ITGB6-Knockout Suppresses Cholangiocarcinoma Cell Migration and Invasion with Declining PODXL2 Expression.

Intrahepatic cholangiocarcinoma (iCCA) is a heterogeneous bile duct cancer with a poor prognosis. Integrin αvß6 (ß6) has been shown to be upregulated in iCCA and is associated with its subclassification and clinicopathological features. In the present study, two ITGB6-knockout HuCCT1 CCA cell lines (ITGB6-ko cells) were established using the clustered regulatory interspaced short palindromic repeats (CRISPR), an associated nuclease 9 (Cas9) system, and single-cell cloning. RNA sequencing analysis, real-time polymerase chain reaction (PCR), and immunofluorescent methods were applied to explore possible downstream factors. ITGB6-ko cells showed significantly decreased expression of integrin ß6 on flow cytometric analysis. Both cell lines exhibited significant inhibition of cell migration and invasion, decreased wound-healing capability, decreased colony formation ability, and cell cycle dysregulation. RNA sequencing and real-time PCR analysis revealed a remarkable decrease in podocalyxin-like protein 2 (PODXL2) expression in ITGB6-ko cells. Colocalization of PODXL2 and integrin ß6 was also observed. S100 calcium-binding protein P and mucin 1, which are associated with CCA subclassification, were downregulated in ITGB6-ko cells. These results describe the successful generation of ITGB6-ko CCA cell clones with decreased migration and invasion and downregulation of PODXL2, suggesting the utility of integrin ß6 as a possible therapeutic target or diagnostic marker candidate.

MRTF-A regulates proliferation and survival properties of pro-atherogenic macrophages.

We have previously reported that promoter polymorphism of myocardin-related transcription factor A (MRTF-A) is associated with coronary atherosclerosis. However, the contribution of MRTF-A to the development of atherosclerosis remains unknown. Macrophages are known to be important mediators of atherosclerosis. It has been demonstrated that local proliferation and survival of macrophages are atherogenic. In this study, we found that MRTF-A was highly expressed in lesional macrophages in human carotid atherosclerotic plaque. We then investigated the role of macrophagic MRTF-A in the pathogenesis of atherosclerosis. ApoE null MRTF-A transgenic mice (ApoE-/-/MRTF-Atg/+), in which human MRTF-A was specifically overexpressed in monocytes/macrophages, were established and fed with normal diet to examine the progression of atherosclerosis. We found that ApoE-/-/MRTF-Atg/+ aggravated atherosclerosis and lesional macrophages were more prominently accumulated in the aortic sinus of ApoE-/-/MRTF-Atg/+ than in that of ApoE-/- littermates. We also found that MRTF-A promoted proliferation and mitigated apoptosis of macrophages both in vitro and in vivo, and down regulated the expression of cyclin-dependent kinase inhibitors. From these findings, we conclude that MRTF-A modulates functional properties of pro-atherogenic macrophages. Our study may play a valuable role in understanding the pathological role of macrophagic MRTF-A in the progression of atherosclerosis.

Fumarate accumulation involved in renal diabetic fibrosis in Goto-Kakizaki rats.

The Goto-Kakizaki (GK) rat is a spontaneous animal model of type 2 diabetes and early stage of diabetic nephropathy. However, the pathophysiological mechanisms contributing to the progression of diabetic nephropathy in GK rats remain unclear. Kidneys from 15-week old male diabetic GK/Jcl rats and age-matched Wistar rats, which have the same genetic background as GK rats, were used. Proteomic analyses of GK and Wistar kidneys were performed using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Differentially expressed proteins in GK rats were subjected to pathway analysis, and expression levels of hypoxia inducible factor 1α (HIF-1α) and transforming growth factor-ß1 (TGF-ß1), and fumarate accumulation in GK kidneys were examined. Azan staining and immunohistochemical staining of α-smooth muscle actin were performed in relation to fibrosis in GK kidneys. Proteomic analysis using 2D-DIGE, analysis of fumarate content, and expression analysis of HIF-1α, TGF-ß1, and α-smooth muscle actin of GK rat's kidney, suggested the mechanism of fibrosis characterized as two stages in diabetic nephropathy of GK rats. Abnormalities of glucose metabolism such as elevated levels of 2-oxoglutarate dehydrogenase and reduction of fumarate hydratase caused the accumulation of fumarate followed by the upregulation of HIF-1α and TGF-ß1 leading to fibrosis in diabetic nephropathy. Alterations in proteins involved in the tricarboxylic acid cycle are associated with fibrosis through fumarate accumulation in diabetic nephropathy of GK rats.

