features characterized by the deposition of IgA-IgG immune complexes and complement factors in the kidney glomeruli. Ap1m2 deficiency markedly enhanced aberrantly glycosylated IgA in the serum owing to downregulation and mis-sorting of polymeric immunoglobulin receptors in IECs. Furthermore, Ap1m2 deficiency caused intestinal dysbiosis by attenuating IL-22-STAT3 signaling. Intestinal dysbiosis contributed to the pathogenesis of IgAN because antibiotic treatment reduced aberrantly glycosylated IgA production and renal IgA deposition in Ap1m2ΔIEC mice. INTERPRETATION: IEC barrier dysfunction and subsequent dysbiosis by AP-1B deficiency provoke IgA deposition in the mouse kidney. Our findings provide experimental evidence of a pathological link between LGS and IgAN. FUNDING: AMED, AMED-CREST, JSPS Grants-in-Aid for Scientific Research, JST CREST, Fuji Foundation for Protein Research, and Keio University Program for the Advancement of Next Generation Research Projects.

Background: Pancreatic islet endocrine cell-supporting architectures, including islet encapsulating basement membranes (BMs), extracellular matrix (ECM), and possible cell clusters, are unclear. Procedures: The architectures around islet cell clusters, including BMs, ECM, and pancreatic acinar-like cell clusters, were studied in the non-diabetic state and in the inflamed milieu of fulminant type 1 diabetes in humans. Result: Immunohistochemical and electron microscopy analyses demonstrated that human islet cell clusters and acinar-like cell clusters adhere directly to each other with desmosomal structures and coated-pit-like structures between the two cell clusters. The two cell-clusters are encapsulated by a continuous capsule composed of common BMs/ECM. The acinar-like cell clusters have vesicles containing regenerating (REG) I alpha protein. The vesicles containing REG I alpha protein are directly secreted to islet cells. In the inflamed milieu of fulminant type 1 diabetes, the acinar-like cell clusters over-expressed REG I alpha protein. Islet endocrine cells, including beta-cells and non-beta cells, which were packed with the acinar-like cell clusters, show self-replication with a markedly increased number of Ki67-positive cells. Conclusion: The acinar-like cell clusters touching islet endocrine cells are distinct, because the cell clusters are packed with pancreatic islet clusters and surrounded by common BMs/ECM. Furthermore, the acinar-like cell clusters express REG I alpha protein and secrete directly to neighboring islet endocrine cells in the non-diabetic state, and the cell clusters over-express REG Ia in the inflamed milieu of fulminant type 1 diabetes with marked self-replication of islet cells.

Cancer grading has become an important topic in the field of image interpretation-based computer aided diagnosis systems. This paper proposes a novel feature descriptor to observe the characteristics of histopathological textures in a discriminative manner. The proposed feature descriptor utilizes fractal geometric analysis with four multifractal measures to construct an eight dimensional feature space. The proposed method employed a bag-of-feature-based classification model to discriminate a set of hepatocellular carcinoma images into five categories according to Edmondson and Steiner's grading system. Three feature selection methods were utilized to obtain the most discriminative features of codeword dictionary (codebook). Furthermore, we incorporated four other textural feature descriptors: Gabor-filters, LM-filters, local binary patterns, and Haralick, to obtain a benchmark of the accuracy of the classification. Two experiments were performed: (i) classifying non-neoplastic tissues and tumors and (ii) grading the hepatocellular carcinoma images into five classes. Experimental results indicated the significance of the multifractal features for describing the histopathological image texture because it outperformed other four feature descriptors. We graded a given ROI image by defining a threshold-based majority-voting rule and obtained an average correct classification rate around 95% for five classes classification. (c) 2012 Elsevier Ltd. All rights reserved.

The diagnosis of prostate cancer is based on the results of ultrasonography-guided needle biopsy of the prostate, but cancer foci are often not visible in conventional transrectal ultrasonography. Sonazoid is a new microbubble contrast agent. The purpose of our study was to compare areas of contrast material enhancement in the prostate at ultrasonography with whole-mount radical prostatectomy specimens to determine if the use of Sonazoid improves the detection rate of prostate cancer. Fifty patients with biopsy-proven cancer of the prostate who were scheduled to undergo radical prostatectomy were recruited for this study. The day before the operation, each patient was evaluated with ultrasonography at baseline and again during intravenous infusion of Sonazoid. A map of ultrasonography findings was created prospectively at the time of imaging. Following radical prostatectomy, independent mapping of the pathologic results was performed and the maps were compared. Ultrasonography evaluation at baseline demonstrated that at least one focus of cancer was identified in 20 of the 50 subjects (40.0%). Meanwhile at least one cancer focus was enhanced in 31 of the 50 patients (62.0%) when Sonazoid was used. The combination of baseline grayscale imaging and contrast-enhanced imaging allowed identification of at least one focus of cancer in 40 patients (80.0%). Contrast-enhanced ultrasonography can improve sensitivity, especially for the detection of large cancer, peripheral zone cancer and highly malignant cancer. Our study has demonstrated significantly improved detection of prostate cancer with the combination of baseline grayscale imaging and contrast-enhanced imaging compared with conventional ultrasonography techniques only, and this technique may be applicable to targeted biopsy.

