A novel surfactant protein C L55F mutation associated with interstitial lung disease alters subcellular localization of proSP-C in A549 cells.

BACKGROUND: Heterozygous mutations of SFTPC, the gene-encoding surfactant protein C (SP-C), result in interstitial lung disease (ILD). However, characterization of mutations located in the mature domain of precursor SP-C (proSP-C) is limited. This study examined the molecular pathogenesis of such a mutation of ILD. METHODS: We employed sequencing of SFTPC and established A549 cells stably expressing several proSP-C mutants. Histopathology and transmission electron microscopy (TEM) of lung tissue from a pediatric patient with ILD were assessed. Effects of mutant proSP-C were evaluated by western blotting, immunofluorescence, and TEM. RESULTS: Sequencing of SFTPC revealed a novel heterozygous mutation, c.163C>T (L55F). In lung tissue, abnormal localization of proSP-C was observed by immunohistochemistry, and small and dense lamellar bodies (LBs) in type II alveolar epithelial cells (AECs) were detected by TEM. TEM of A549 cells stably expressing proSP-C(L55F) displayed abnormal cytoplasmic organelles. ProSP-C(L55F) exhibited a band pattern similar to that of proSP-C(WT) for processed intermediates. Immunofluorescence studies demonstrated that proSP-C(L55F) partially colocalized in CD63-positive cytoplasmic vesicles of A549 cells, which was in contrast to proSP-C(WT). CONCLUSION: We detected a novel c.163C>T mutation located in the mature domain of SFTPC associated with ILD that altered the subcellular localization of proSP-C in A549 cells.

A thin carbon-fiber web as a scaffold for bone-tissue regeneration.

Due to the rapid progress being made in tissue regeneration therapy, biomaterials used as scaffolds are expected to play an important role in future clinical application. We report the development of a 3D web (sheet) consisting of high-purity carbon fibers in a nanoscale structure. When the thin carbon-fiber web (TCFW) and recombinant human bone morphogenetic protein 2 (rhBMP-2) composite is implanted in the murine back muscle, new ectopic bone is formed, and the values of the bone mineral content and bone mineral density are significantly higher than those obtained with a collagen sheet. Observation of the interface between the carbon fibers and bone matrix reveal that the fibers are directly integrated into the bone matrix, indicating high bone-tissue compatibility. Further, the rhBMP-2/TCFW composite repairs a critical-size bone defect within a short time period. These results suggest that the TCFW functions as an effective scaffold material and will play an important role in tissue regeneration in the future.

Localization of Liv2 as an immature hepatocyte marker in EB outgrowth.

The objective of this study was to establish Liv2, a surface marker of mouse immature hepatocytes (hepatoblasts), as a selection tool for embryonic stem (ES) cell-derived immature hepatocytes by acquiring basic data on Liv2 in normal mouse embryos and by confirming Liv2 expression in mouse ES-derived cells. The estimated molecular weight of Liv2 was 40-45 kDa, and immunoreactivity was definitively detected in the cell membrane of fetal hepatocytes on embryonic day (E) 9.5, declined gradually until E12.5,and subsequently became undetectable. Liv2 was localized on and close to the cell membrane. Embryoid bodies (EB) were formed from mouse ES cells whose undifferentiated state was confirmed with immunostaining of Nanog by the hanging drop method. A few Liv2-positive cells occurred as a cluster in EB outgrowth on day 7, but only some of these were albumin (ALB)-positive on day 13. These cells had the same pattern of immunoreactivity, i.e., localization on the cell membrane, as immature hepatocytes in the developing liver, although there were other types of cells with a different pattern of immunoreactivity that were seen only as a granular pattern in the cytoplasm and without ALB or the neuronal marker nestin. These results suggest thatLiv2 may be useful as a surface marker for immature hepatocytes derived from ES cells.This application would allow for the sole selection of immature hepatocytes and provide a useful tool for regenerative medicine.

Critical roles of VEGF-C-VEGF receptor 3 in reconnection of the collecting lymph vessels in mice.