Optimization of trypsin treatment condition utilizing immunohistochemistry for chromogenic in situ hybridization.

Chromogenic in situ hybridization (CISH) is a molecular technique used to visualize specific genes. Both heat treatment and protease treatment play important roles for the success of CISH on formalin-fixed paraffin-embedded (FFPE) tissue sections. In contrast to heat treatment, the optimal condition of protease treatment may vary depending on each sample. Because trypsin has a substrate specificity to cleave lysine and arginine, we hypothesized that trypsin could effectively degrade histones rich in lysine and arginine and that the removal of histones from DNA following heat treatment could improve CISH results. We selected 21 patients with lung adenocarcinoma previously known to be positive or negative for anaplastic lymphoma kinase (ALK) gene rearrangement and used FFPE tissue sections collected from these patients. Then, we assessed histone degradation among the following protease treatments; trypsin, pepsin, and proteinase K, and compared the ALK CISH results with results obtained using commercially available kits and these protease treatments. The results showed that trypsin effectively degraded histones. Additionally, compared with the other treatments, ALK CISH with trypsin treatment showed the most evaluable cells and the smallest standard deviation. Our study suggests that the degradation of histones by trypsin subsequent to heat treatment might improve CISH results.

Frequent appearance of club cell (Clara cell)-like cells as a histological marker for ALK-positive lung adenocarcinoma.

Anaplastic lymphoma kinase-rearranged (ALK+ ) lung cancers show characteristic histological features, such as solid signet ring cell patterns and mucinous cribriform patterns; however, these features are not always observed in ALK+ lung cancers. We noticed that club cell (Clara cell)-like cells (CLCs) were frequently present in the papillary portion of ALK+ lung adenocarcinomas. In this study, we investigated the importance of CLCs in papillary patterns of ALK+ lung cancers. We compared the histological features of 18 ALK+ cases with 62 control cases (22 epidermal growth factor receptor-positive (EGFR+ ) and 40 ALK- and EGFR-negative (ALK- /EGFR- ) cases). The present study analyzed presence of papillary pattern, proportion of papillary pattern area, presence of micropapillary pattern, frequency of CLCs and lengths of snout. The frequency of CLCs in ALK+ cases was significantly higher than that in EGFR+ cases and ALK- /EGFR- cases. Micropapillary pattern was more frequently observed in ALK+ cases than that in ALK- /EGFR- cases (P < 0.001). The present study indicated that the high frequency of CLCs in papillary patterns was significantly associated with ALK+ cases. When solid signet ring cell patterns and mucinous cribriform patterns are absent, the high frequency of CLCs in papillary adenocarcinoma could be a useful histological marker for ALK+ lung cancers.

ß4 and ß6 Integrin Expression Is Associated with the Subclassification and Clinicopathological Features of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a heterogeneous group of cancers of the intrahepatic biliary tract. However, few studies have evaluated integrin expression according to an ICC subgroup. We immunohistochemically investigated α6ß4 (ß4) and αvß6 (ß6) integrin expressions in 48 ICCs, and evaluated their relationship with clinical and pathological parameters and ligand expression, as well as transforming growth factor (TGF)-ß1. ß4 and ß6 expressions were detected in 46 (96%) and 35 (73%) ICC cases, respectively. We classified ICC into negative, low (ß4, 29 cases; ß6, 36 cases), or high (ß4, 19 cases; ß6, 12 cases) integrin expression groups. ß4 and ß6 integrin levels were higher in the non-peripheral central localization type ICC than in the peripheral localization type; they were also higher in the periductal-infiltrating or intraductal-growth types than in the mass-forming type ICC; lastly, they were higher in the well-differentiated type than in the poorly-differentiated type ICC. High expression was related to bile duct invasion. In addition, ß4 and ß6 expressions were associated with mucin production and the expression of cytoplasmic epithelial membrane antigen, laminin-5, and tenascin-C. TGF-ß1 was correlated with ß6 expression and poor overall survival. These results suggest that integrin expression is associated with subclassification and clinicopathological features of ICC through the coincident expression of their ligands and TGF-ß1.