In this study, we describe a phage display strategy to obtain human monoclonal single-chain Fv (scFv) antibodies binding target cancer cell surface proteins. By developing a cancer cell immunization protocol for SCID mice engrafted with human peripheral blood lymphocytes in combination with an antibody phage display method, we have isolated phage antibodies binding small-cell lung cancer cell line H889 by subtractive selection. One of the isolated scFv antibodies, 12EAb, recognized the E2 component of pyruvate dehydrogenase complex (PDC-E2) by immunoprecipitation according to MALDI-TOF MS analysis. Furthermore, we have confirmed the plasma membrane localization of PDC-E2 in small-cell lung cancer cells by immunocytochemistry and cell surface protein biotinylation, although PDC-E2 is usually located in the mitochondrial matrix. These results, including unique localization of identified antigens, were obtained by proteomic approaches. The present methods can be applied to generate human monoclonal scFv antibodies against tumor cells and to identify new molecular targets for immunotherapy and markers for diagnosis.

This paper proposes a digital image analysis method to support quantitative pathology by automatically segmenting the hepatocyte structure and quantifying its morphological features. To structurally analyze histopathological hepatic images, we isolate the trabeculae by extracting the sinusoids, fat droplets, and stromata. We then measure the morphological features of the extracted trabeculae, divide the image into cords, and calculate the feature values of the local cords. We propose a method of calculating the nuclear-cytoplasmic ratio, nuclear density, and number of layers using the local cords. Furthermore, we evaluate the effectiveness of the proposed method using surgical specimens. The proposed method was found to be an effective method for the quantification of the Edmondson grade.

The “preconditioning effect” in AKI is a phenomenon in which an episode of ischemia-reperfusion results in tolerance to subsequent ischemia-reperfusion injury. However, its relationship between DNA damage repair has not been elucidated. Here, we show the role of KAT5 in the preconditioning effect. Preconditioning attenuated DNA damage in proximal tubular cells with elevated KAT5 expression. Ischemia-reperfusion (IR) injuries were exacerbated, and preconditioning effect vanished in proximal tubular-cell-specific KAT5 knockout mice. Investigation of tubuloglomerular feedback (TGF) by MALDI-IMS and urinary adenosine revealed that preconditioning caused attenuated TGF at least in part via KAT5. In addition, K-Cl cotransporter 3 (KCC3) expression decreased in damaged proximal tubular cells, which may be involved in accelerated TGF following IR. Furthermore, KAT5 induced KCC3 expression by maintaining chromatin accessibility and binding to the KCC3 promoter. These results suggest a novel mechanism of the preconditioning effect mediated by the promotion of DNA repair and attenuation of TGF through KAT5.

Liver cancer is one of the frequent causes of death in the world. Hepatocellular carcinoma (HCC) is the most common histological type of primary liver cancer. HCC can be graded according to the malignancy of the tumors. Generally, a HCC grade is determined based on the characteristics of liver cell nuclei. This paper illustrates a methodology for classifying liver cell nuclei and grading HCC histological images quantitatively. The liver cell nuclei are classified in three consecutive tasks: nuclear segmentation, fibrous region detection, and nuclear classification. Each task utilizes the pixel-based textural features that are obtained through multifractal computation on digital images. First, the system segments every possible type of nuclei and excludes the nuclei within fibrous regions. Then, it classifies the rest of the nuclei to discriminate liver cell nuclei. For tumor grading, this method utilizes the following four categories of nuclear features: inner texture, geometry, spatial distribution, and surrounding texture. The proposed method was employed to classify a set of HCC histological images into five.