Molecular mechanisms of reconnection of collecting lymph vessels were analyzed by using murine popliteal prenodal lymph vessels. At 1 and 2 weeks after being divided by cutting the lymph vessel, lymphatic reconnection was frequently observed accompanied by mesh-like lymphatic channels. Electron microscopic study also showed a monolayer of endothelial cells in the newly developed lymph vessels. Smooth muscle markers were immunofluorescently demonstrated in the wall of the new vessels. At 1 week after the procedure of cutting, augmented expressions of VEGF receptors 1, 2 and 3 were found immunohistochemically at the site of the reconnected lymph vessels. The expression of mRNA for VEGF receptor 3 was enhanced at 5 days and 1 week in small pieces of the tissues containing the reconnected lymph vessels, compared with that in the corresponding tissues obtained with sham operated ones. The administration of VEGF-C at the cutting site of the collecting lymph vessel significantly increased the rate of the reconnected lymph vessels, whereas additional treatment with Flt4/Fc chimera protein significantly reduced the rate of the reconnected ones. These results suggest that activation of VEGF-C-VEGF receptor 3 has critical roles in reconnection of the collecting lymph vessels in adult mice.

A Comparative Immunohistochemical Study of Anal Canal Epithelium in Humans and Swine, Focusing on the Anal Transitional Zone Epithelium and the Anal Glands.

To better understand the cellular origins and differentiation of anal canal epithelial neoplasms, the immunohistochemical profiles of the anal canal epithelium in humans and swine were evaluated. Formalin-fixed tissue sections were immunostained for mucin (MUC: MUC2, MUC5AC, MUC5B), desmoglein 3 (DGS3), p63, CDX2, SOX2, and α-smooth muscle actin (α-SMA). The anal transitional zone (ATZ) epithelium covered the anal sinus and consisted of a stratified epithelium with mucous cells interspersed within the surface lining. Anal glands opened into the anal sinus. Ducts and acini of intraepithelial or periepithelial mucous type were the main structures of human anal glands, whereas those of swine were compound tubuloacinar mixed glands. Distal to the ATZ epithelium, non-keratinized stratified squamous epithelium merged with the keratinized stratified squamous epithelium of the perianal skin. MUC5AC expression predominated over MUC5B expression in the ATZ epithelium, while MUC5B expression was higher in the anal glands. SOX2 was positive in the ATZ epithelium, anal glands, and squamous epithelium except in the perianal skin. In humans, DGS3 was expressed in the ATZ epithelium, anal gland ducts, and squamous epithelium. p63 was detected in the ATZ epithelium, anal glands, and squamous epithelium. Myoepithelial cells positive for α-SMA and p63 were present in the anal glands of swine. Colorectal columnar cells were MUC5B+ /MUC2+ /CDX2+ /MUC5AC- /SOX2- . The ATZ epithelium seems to be a distinctive epithelium, with morphological and functional features allowing smooth defecation. The MUC5AC+ /SOX2+ /MUC2- /CDX2- profile of the ATZ epithelium and anal glands is a useful feature for diagnosing adenocarcinoma arising from these regions. Anat Rec, 301:796-805, 2018. © 2017 Wiley Periodicals, Inc.

Characterization of clinically isolated thymidine-dependent small-colony variants of Escherichia coli producing extended-spectrum ß-lactamase.

PURPOSE: Thymidine-dependent small-colony variants (TD-SCVs) are difficult to detect or test for antimicrobial susceptibility. We investigated the characteristics of clonal TD-SCVs of Escherichia coli, both with and without blaCTX-M-3, isolated from a patient. METHODOLOGY: Mutation in the thyA gene was analysed by sequencing, and morphological abnormalities in the colonies and cells of the isolates were examined. Additionally, conjugational transfer experiments were performed to prove the horizontal transferability of plasmids harbouring resistance genes. RESULTS: The TD-SCVs contained a single nucleotide substitution in the thyA gene, c.62G>A, corresponding to p.Arg21His. Morphologically, their colonies were more translucent and flattened than those of the wild-type strain. In addition, cells of the TD-SCVs were swollen and elongated, sometimes with abnormal and incomplete divisions; a large amount of cell debris was also observed. Changing c.62G>A back to the wild-type sequence reversed these abnormalities. Conjugational transfer experiments showed that the TD-SCV of E. coli with blaCTX-M-3 failed to transfer blaCTX-M-3 to E. coli CSH2. However, the TD-SCV of E. coli without blaCTX-M-3 experimentally received the plasmid encoding blaSHV-18 from Klebsiella pneumoniae ATCC 700603 and transferred it to E. coli CSH2. CONCLUSION: Mutation in the thyA gene causes morphological abnormalities in the colonies and cells of E. coli, as well as inducing thymidine auxotrophy. In addition, TD-SCVs horizontally transmit plasmids encoding resistance genes. It is important to detect TD-SCVs based on their characteristics because they serve as reservoirs of transferable antibiotic resistance plasmids.