Clinical and pathological significance of myeloid differentiation factor 88 expression in human hepatocellular carcinoma tissues.

The innate immune system, which includes toll-like receptor (TLR) signaling, plays an important role in inflammation and oncogenesis. Although TLR common adaptor myeloid differentiation factor 88 (MyD88) is known to have multiple effects on carcinogenesis, the role of MyD88 in hepatocarcinogenesis remains unknown. In this study, MyD88 expression was examined in 105 samples of human hepatocellular carcinoma (HCC) tissue by immunohistochemistry, Western blot, and quantitative reverse-transcriptase polymerase chain reaction methods. The relationships between MyD88 expression and clinical and pathological parameters were analyzed. The results showed that attenuated expression of MyD88 in HCC tissue tumor cells was significantly related to hepatitis B virus infection, large tumor size, positive vascular invasion, and intrahepatic metastasis (P < 0.05). Western blot analysis of MyD88 protein in nine normal livers and 28 HCCs showed gender disparity (P < 0.01, P < 0.05), and attenuated expression in cirrhotic livers (P < 0.05). Low expression of MyD88 mRNA was evident in HCCs with vascular invasion (P < 0.01). In contrast to previous reports, these results suggest that attenuated expression of MyD88 in HCC is associated with tumor progression.

High-fat diet increases vulnerability to atrial arrhythmia by conduction disturbance via miR-27b.

BACKGROUND: Lifestyle-related diseases, such as obesity and dyslipidemia are important risk factors for atrial fibrillation (AF). However, the underlying mechanism linking these diseases and AF has not been fully investigated. METHODS: Adult male mice were fed a high-fat diet (HFD) or vehicle (NC) for 2 months. Electrocardiography and in vivo electrophysiological study were performed. Mice were then sacrificed for quantification of mRNA, microRNA, and protein in atria, in addition to histological analysis. Conduction velocity (CV) in right atrium was measured by optical mapping in Langendorff perfused hearts. Cultured atrial cardiomyocytes were treated with palmitate with or without a specific microRNA inhibitor. Twelve hours after stimulation, cells were lysed, and subjected to analysis with qPCR and Western blotting. RESULTS: HFD mice showed prolonged P wave duration, increased inducibility of sustained atrial tachycardia, and reduced atrial CV than NC mice. HFD mice also showed increased expression in inflammatory cytokines, whereas fibrotic area and signals relating fibrosis were not changed. HFD mice demonstrated reduced expression of Cx40 in mRNA and protein levels, and its lateralized expression in atria. MicroRNA array analysis revealed that miR-27b expression was up-regulated in HFD mice, and luciferase assay confirmed the direct interaction between miR-27b and Cx40 3'UTR. In palmitate-stimulated atrial cardiomyocytes, miR-27b up-regulation and Cx40 down-regulation were observed, while expression of inflammatory cytokines was not altered. Inhibition of miR-27b with antisense oligonucleotides reversed the alteration caused by palmitate stimulation. CONCLUSION: HFD may increase the vulnerability to atrial arrhythmia by down-regulation of Cx40 via miR-27b, rather than fibrosis, which is independent of inflammation.

Preserved or enhanced OATP1B3 expression in hepatocellular adenoma subtypes with nuclear accumulation of ß-catenin.

AIM: Recent studies have indicated that hepatocellular adenoma (HCA) is a heterogenous group of benign tumors with various genetic and clinicopathological characteristics. We delineated the clinicopathological characteristics of HCA subtypes and evaluated the expression of transporter protein OATP1B3 in HCA. METHODS: HCA in 34 Japanese patients were investigated immunohistochemically and classified into four subtypes (HNF1α-inactivated type, H-HCA; ß-catenin-activated type, b-HCA; inflammatory type, I-HCA; unclassified type, u-HCA). Immunostaining of OATP1B3 protein in HCA tissue sections was performed to determine the association between OATP1B3 expression and HCA subtypes. RESULTS: HCA was categorized into the following four subtypes and two combined subtypes: 10 H-HCA (29%), 10 I-HCA (29%), seven b-HCA (21%), two b-HCA/H-HCA (6%), two b-HCA/I-HCA (6%) and three u-HCA (9%). The male-to-female ratio was 18:16. Oral contraceptive use was rare but seven HCA were found in patients with glycogen storage disease, congenital absence of the portal vein and idiopathic portal hypertension. OATP1B3 expression was decreased in 24 HCA but was preserved or increased in 10 HCA. All nine HCA with nuclear staining for ß-catenin showed preserved or enhanced OATP1B3 expression, indicating a significant association between nuclear ß-catenin accumulation and OATP1B3 expression in HCA. CONCLUSION: HCA subtype classification was validated in 91% of our Japanese subjects although their clinical backgrounds including rare contraceptive use were different from European subjects. A close association between preserved or enhanced OATP1B3 expression and b-HCA subtype indicated important modalities for clinical decisions in the treatment and follow up of patients with HCA.

