Generation of Otic Lineages from Integration-Free Human-Induced Pluripotent Stem Cells Reprogrammed by mRNAs.

Damage to the sensory hair cells and the spiral ganglion neurons of the cochlea leads to deafness. Induced pluripotent stem cells (iPSCs) are a promising tool to regenerate the cells in the inner ear that have been affected by pathology or have been lost. To facilitate the clinical application of iPSCs, the reprogramming process should minimize the risk of introducing undesired genetic alterations while conferring the cells the capacity to differentiate into the desired cell type. Currently, reprogramming induced by synthetic mRNAs is considered to be one of the safest ways of inducing pluripotency, as the transgenes are transiently delivered into the cells without integrating into the genome. In this study, we explore the ability of integration-free human-induced pluripotent cell lines that were reprogrammed by mRNAs, to differentiate into otic progenitors and, subsequently, into hair cell and neuronal lineages. hiPSC lines were induced to differentiate by culturing them in the presence of fibroblast growth factors 3 and 10 (FGF3 and FGF10). Progenitors were identified by quantitative microscopy, based on the coexpression of otic markers PAX8, PAX2, FOXG1, and SOX2. Otic epithelial progenitors (OEPs) and otic neuroprogenitors (ONPs) were purified and allowed to differentiate further into hair cell-like cells and neurons. Lineages were characterised by immunocytochemistry and electrophysiology. Neuronal cells showed inward Na+ (I Na) currents and outward (I k) and inward K+ (I K1) currents while hair cell-like cells had inward I K1 and outward delayed rectifier K+ currents, characteristic of developing hair cells. We conclude that human-induced pluripotent cell lines that have been reprogrammed using nonintegrating mRNAs are capable to differentiate into otic cell types.

Simple Silica Column-Based Method to Quantify Inorganic Polyphosphates in Cartilage and Other Tissues.

OBJECTIVE: Inorganic polyphosphates (polyP) play a multitude of roles in mammalian biology. PolyP research is hindered by the lack of a simple and sensitive quantification method. The aim of this study was to develop a robust method for quantifying the low levels of polyP in mammalian tissue such as cartilage, which is rich in macromolecules that interfere with its determination. DESIGN: Native and in vitro formed tissues were digested with proteinase K to release sequestrated polyP. The tissue digest was loaded on to silica spin columns, followed by elution of bound polyP and various treatments were assessed to minimize non-polyP fluorescence. The eluent was then quantified for polyP content using fluorometry based on DAPI (4',6-diamidino-2-phenylindole) fluorescence shift occurring with polyP. RESULTS: Proteinase K pretreatment reduced the inhibitory effect of proteins on polyP recovery. The eluent was contaminated with nucleic acids and glycosaminoglycans, which cause extraneous fluorescence signals. These were then effectively eliminated by nucleases treatment and addition of concentrated Tris buffer. PolyP levels were quantified and recovery ratio determined using samples spiked with a known amount of polyP. This silica spin column method was able to recover at least 80% of initially loaded polyP, and detect as little as 10-10 mol. CONCLUSIONS: This sensitive, reproducible, easy to do method of quantifying polyP will be a useful tool for investigation of polyP biology in mammalian cells and tissues. Although the protocol was developed for mammalian tissues, this method should be able to quantify polyP in most biological sources, including fluid samples such as blood and serum.

Pelvic Limb Amputation for the Treatment of a Soft-tissue Sarcoma of the Tibiotarso-tarsometatarsal Joint in a Blue-and-gold Macaw (Ara ararauna).

An unsexed, 16-year-old blue-and-gold macaw (Ara ararauna) was presented for evaluation of rapidly growing subcutaneous masses at the left tibiotarso-tarsometatarsal joint. Results of incisional biopsy were diagnostic for an intermediate-grade soft-tissue sarcoma. A distal-femoral amputation was performed and the leg was submitted for histopathology. Histopathologic examination confirmed the biopsy diagnosis and revealed neoplastic spread into the bone marrow cavity of the tibiotarsus. Excisional margins were complete. The macaw recovered and did well until it died suddenly 32 months after surgery. At necropsy, death was attributed to acute hepatic hemorrhage. No recurrence or metastasis of the sarcoma was identified.

Generation of human iPSC line GRX-MCiPS4F-A2 from adult peripheral blood mononuclear cells (PBMCs) with Spanish genetic background.

We have generated iPSCs from peripheral blood mononuclear cells (PBMCs) of a healthy man using heat sensitive and non-integrative Sendai virus containing Sox2, Oct3/4, c-Myc and Klf4. Human GRX-MCiPS4F-A2 cell line was established and characterized through this study.

Antimicrobial effects of coprisin on wounds infected with Staphylococcus aureus in rats.