The fibrous form of intracellular inclusion bodies in recombinant variant fibrinogen-producing cells is specific to the hepatic fibrinogen storage disease-inducible variant fibrinogen.

Fibrinogen storage disease (FSD) is a rare disorder that is characterized by the accumulation of fibrinogen in hepatocytes and induces liver injury. Six mutations in the γC domain (γG284R, γT314P, γD316N, the deletion of γG346-Q350, γG366S, and γR375W) have been identified for FSD. Our group previously established γ375W fibrinogen-producing Chinese hamster ovary (CHO) cells and observed aberrant large granular and fibrous forms of intracellular inclusion bodies. The aim of this study was to investigate whether fibrous intracellular inclusion bodies are specific to FSD-inducible variant fibrinogen. Thirteen expression vectors encoding the variant γ-chain were stably or transiently transfected into CHO cells expressing normal fibrinogen Aα- and Bß-chains or HuH-7 cells, which were then immunofluorescently stained. Six CHO and HuH-7 cell lines that transiently produced FSD-inducible variant fibrinogen presented the fibrous (3.2-22.7 and 2.1-24.5%, respectively) and large granular (5.4-25.5 and 7.7-23.9%) forms of intracellular inclusion bodies. Seven CHO and HuH-7 cell lines that transiently produced FSD-non-inducible variant fibrinogen only exhibit the large granular form. These results demonstrate that transiently transfected variant fibrinogen-producing CHO cells and inclusion bodies of the fibrous form may be useful in non-invasive screening for FSD risk factors for FSD before its onset.

Glucagon-like peptide-1 differentiation of primate embryonic stem cells into insulin-producing cells.

The present study was performed to determine whether glucagon-like peptide-1 (GLP-1) stimulates differentiation of nestin-selected embryonic stem cells into insulin-producing cells. Our experimental strategy began with the production of a highly enriched population of nestin-positive cells from embryoid bodies. These cells differentiated into insulin-producing cells after addition of GLP-1. Islet-like cell clusters (ICCs) formed in inducing culture. These nestin-positive cell-derived ICCs expressed numerous beta-cell lineage genes, including insulin; Glut-2; pancreatic duodenal homebox-1 protein (PDX-1); islet amyloid polypeptide (IAPP); neurogenin 3 (ngn3); and alpha, gamma, and delta cell gene markers. Cells of ICCs showed increased insulin protein expression, glucose-dependent insulin release, and coexpression of insulin and C-peptide. In addition, ICCs were characterized by coexpression of nestin/insulin and nestin/PDX-1. The levels of pancreas-related gene and protein expression and insulin secretion in the GLP-1 group were stronger than those in the normal controls. GLP-1 has been shown to be involved in stimulating the signaling pathways downstream of the transcription factor PDX-1, by increasing its protein and messenger RNA levels. In vivo, ICCs displayed the ability to reverse hyperglycemia in diabetic severe combined immunodeficiency (SCID) mice. We concluded that GLP-1 induced differentiation of nestin-positive progenitor embryonic stem cells into insulin-producing cells, which was achieved by upregulation of PDX-1 expression. This method may have future applications in stem cell therapy of diabetes.

Ultrastructural analysis of TiO2 nanotubes with photodecomposition of water into O2 and H2 implanted in the nude mouse.