Stromal miR-21 is more important than miR-21 of tumour cells for the progression of gastric cancer.

BACKGROUND: Gastric cancer (GC) is a common cancer globally. miRNA-21 (miR-21) appears to be important in the tumourigenesis of almost all types of human cancer. However its precise localization in GC has yet to be clarified. We thus examined miR-21 localization in GC and revealed its clinicopathological importance. METHODS: Tissue arrays of 469 GCs from 454 patients were examined for miR-21 using in situ hybridization (ISH). The positivity was evaluated separately in tumour cells and stromal cells. Conventional sections of 10 GCs were also stained. Eight cases were examined by quantitative RT-PCR (qRT-PCR). RESULTS: miR-21 was highly expressed in tumour cells of 44% of cases and in cancer stroma of 51% of cases. miR-21 of tumour cells was not related to clinicopathological factors, whereas stromal miR-21 was related to many factors including tumour stage, size, and nodal metastasis. Stromal miR-21 gradually increased during tumour progression. ISH of whole sections showed stronger stromal positivity in invasive areas with desmoplastic reaction. Cancer stroma also showed higher miR-21 expression than tumour and non-tumourous tissue in the qRT-PCR study. CONCLUSION: Stromal miR-21 is closely related to tumour progression in GC. Stromal miR-21 of tumours might be a target of treatment.

Integrins αvß6, α6ß4 and α3ß1 are down-regulated in cholangiolocellular carcinoma but not cholangiocarcinoma.

AIM: The aim of this study was to evaluate integrin expression and immunolocalization of the extracellular matrix proteins in cholangiolocellular carcinoma (CoCC). METHODS: Tissue specimens of 23 CoCC, 28 cholangiocarcinomas (CCC), 42 hepatocellular carcinomas (HCC) and 11 classical type combined hepatocellular cholangiocarcinomas (CHC) were immunostained for ß6, ß4 and α3 integrins, fibronectin and laminin. ITGB6, B4 and A3 mRNA levels in six HCC cell lines, five CCC cell lines and two CHC cell lines were quantified by quantitative reverse transcription polymerase chain reaction. RESULTS: Little or no positivity for ß6, ß4 and α3 integrins was shown in 91%, 91% and 52% of CoCC and 100%, 98% and 81% of HCC, respectively, according to immunostaining, whereas intense positive staining for these integrins was demonstrated in 64%, 96% and 75% of CCC, respectively. There was a close correlation between ß4 and α3 integrin expression and intracytoplasmic laminin in CoCC, CCC and HCC, but not between ß6 expression and its ligand, fibronectin. Integrin mRNA levels were increased in four of five CCC cell lines, but nearly undetectable in five of six HCC cell lines and one CHC cell line. Tubular differentiation of a CHC cell line cultured in collagen gel matrix induced upregulation of these integrins. CONCLUSION: Our results first indicated downregulation of αvß6, α6ß4 and α3ß1 integrins in CoCC, in contrast to its high expression in CCC, suggesting a diagnostic value of integrins in the differential diagnosis of CoCC and CCC, as well as a useful inducible marker of the intermediate features of CoCC.

Japanese herbal medicines shosaikoto, inchinkoto, and juzentaihoto inhibit high-fat diet-induced nonalcoholic steatohepatitis in db/db mice.