BACKGROUND: We require a clinicopathological risk stratification method for immunoglobulin A nephropathy (IgAN) to predict kidney outcomes. We examined a renal failure risk group (RF-RG) classification system created following a prior multicentre, retrospective study to determine if RF-RG could predict kidney outcomes. METHODS: We collected data from Japanese patients with IgAN registered between 1 April 2005 and 31 August 2015. The primary outcome was a composite 50% increase in serum creatinine from baseline or dialysis induction. The secondary outcomes were times to proteinuria remission (ProR) and haematuria remission (HemR). RESULTS: The enrolled 991 patients from 44 facilities were followed for a median of 5.5 years (interquartile range 2.5-7.5), during which 87 composite events (8.8%) occurred. RF-RG was significantly associated with the primary outcome {hazard ratio [HR] II 2.78 [95% confidence interval (CI) 1.12-6.93], III 7.15 (2.90-17.6), IV 33.4 (14.1-79.0), I as a reference, P < .001}.The discrimination performance was good [C-statistic 0.81 (95% CI 0.76-0.86)] and the time-dependent C-statistics exceeded 0.8 over 10 years. Among the 764 patients with proteinuria and 879 patients with haematuria at baseline, 515 and 645 patients showed ProR and HemR, respectively. ProR was significantly less frequent in patients with advanced disease [subdistribution HR: II 0.79 (95% CI 0.67-0.94), III 0.53 (0.41-0.66), IV 0.15 (0.09-0.23), I as a reference, P < .001]. We also observed an association between HemR and RF-RG. CONCLUSIONS: RF-RG demonstrated good predictive ability for kidney outcomes.

PURPOSE: Prostate cancer generally occurs multifocally. The lesions of the largest size and highest-grade are often concordant, and defined as an index tumor. However, these factors sometimes do not coincide within one lesion. In such discordant cases, not the largest size lesion but the highest-grade lesion is known to determine the prognosis. We focused on the multiparametric magnetic resonance imaging (mpMRI) detectability of the highest-grade tumors in discordant cases. MATERIALS AND METHODS: We investigated the detectability of the highest-grade tumor using preoperative mpMRI in 50 discordant patients who underwent radical prostatectomy. The radiologist was informed of the tumor location on the pathological tumor map, and mpMRI interpretation for each tumor was performed. RESULTS: Prostate Imaging-Reporting and Data System (PI-RADS) scores of 1, 2, 3, 4, and 5 on preoperative mpMRI were assigned to 13, 1, 9, 16, and 11 of the largest tumors, respectively. On the other hand, scores of 1, 2, 3, 4, and 5 were assigned to 23, 0, 7, 19, and 1 of the highest-grade tumors, respectively. The difference between them was statistically significant (p=0.007). We also found that the largest anterior tumor frequently hid the ipsilateral posterior highest-grade tumor; the detection rate of the highest-grade tumor in this pattern was 42.1% (8 of 19 cases) CONCLUSION: We found that mpMRI detectability of the highest-grade tumor in discordant cases was inferior to that of the largest tumor with low malignant potential. Our results suggest that the risk of high-grade tumors which determine patient prognosis being overlooked.

The analysis of hepatic tissue structure is required for quantitative assessment of liver histology. Especially, a cord-like structure of liver cells, called trabecura, has important information in the diagnosis of hepatocellular carcinoma (HCC). However, the extraction of trabeculae is thought to be difficult because liver cells take on various colors and appearances depending on tissue conditions. In this paper, we propose an approach to extract trabeculae from images of hematoxyline and eosin stained liver tissue slide by extracting the rest of trabeculae: sinusoids and stromal area. The sinusoids are simply extracted based on the color information, where the image is corrected by an orientation selective filtering before segmentaion. The stromal area mainly consists of fiber, and often includes lymphocytes densely. Therefore, in the proposed method, fiber region and lymphocytes are extracted separately, then, stromal region is determined based on the extracted results. The determination of stroma is performed based on superpixels, to obtain precise boundaries. Once the regions of sinusoids and stroma are obtained, trabeculae can be segmented as the remaining region. The proposed method was applied to 10 test images of normal and HCC liver tissues, and the results were evaluated based on the manual segmentation. As a result, we confirmed that both sensitivity and specificity of the extraction of trabeculae reach around 90%.