To analyze the biocompatibility and O2 generation of TiO2 nanotubes via photodecomposition of water into O2 and H2 in vivo, samples were implanted under the inguinal skin of the nude mouse. Venous oxygen saturation (SvO2) of the inguinal skin over the implanted region was measured with a tissue oximeter and the ultrastructures were examined with an electron microscope. Four weeks after the implantation, SvO2 of the inguinal skin of the groups with TiO2 nanotubes was 30-40% higher than that of the opposite control region (54%). SvO2 of the other groups, comprising splenic autografts, fetal cardiac tissue transplantation and surgical procedure without TiO2 nanotubes, was roughly the same as that of controls. Ultrastructurally, TiO2 nanotubes were phagocytized by the macrophage and promoted filament formation in its cytoplasm. Neither death of the cell nor destruction of the tissue was recognized. These findings indicate excellent biocompatibility and O2 generation of TiO2 nanotubes in vivo.

Natriuretic peptides in ectopic myocardial tissues originating from mouse embryonic stem cells.

In a previous report we described the survival and contractile function of mouse embryonic stem cell-derived cardiomyocytes in the host retroperitoneum. To further understand the nature of embryonic stem cell-derived cardiomyocytes, the study assessed the synthesis of natriuretic peptides in ectopic myocardial tissues of embryonic stem cell origin. Cardiomyocytes formed in embryoid body outgrowths were transplanted into the retroperitoneum of adult nude mice, and the myocardial tissues that developed were characterized by RT-PCR and immunohistochemistry concerning atrial and brain natriuretic peptides (ANP, BNP). In the outgrowths of embryoid bodies in vitro, gene expression of ANP and BNP was detected by RT-PCR and granules positive for the peptides were identified in a few cardiomyocytes by light and electron microscopic immunocytochemistry. Seven days after transplantation the transplants exhibited multidifferentiated teratoma tissues. Developing chamber myocardial tissues positive for cardiac troponin I, cadherin, and connexin 43 were evident in the transplants, which contained ANP-positive cardiomyocytes. Transplants with beating bundles were observed 30 days after transplantation, in which gene expression of both natriuretic peptides was detected. Myocardial tissues with abundant ANP-immunoreactivity, as well as with BNP-immunoreactivity to a lesser extent, were evident in the transplants. Also, myocardial tissues without immunoreactivity for natriuretic peptides were observed. Immunoelectron microscopy showed discernible secretory granules containing ANP and/or BNP in the cardiomyocytes. These results showed that part of the cardiomyocytes in embryonic stem cell-derived ectopic myocardial tissues are capable of producing natriuretic peptides, which suggests that they may be used as an endocrine source for cardiac hormones.

Phenotype-specific cells with proliferative potential are produced by polyethylene glycol-induced fusion of mouse embryonic stem cells with fetal cardiomyocytes.

Because cardiomyocytes lose the ability to divide upon differentiation, myocardial failure is assumed to be generally irreversible. For terminal cardiac insufficiency, the potential for regenerative treatment by stem cells, especially embryonic stem (ES) cells, offers hope for the future. Recent studies showed that stem cells fuse spontaneously with cells remaining in damaged tissues, and restore tissue function. To imitate spontaneous fusion in vivo, we used polyethylene glycol (PEG) in vitro to fuse mouse ES cells and fetal cardiomyocytes and analyzed the cytochemical properties of the fused cells. Confocal laser scanning microscopy coupled with lipophilic dye labeling of the living cell membranes showed that there were fused cells of ES cells and cardiomyocytes after PEG treatment. By flow cytometry, the fusion efficiency between ES cells and cardiomyocytes was estimated to be about 45% of the total resulting cells. When green fluorescent protein (GFP)-expressing ES cells were fused with cardiomyocytes, the fused cells had immunoreactivity for GFP in their cytoplasm and cardiac troponin I in their myofibrils. Some of these cells also expressed proliferating cell nuclear antigen up to 11 days after fusion, the last time point examined. This study shows that PEG-induced fusions of mouse ES cells and cardiomyocytes have the cardiomyocyte phenotype and proliferation potential.

Stimulation of P19CL6 with multiple reagents induces pulsating particles in vivo.