Few studies have investigated the effects of Japanese herbal medicines on nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). To the best of our knowledge, only one study has examined whether high-fat (HF) diet-fed db/db mice are appropriate animal models of NASH. We investigated the effects of four types of Japanese herbal medicines (shosaikoto (TJ-9), inchinkoto (TJ-135), juzentaihoto (TJ-48), and keishibukuryogan (TJ-25)) on hepatic lesions of HF diet-fed db/db mice. Db/db mice were divided into six groups: control diet (control); HF diet (HF); and HF diet supplemented with TJ-9, TJ-135, TJ-48, or TJ-25 (TJ-9, TJ-135, TJ-48, and TJ-25, respectively). Mice were killed after 6 weeks of treatment, and biochemical and pathological analyses were performed. Mice in the HF group consistently developed histopathological features consistent with definite NASH, and marked necroinflammation occurred. Serum alanine aminotransferase levels in the TJ-9, TJ-135, and TJ-48 groups were significantly improved compared with those in the HF group. With regard to liver histology, TJ-9 and TJ-48 significantly improved lobular inflammation, and TJ-135 significantly improved ballooning degeneration. We have shown that HF diet-fed db/db mice are animal models that correctly recapitulate the histopathology of human NASH and that TJ-9, TJ-135, and TJ-48 inhibit necroinflammatory activity in this model.

Long-term surgical outcomes of patients with type 1 autoimmune pancreatitis.

BACKGROUND: Although pancreatectomy has sometimes been performed for patients with autoimmune pancreatitis (AIP) presenting atypical radiologic findings under the diagnosis of pancreatobiliary malignancy, the long-term surgical outcome of these patients had not yet been fully elucidated. METHODS: The long-term surgical outcomes of 13 patients with pathologically diagnosed type 1 AIP with immunohistochemical staining for immunoglobulin G4 (IgG4) were retrospectively compared with those of 34 patients with conventional chronic pancreatitis to evaluate the residual pancreatic function. RESULTS: A definite relapse of AIP in terms of the clinical manifestations and diagnostic imaging was not found in any of the patients, although one patient experienced an attack of acute pancreatitis caused by pancreatic stones, and stricture of the hepaticojejunostomy occurred in one patient. The overall body weight decreased significantly more in patients with AIP than in patients with conventional chronic pancreatitis (p < 0.05); however, there was no difference in the preoperative nondiabetic patients. Refractory diarrhea occurred in only one patient with AIP. The cumulative new-onset rates of diabetes mellitus of the ten patients with AIP and 24 patients with conventional chronic pancreatitis at 5 years after the surgery were 32.5 and 26.1 %, respectively (p = 0.70). CONCLUSIONS: Careful long-term follow-up is needed for patients undergoing pancreatectomy for type 1 AIP because remnant pancreatic function can deteriorate as severely as that of patients who undergo pancreatectomy for conventional chronic pancreatitis. In the present series, however, there were few definite manifestations indicating relapse or the persistent existence of AIP.

Development of a new histological identification method of human sinoatrial node suitable for immunohistochemical study.

Histological identification of the human sinoatrial node (SAN) remains a challenge. Conventional identification methods, such as Lev's method, have certain limitations. The aim of our study was to develop a new histological identification method that could properly identify the sinoatrial node, applicable to the immunohistochemical study of intra-nodal structures. Thirty-nine human autopsied hearts were included in this study. The cases included 23 men and 16 women ranging in age from 20 to 99 years. The sinoatrial area from eight control samples was cut in the vertical section using the conventional Lev's method. In our new method, called the "En face one-block method," the sinoatrial node was cut in "En face" at the junction of the right border of the right appendage and superior vena cava, placed in one long cassette, and serially cut using a microtome. Immunostaining was performed using primary antibodies against CD31, podoplanin (D2-40), S-100, and other proteins. The average area of the SAN on the slide glass in our new method was 32.2 mm2, which was significantly larger than that (3.59 mm2) of the control samples by Lev's method. The SAN area was positively correlated with age (r = 0.357; p = 0.026), especially in women (r = 0.626; p = 0.0095). The SAN group had significantly lower percentage of CD31-positive blood capillaries, higher percentage of podoplanin-positive lymphatic channels, and S-100-positive peripheral nerves. We successfully developed a novel cutting method applicable to immunohistochemical studies, with which we could provide a bird's-eye view of the sinoatrial nodes.

Gross Cystic Disease Fluid Protein-15 (GCDFP-15) Expression Characterizes Breast Mucinous Carcinomas in Older Women.