Proteinuria selectivity index (PSI) is a potential tool for histological classification and prediction of treatment response in nephrotic syndrome, but evidence is insufficient. Clinical relevance of fractional excretion of sodium (FENa) in nephrotic syndrome remains largely unexplored. This multicenter retrospective study included patients with nephrotic syndrome who underwent kidney biopsy between January 2012 and June 2022. Optimal cutoffs for predicting complete remission based on PSI and FENa were determined using receiver operating characteristic curves. Patients were divided into two groups using these cutoffs and followed until complete remission. Of the 611 patients included, 177 had minimal change disease (MCD), 52 had focal segmental glomerulosclerosis (FSGS), and 149 had membranous nephropathy (MN). Median (interquartile range) PSI were 0.14 (0.09-0.19) for MCD, 0.33 (0.23-0.40) for FSGS, and 0.20 (0.14-0.30) for MN. FENa were 0.24 (0.09-0.68), 1.03 (0.50-2.14), and 0.78 (0.41-1.28). Patients with low PSI and FENa had a higher incidence of complete remission. Cox regression analyses demonstrated that both parameters were associated with achieving complete remission (HR 2.73 [95% CI 1.97-3.81] and HR 1.93 [95% CI 1.46-2.55], respectively). PSI and FENa may be useful for histological classification and predicting remission in nephrotic syndrome.

We have been developing a prototype system for the automatic detection of hepatocellular carcinoma (HCC) from whole slide images (WSIs) of liver biopsy based on image analysis techniques. In this paper, we present two color-related topics of this system: color correction and the calculation of color-related features of cytoplasm. A WSI-basis color correction method was implemented for the prototype system. We tested the color correction using more than 300 WISs, and it was confirmed that the color correction works well and stably. In addition, it was found that the success rate of nuclei detection significantly increaseed due to color correction. As for color-related features, we propose a method to calculate the representative color of cytoplasm and the clearness of cytoplasm. It was found that there was slight difference between the distributions of the representative colors of HCC and non-cancer tissues. In addition, there was slight correlation between the clearness of cytoplasm and nuclei density, which implies a promising role of the clearness index in a nuclei-based HCC detection.

An in vitro model of apoptosis and differentiation in human embryonal carcinoma (EC) cells was developed to study the mode of cell death and mechanisms of cell death in early development. Death of these cells was induced by treatment with retinoic acid (RA) under the same conditions as those for induction of differentiation. The manner of this cell death was apoptosis, not necrosis, with the morphological criterion for apoptosis. Serum deprivation likewise caused apoptosis in both undifferentiated and differentiated EC cells. In differentiated EC cells, DNA fragmentation was observed in a smear pattern lacking the ladder pattern typically associated with apoptosis. However, in differentiated EC cells, DNA fragmentation occurred in various sizes. The expression of a carbohydrate antigen, Le(Y), a reported marker of apoptotic cancer cells, was increased by the treatment with RA. However, two-color analysis by flow cytometry with nick end labelling method revealed that Le(Y) expression was closely correlated with cellular differentiation but not apoptosis after RA treatment in the human EC cell system. Collection of Le(Y) positive cells by the magnetic bead method demonstrated that this expression was not due to apoptosis but rather to differentiation. On the other hand, Le(Y) expression associated with apoptosis was induced by serum deprivation in both undifferentiated and differentiated EC cells. These results show that a subpopulation of undifferentiated EC cells takes the apoptotic pathway by induction of differentiation. The results also suggest that the population of cells taking an apoptotic pathway differs from a population of cells taking a differentiation pathway. This in vitro system for apoptosis in human EC cells will be useful for studies concerning apoptosis or programmed cell death in human developmental biology.

Objective. The functions of a small GTP-binding protein, Rho, in human leukemic cell invasion was investigated in vivo and in vitro. Materials and Methods. Human leukemic KM3 and Reh cells (derived from B-cell-type common acute lymphoid leukemias) were inoculated into severe combined immundeficiency (SCID) mice, Alteration of invasion in SCID mice inoculated with KM3 cells that were introduced with the expression vector for Rho Val14 (Rho V14), an activated mutant form of Rho, was observed. Results. SCID mice inoculated with KM3 and Reh cells developed paraplegia 21 days after inoculation. All died by day 26-27, The leukemic cells were localized to bone marrow and around the spinal cord, with no infiltration into peripheral blood, spleen, liver, thymus, or lymph nodes. SCID mice inoculated with Rho V14-transfected KM3 cells showed a 5-day reduction in the time to paraplegia and death compared with SCID mice inoculated with hygromycin-resistance gene-transfected KM3 (hyg(r)) cells. In addition, the mice inoculated with Rho V14 cells showed leukemic cell infiltration, not only into bone marrow and around the spinal cord but also into peripheral blood, liver, and spleen. There were no in vitro or in vivo differences in growth rates of Rho V14 and hyg(r) cells. However, the Rho V14 cells showed markedly increased cell adhesion compared to the hygr cells. Conclusion. Results suggest that Rho activation accelerates human leukemic cell invasion via augmentation of cell adhesion. (C) 2000 International Society for Experimental Hematology, Published by Elsevier Science Inc.