OBJECTIVE: Injection of stem cells into ischaemic areas of the heart is expected to be an effective method for myocardial regeneration. The embryogenic carcinoma (EC) cell line P19CL6 is known to differentiate into cardiomyocytes when cultured with dimethyl sulfoxide (DMSO) and is expected to be a promising source for regenerative therapy in cardiac disease. To establish a high-yield method of cardiomyocyte differentiation, P19CL6 cells were double-stimulated with 5-azacytidine. Double stimulation-induced cardiomyocytes were also transplanted into ectopic sites in mice and their function evaluated. METHODS AND RESULTS: To induce differentiation under adherent conditions, P19CL6 cells were incubated in growth medium with 10 microM 5-azacytidine for 24 h. After 5-azacytidine treatment, P19CL6 cells were incubated with 1% DMSO for nine days until they began to pulsate. Prior to transplantation, cells were treated again with 5-azacytidine. Differentiated cells were injected into the greater omentum, para-aorta region of the retroperitoneum and peri-femoral artery of adult BALB/c nude mice. Nine days after transplantation, irregularly pulsating tissues at a rate slower than the host heart were observed in the transplanted sites. Light microscopy showed formation of cardiac muscle tissues originating from P19CL6 cells. Differentiated cardiomyocytes were positive for cardiac troponin I, cadherin and alpha-smooth muscle actin, and the expressions of Csx/Nkx2.5 and GATA4 mRNAs were up-regulated. Electron microscopy demonstrated components specific to cardiomyocytes, such as Z-bands, desmosomes, fasciae adherens, myofibrils and mitochondria, which confirmed successful heterotopic cardiac muscle differentiation from P19CL6 cells. CONCLUSION: This study demonstrated high-yield cardiac muscle differentiation of P19CL6 by 5-azacytidine and DMSO double stimulation and successful formation of cardiac muscle-like tissue by ectopic transplantation of cardiomyocytes derived from P19CL6 into the retroperitoneal area as well as into the peripheral vessel area.

Survival and function of mouse embryonic stem cell-derived cardiomyocytes in ectopic transplants.

OBJECTIVE: Embryonic stem cell-derived cardiomyocytes are a useful source for cell transplantation into the heart, as well as for tissue engineering of the extracardiac vascular system. The present study was designed to investigate the survival and contractile function of embryonic stem cell-derived cardiomyocytes around large blood vessels to assess the feasibility of their ectopic use for future engineering of cardiovascular tissues. METHODS: The mouse embryonic stem cell-derived cardiomyocytes were transplanted into the retroperitoneum of the adult nude mice, and the myocardial tissues that developed were characterized by electrophysiological and histological techniques. RESULTS: Macroscopic and electrophysiological analyses showed spontaneously contracting transplants in the host retroperitoneum 7 and 30 days after transplantation. Immunohistochemistry detected developing cardiomyocytes in the transplants on Day 7, which formed the myocardial tissues. They were positive for cardiac troponin I, cadherin, connexin 43, and proliferating cell nuclear antigen, but negative for alpha-smooth muscle actin. Vascular formation was discernible in the transplant tissues. By Day 30, more mature myocardial tissues had been established in the transplants. Electron microscopic study emphasized that the transplant tissues comprised cardiomyocytes, in which myofibrils with organized sarcomeres were observed. Desmosomes, fasciae adherens and gap junctions were evident in the cellular junctions. CONCLUSIONS: The cardiomyocytes derived from the mouse ES cells were demonstrated to be viable and function in the ectopic site of the host retroperitoneum up to Day 30, following a process of proliferation and differentiation. Vascularization and host perfusion beneficial for the survival of the cardiomyocytes occurred in the transplants.

Spatial distribution and initial changes of SSEA-1 and other cell adhesion-related molecules on mouse embryonic stem cells before and during differentiation.