The predominant histological subtype of breast mucinous carcinoma in older women is type B (hypercellular type), and, in younger women, it is type A (hypocellular type). The characteristics of mucinous carcinomas of the same histological subtype may differ between older and younger women. This study aims to systematically clarify the pathological/immunohistochemical features of mucinous carcinomas. A total of 21 surgical cases of mucinous carcinoma (type A/B: 9/12 cases) in the older group (≥65 years) and 16 cases (type A/B: 14/2 cases) in the younger group (≤55 years) (n = 37) were included. Gross cystic disease fluid protein-15 (GCDFP-15) and eight other markers were used for immunostaining. The GCDFP-15-positive rate in the older group was high regardless of the histological subtype (type A, 77.8%; type B, 91.7%). The GCDFP-15 positivity in the older group was significantly higher than that in the younger group (p < 0.001 for Allred score). Among type A, GCDFP-15 positivity was significantly higher in the older group than in the younger group (p = 0.042 for the Allred score and p = 0.007 for the positivity rate). The present results suggest that GCDFP-15 expression characterizes mucinous carcinomas in older women.

Proteome analysis demonstrates involvement of endoplasmic reticulum stress response in human myocardium with subclinical left ventricular diastolic dysfunction.

AIM: Heart failure is increasing in Japan, in particular that with preserved ejection fraction (HFpEF) prevalent in older-aged patients. The purpose of this study was to investigate the pathophysiology during the early stage of left ventricular (LV) diastolic dysfunction by the quantitative proteome analysis of human myocardium. METHODS: Among 331 post-mortem autopsy patients, we selected 23 patients (aged 79 ± 9.6 years) with echocardiographic data and without major comorbidities, except hypertension. Cryopreserved autopsy tissue of the LV myocardium was subjected to proteome analysis. LV diastolic function was evaluated by echocardiographic data. Thirteen patients were classified into the impaired diastolic function (IDF) group, and 10 the normal cardiac function group. We performed comparative proteome analysis between the IDF and normal groups by isobaric tags for relative and absolute quantitation (iTRAQ) using nano-liquid chromatography-tandem mass spectrometry. RESULTS: The iTRAQ-based proteome analysis revealed 57 differentially expressed proteins in the IDF group. Molecular network analysis of differentially expressed proteins indicated that endoplasmic reticulum (ER) stress was a potentially important event. Furthermore, the expressions of proteins associated with the ER stress response, such as glucose-regulated protein 78 kDa, inositol-requiring kinase 1α and spliced X-box binding protein 1, were significantly decreased in the IDF group. CONCLUSIONS: This study suggested that reduced ER stress responses were involved during the early stage of LV diastolic dysfunction. Geriatr Gerontol Int ••; ••: ••-•• Geriatr Gerontol Int 2021; 21: 577-583.

A missense mutation in the RSRSP stretch of Rbm20 causes dilated cardiomyopathy and atrial fibrillation in mice.

Dilated cardiomyopathy (DCM) is a fatal heart disease characterized by left ventricular dilatation and cardiac dysfunction. Recent genetic studies on DCM have identified causative mutations in over 60 genes, including RBM20, which encodes a regulator of heart-specific splicing. DCM patients with RBM20 mutations have been reported to present with more severe cardiac phenotypes, including impaired cardiac function, atrial fibrillation (AF), and ventricular arrhythmias leading to sudden cardiac death, compared to those with mutations in the other genes. An RSRSP stretch of RBM20, a hotspot of missense mutations found in patients with idiopathic DCM, functions as a crucial part of its nuclear localization signals. However, the relationship between mutations in the RSRSP stretch and cardiac phenotypes has never been assessed in an animal model. Here, we show that Rbm20 mutant mice harboring a missense mutation S637A in the RSRSP stretch, mimicking that in a DCM patient, demonstrated severe cardiac dysfunction and spontaneous AF and ventricular arrhythmias mimicking the clinical state in patients. In contrast, Rbm20 mutant mice with frame-shifting deletion demonstrated less severe phenotypes, although loss of RBM20-dependent alternative splicing was indistinguishable. RBM20S637A protein cannot be localized to the nuclear speckles, but accumulated in cytoplasmic, perinuclear granule-like structures in cardiomyocytes, which might contribute to the more severe cardiac phenotypes.

A 3-Dimensional Model of the Human Round Window Membrane.