A multicenter case-control study on IgA nephropathy (IgAN) was conducted to develop an evidence-based clinicopathologic classification of IgAN for predicting long-term renal outcome. Methods: Two hundred and eighty-seven patients including those with isolated hematuria or very mild proteinuria were enrolled. During a median follow-up of 9.3 years after biopsy, 49 patients (17%) progressed to end stage renal disease (ESRD). The associations between pathological variables and the need for chronic dialysis was examined by multivariate logistic regression analysis separately in patients who required dialysis earlier than 5 years (Early Progressors) and those who required dialysis within 5 to 10 years (Late Progressors) after biopsy. Results: Independent pathological variables predicting progression to ESRD were global sclerosis, segmental sclerosis and fibrous crescents for Early Progressors, and global sclerosis and cellular/fibrocellular crescents for Late Progressors. Four histological grades, HG 1, HG 2, HG 3 and HG 4, were established corresponding to < 25%, 25-49%, 50-74% and ≥75% of glomeruli exhibiting cellular or fibrocellular crescents, global sclerosis, segmental sclerosis or fibrous crescents. Eleven (7%) patients in HG 1, 12 (16%) in HG 2, 13 (31%) in HG 3 and 13 (68%) in HG 4 progressed to ESRD. Multivariate logistic analysis revealed that the risk of progression to ESRD was significantly higher in HG 2, 3 and 4 than in HG 1 (odds ratio, 2.4, 5.7 and 27.6 vs. 1.0). Conclusions: Our evidence-based histologic classification can identify the magnitude of the risk of progression to ESRD and is useful for predicting longterm renal outcome in IgAN. © 2012 Società Italiana di Nefrologia.

We report a case of membranous nephropathy with primary biliary cirrhosis (PBC). A 76-year-old Japanese man had been treated for PBC and was referred to our department because of acute severe proteinuria (10 g/day). Renal biopsy was performed and was compatible with a diagnosis of membranous nephropathy. The patient was treated with cyclosporine A (CsA; Neoral (R)), which was followed by normalization of liver function and partial remission of proteinuria (0.68 g/day) within six months without any significant side effects. This case suggests that CsA monotherapy is effective for the treatment of both liver dysfunction and membranous nephropathy associated with PBC.

Objectives: To evaluate the correlation between preoperatively predicted and pathologically measured prostate cancer volumes and to investigate the clinical use of preoperatively predicted cancer volume in predicting pathological stage. Methods: Correlations between pathological findings and various preoperative parameters, including the cancer volumes as predicted by using two methods (Vca and estimated PCvol), were analyzed in 196 patients who underwent radical prostatectomy for clinically localized prostate cancer. Results: Pathologically measured prostate cancer volume was significantly correlated with the Vca and estimated PCvol, but the correlation coefficients were respectively only 0.46 and 0.35. Prostate-specific antigen (PSA), PSA density (PSAD), primary Gleason score, Vca, Vca fraction (Vcafx), and estimated PCvol were significantly higher in 82 patients with extraprostatic cancer than in 114 patients with organ-confined cancer. Magnetic resonance imaging (MRI) findings were significantly correlated with pathological stage. Multivariate logistic regression analysis indicated that the Vcafx and MRI findings were significant predictors of extraprostatic cancer, but receiver operating characteristic analysis revealed that the combination of Vcafx and MRI findings had no advantage over the combination of Gleason score, PSAD, and MRI findings. Conclusions: Vca and estimated PCvol are significantly correlated with the pathologically measured cancer volume but their ability to accurately predict cancer volume is limited. Vcafx and MRI findings were statistically significant predictors of extraprostatic cancer but their combination was not superior to the combination of Gleason score, PSAD, and MRI findings.

POEMS (Polyneuropathy, Organomegaly, Endocrinopathy, M-protein, Skin changes) syndrome is a rare hematological disease associated with overproduction of pro-inflammatory cytokines. Under the current nomenclature and diagnostic criteria for POEMS syndrome, the presence of characteristic polyneuropathy is required for diagnosis. We report a 43-year-old Japanese woman with organomegaly, endocrinopathy, M-protein, skin lesions, as well as typical renal lesions and sclerotic bone lesions. Of note, neurological examinations and peripheral nerve conduction tests were normal in this patient. In view of the overwhelming number of otherwise characteristic signs and symptoms, we made a provisional diagnosis of 'atypical POEMS syndrome without polyneuropathy'. If further similar cases are reported in the future, reconsideration of the nomenclature and/or diagnostic criteria for POEMS syndrome may be required.