We examined the distribution of cell adhesion-related molecules (CAMs) among mouse embryonic stem (ES) cells and the spatial distribution on cell surfaces before and during differentiation. The cell-cell heterogeneity of SSEA-1, PECAM-1, and ICAM-1 among the undifferentiated cells in the ES cell colonies was evident by immunohistochemistry and immuno-SEM, supporting the flow cytometry findings. In contrast, most undifferentiated ES cells strongly expressed CD9. SSEA-1 was located preferentially on the edge of low protuberances and microvilli and formed clusters or linear arrays of 3-20 particles. PECAM-1 and ICAM-1 were randomly localized on the free cell surfaces, whereas CD9 was preferentially localized on the microvilli or protuberances, especially in the cell periphery. Both the SSEA-1(+) fraction and the SSEA-1(-) fraction of magnetic cell sorting (MACS) formed undifferentiated colonies after plating. Flow cytometry showed that these populations reverted separately again to a culture with a mixed phenotype. Differentiation induced by retinoic acid downregulated the expression of all CAMs. Immuno-SEM showed decreases of SSEA-1 in the differentiated ES cells, although some clustering still remained. Our findings help to elucidate the significance of these molecules in ES cell maintenance and differentiation and suggest that cell surface antigens may be useful for defining the phenotype of undifferentiated and differentiated ES cells.

Immunohistochemical localization of hepatocyte growth factor activator (HGFA) in developing mouse liver tissues. Heterogeneous distribution of HGFA protein.

Hepatocyte growth factor activator (HGFA) can activate the single-chain hepatocyte growth factor (HGF) required for embryonic development. We studied the immunohistochemical (IHC) localization of HGFA in adult mouse liver and its developmental changes from embryonic day 12 to postnatal day 30. A heterogeneous distribution of HGFA was observed in adult liver tissues. The hepatocytes around the hepatic veins were preferentially positive for HGFA, whereas those in other areas were negative. Depending on the vascular diameter, the hepatic veins were bordered by a one- to three-cell-thick layer of hepatocytes positive for HGFA, which showed evidence of cell-cell heterogeneity in staining intensity. Immunoelectron microscopy detected ubiquitous distribution of the gold particle reaction product for HGFA in the cytoplasm of these hepatocytes, especially in the rough endoplasmic reticulum. Developmental analysis indicated that there was hardly any staining of HGFA until postnatal day 0 and that noticeable staining was initially detected in the pericentral hepatocytes on postnatal day 3. Subsequently, immunoreactivity increased and the distinct staining pattern had been established by postnatal day 30. These results suggest that HGFA proteins are produced in the hepatocytes surrounding the efferent hepatic veins in the mouse and that development of the unique distributing pattern takes place postnatally.

Embryonic stem cell-derived embryoid bodies in three-dimensional culture system form hepatocyte-like cells in vitro and in vivo.

Pluripotent embryonic stem (ES) cells can be a source of hepatocytes for bioartificial livers or transplantation. In this study, embryoid bodies (EBs) were formed from ES cells cultured in polypropylene conical tubes. The EBs were then inserted into a collagen scaffold three-dimensional culture system and stimulated with exogenous growth factors and hormones to induce hepatic histogenesis. The EB-derived cells expressed liver-specific genes, and albumin-positive cells formed cord-like structures that were not present in two-dimensional monolayer culture systems. However, these albumin- positive cells were not cytokeratin 18 positive. Electron microscopy showed immature hepatocyte- like cells having tight junctions, rough endoplasmic reticulum, and intercellular canaliculi. The scaffold including EB-derived hepatocyte-like cells was transplanted into the median lobes of partially hepatectomized nude mice. After 7 and 14 days, cells positive for both albumin and cytokeratin 18 appeared in the transplant and formed clustered aggregates. Thus the collagen scaffold three-dimensional culture system and the liver regeneration environment induced hepatocyte-like cells and hepatic lobule-like aggregates from EBs. Therefore, differentiating EBs in the scaffold culture system may be useful in developing bioartificial livers, secondary livers, and as pharmaceutical models.

Formation of the biopulsatile vascular pump by cardiomyocyte transplants circumvallating the abdominal aorta.