OBJECTIVES: The round window membrane (RWM) is small in size, making it difficult to clarify its shape and structure. The authors examined a 40x magnified 3-dimensional model of the human RWM to clarify its morphologic aspects and characteristics. METHODS: An RWM specimen was obtained from an archival, formalin-fixed, decalcified, left temporal bone of an 84-year-old female cadaver. The data obtained by laser scanning microscopy were input into a 3-dimensional printer. After a model of the RWM was created, the following features were examined: striae on the surfaces, curvatures, thickness, and areas. Cross sections of the original specimen were made for histological observations. RESULTS: The contour of this RWM model was approximately elliptic, with a saddle shape. When illuminated from the scala tympani side, the surface facing the fossula exhibited dark anterior and clear posterior portions. A borderline appeared where the 2 portions were bound along the short axis of the ellipse. This borderline was identified as the line of inflection. Collagen fibers were shown to run parallel to the borderline in the posterior portion but were fanned out in the anterior portion. CONCLUSIONS: The magnified 3-dimensional model clarified gross anatomy and characteristics of the RWM. It is good teaching material for small tissues, such as the RWM.

Age-associated proteomic alterations in human aortic media.

AIM: Human vascular senescence, which mainly occurs in media, is not completely understood. Here, we used proteomic approaches to investigate age-associated changes in human aortic media with the goal of understanding the molecular mechanisms underlying vascular senescence. METHOD: Cryopreserved autopsy samples of aortic media from older-aged (aged 70-100 years, n = 25), middle-aged (aged 49-68 years, n = 24), and young (aged 21-39 years, n = 12) subjects were collected. We used two proteomic techniques, two-dimensional differential gel electrophoresis and isobaric tags for relative and absolute quantitation, and we subjected differentially-expressed proteins among age groups to immunohistochemical analyses. RESULTS: Proteomic analyses showed that the expression of lactadherin, which produces medin, was elevated in aortic media of older-aged individuals. Immunohistochemical and Congo red staining showed that lactadherin and apolipoprotein E were deposited, and that amyloidosis was enhanced in older-aged aortic media. Furthermore, the markers of oxidative damage (8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal) were significantly elevated in aortic media of middle-aged or older-aged individuals. The immunohistochemical expression of anti-oxidant proteins (thioredoxin and extracellular superoxide dismutase) was also high in middle-aged and older-aged groups. Oxidative damage might induce the disruption of smooth muscle cells, resulting in the decrement of α-actin, a highly-expressed protein in smooth muscle cells, and matrix remodeling, in which several proteins associated with extracellular matrix were altered with aging. CONCLUSIONS: Proteomic approaches showed that the elevated expression of lactadherin might contribute to amyloid deposition, enhancement of oxidative stress, induction of antioxidant proteins and matrix remodeling in older-aged aortic media. Geriatr Gerontol Int 2019; 19: 1054-1062.

Useful Application of Immunostaining to Malignant Pleural Effusion among Lao People in Vientiane Capital, Lao PDR

Background: Pathology and laboratory medicine (PALM) services are limited in low-resource countries, such as Lao PDR. Patients with malignant pleural effusion (MPE) are not properly diagnosed and treated in these situations. The purpose of this study is to confirm the usefulness of immunocytochemistry in MPE to identify the histological type and probable primary site of cancer of MPE and to discuss its usefulness in low-resource countries, such as Laos. Methods: We retrospectively reviewed glass slides of pleural effusion sent to the Department of Pathology at the University of Health Sciences from the central hospitals for cytological screening from January 2012 to December 2016. The cytological review, cell transfer and immunocytochemical staining were performed at Tokyo Medical and Dental University. Among 81 cases of MPE from Laos, 66 cases of malignant tumors that contained enough tumor cells were included in this study, and the slides were screened with 14 primary antibodies to classify the histological type and identify the probable primary site of carcinoma. Results: Among the 66 cases, 34 cases (52%) were of female patients, and 32 cases (48%) were of male patients. The patients' ages ranged from 28 to 88 years with an average of 58 years. The immunocytochemical study identified 32 cases (49%) of primary lung adenocarcinoma, two cases (3%) of malignant mesothelioma, one case (1.5%) of breast/gynecological carcinoma, one case (1.5%) of T cell lymphoma, and one case (1.5%) of B cell lymphoma. Twenty-nine cases (43.5%) were classified as carcinoma not otherwise specified. Pulmonary small cell carcinoma/squamous cell carcinoma and metastatic colon, prostate, and liver carcinoma were not identified among the cases. Conclusions: Immunocytochemistry is a useful ancillary method in MPE diagnostics. This method could be applied in the pathological laboratories in low- or middle-resource countries, such as Laos.