In spite of the fact that patients with heart diseases requiring heart transplantation are increasing in the world, there are a lack of donors, which makes it hard to offer them these life-saving transplants. As a way to overcome this dilemma, we have researched the addition of the new biopump, which consists of the cultured embryonic cardiomyocytes grafted around the abdominal aorta and contracts spontaneously, which subsequently supports the function of the host heart. Ventricular tissues from ICR 14-day-old embryos were cultured and were injected to BALB/c nude mice (male, 8-week-old) subperitoneally around the abdominal aorta. At 3 and 7 days after implantation, action potential of the grafts was measured. Grafts were prepared for histological study. The grafts survived, showed vigorous angiogenesis, and contracted spontaneously. The cardiomyocytes in the grafts showed irregular arrangement, containing myofibrils with sarcomeres and intercalated disks. It was confirmed by immunohistochemistry that the cardiomyocytes in the grafts matured in accordance with normal development. The grafts were very quickly invaded by small vessels from the surrounding tissues showing the formation of new circulation. Embryonic cardiomyocytes have the ability to remodel the abdominal aorta into a spontaneous pulsating apparatus and to function as a vascular pump.

γ375W fibrinogen-synthesizing CHO cells indicate the accumulation of variant fibrinogen within endoplasmic reticulum.

BACKGROUND: Hepatic endoplasmic reticulum (ER) storage disease (HERSD) associated with hypofibrinogenemia has been reported in patients with four types of heterozygous γ-chain variant fibrinogen in the C terminal region. Of interest, substitution of γR375W induced hypofibrinogenemia and HERSD, whereas γR375G induced dysfibrinogenemia. OBJECTIVES: To analyze the synthesis of variant fibrinogen and morphological characteristics, we established variant fibrinogen-producing cells and compared them with wild-type fibrinogen-synthesizing cells. METHODS: The fibrinogen γ-chain expression vectors coding γ375W and γ375G were altered by oligonucleotide-directed mutagenesis and transfected into Chinese hamster ovary (CHO) cells. Synthesis of fibrinogen (media and cell lysates) was measured by ELISA for each cloned cell line and morphological characteristics were observed by immunofluorescence and transmission electron microscopy. RESULTS: The medium/cell lysate fibrinogen ratio of γ375W-CHO cells was markedly lower than that of the normal cells and γ375G-CHO cells. Immunostaining with anti-fibrinogen antibody showed only γ375W-CHO cells, but revealed two types of cells containing cytoplasmic inclusion bodies, scattered large-granular bodies and fibrous forms. Observation by confocal microscopy indicated that both inclusion bodies were colocalized with fibrinogen and ER-membrane protein; furthermore, transmission electron microscopic observation demonstrated dilatation of the ER by large-granular inclusion bodies and fibrous forms filled with regularly structured fibular materials within the dilated ER. CONCLUSION: These results demonstrated that assembled and non-secreted γ375W fibrinogen was accumulated in the dilated ER and aggregated variant fibrinogen was seen as regularly structured fibular materials, which was similar to the fingerprint-like pattern observed at inclusion bodies in patients' hepatocytes affected with HERSD.

Helicobacter heilmannii sensu stricto-related gastric ulcers: a case report.

A spiral bacterium (SH9), morphologically different from Helicobacter pylori (H. pylori), was found in a 62-year-old woman's gastric mucosa. Gastroscopic examination revealed multiple gastric ulcers near the pyloric ring; mapping gastric biopsy showed mild mononuclear infiltration with large lymphoid follicles in the antrum, without corpus atrophy. Urea breath test and H. pylori culture were negative, but Giemsa staining of biopsies revealed tightly coiled bacteria that immunostained with anti-H. pylori antibody. Sequencing of SH9 16S rRNA and the partial urease A and B subunit genes showed that the former sequence had highest similarity (99%; 1302/1315 bp) to Helicobacter heilmannii (H. heilmannii) sensu stricto (H. heilmannii s.s.) BC1 obtained from a bobcat, while the latter sequence confirmed highest similarity (98.3%; 1467/1493 bp) to H. heilmannii s.s. HU2 obtained from a human. The patient was diagnosed with multiple gastric ulcers associated with H. heilmannii s.s. infection. After triple therapy (amoxicillin, clarithromycin, and lansoprazole) with regimen for eradicating H. pylori, gastroscopy showed ulcer improvement and no H. heilmannii s.s. upon biopsy.