Antimicrobial peptides (AMPs) are naturally produced antibiotics that play important roles in host defense mechanisms. These proteins are found in variety of animal and plant species. The antibiotic effects of AMPs are gaining attention for use in human medicine. In this study, the antimicrobial effects of coprisin, a novel AMP isolated from the dung beetle (Copris tripartitus), were evaluated. The peptide was used to treat rats with wounds infected with Staphylococcus aureus. Coprisin accelerated wound closure both grossly and microscopically compared with the untreated group. Additionally, treatment with this peptide decreased phosphorylated-Smad2/3 (p-Smad2/3) levels, a downstream factor of the transforming growth factor-ß signaling pathway which is believed to inhibit reepithelization, in the nucleus and cytoplasm of regenerating cells. Moreover, increased cell populations and angiogenesis were observed in lesions treated with coprisin, suggesting that this peptide promotes wound healing via its antimicrobial activity against S. aureus. Our results demonstrated that coprisin is a potential therapeutic agent that can possibly replace traditional antibiotics and overcome microbial resistance.

Mucosa-associated lymphoid tissue lymphoma of the third eyelid conjunctiva in a dog.

A 4-year-old, neutered female Cocker Spaniel was presented to the veterinary clinic for protrusion of the left third eyelid. When the third eyelids from both eyes were everted, lobulated masses were present on the bulbar surface. The left third eyelid had a larger protrusion. There was no apparent associated ocular or systemic involvement. The tumor of left third eyelid was removed and referred for histological examination. Histologically, there were proliferations of lymphoid follicles surrounded by lymphoid cells forming a marginal zone. Those lymphoid cells occasionally infiltrated into conjunctival epithelium. A few apoptotic bodies with karyopyknotic and karyorrhexic nuclei were observed in the germinal center of lymphoid follicles. Mitotic figures were rare. On immunohistochemistry, tumor cells expressed CD79a but not CD3. A diagnosis of extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) of the third eyelid was established based on the histological and immunophenotypical features. At the 1-year follow-up, there was no evidence of recurrence of the mass at the area of excision of the left third eyelid and the remaining tumor of the right third eyelid was still a similar size. The dog still showed no significant findings, except those of the tumor, and no evidence of systemic involvement. To the authors' knowledge, this is the first reported case of MALT lymphoma of the third eyelid in a dog.

Vitamin C deficiency increases the binucleation of hepatocytes in SMP30 knock-out mice.

BACKGROUND AND AIMS: The binucleation of hepatocytes, which was known as an important feature of liver growth and physiology, has been reported to be increased during the chronic oxidative injury stage and has been regarded as an age-related change of hepatic structures. Therefore, we investigated the binuclearity pattern in the livers of senescence marker proteins-30 (SMP30) knock-out (KO) mice compared with wild-type (WT) mice and vitamin C-treated KO (KO + VC) mice. METHODS: The WT, KO and KO + VC mice were fed a vitamin C free diet and VC(+) group mice were given vitamin C water containing 1.5 g/L of vitamin C, whereas VC(-) group was given normal drinking water without vitamin C, for 16 weeks. RESULTS: In microscopic examination, the livers of KO mice showed a significantly increased number of binuclear hepatocytes compared with that of WT mice and KO + VC mice. KO mice also showed the most increased expression level of CYP2E1 and PCNA determined by immunohistochemistry and immunoblot analysis. Moreover, KO mice indicated the highest level of serum alanine aminotransferase and aspartate aminotransferase level in serum biochemical analysis. Accordingly, significantly decreased levels of reactive oxygen species, MDA (malondialdehyde) and HAE (4-hydroxyalkenals) were detected in KO + VC mice compared with KO mice. CONCLUSION: Therefore, it is concluded that vitamin C deficiency induces an increase of CYP2E1 expression and elevated ROS production, which causes oxidative liver injury and the elevation of hepatocyte binucleation in SMP30 KO mice.

Molecular basis for age-related changes in ileum: involvement of Bax/caspase-dependent mitochondrial apoptotic signaling.

Previous studies indicate that in the elderly, a morphological change in the small intestine is accompanied by apoptosis. However, currently little information is available on the molecular events leading up to the apoptotic process in aged ileum. Our current study assessed mitochondrial apoptotic signaling along with key factors known to be involved in mitochondrial permeabilization in rat ileum. Experimentations were carried out utilizing Sprague-Dawley rats at 6 and 24 months of age. The histological analysis showed a significant loss in thickness of the intestinal mucosa during aging, which was accompanied by higher reactive species. Molecular analysis revealed the mitochondrial translocation of Bax showed a significant increase with aging. However, the expression of cyclophilin D, adenine nucleotide translocator, and the voltage-dependent anion channel that regulates the mitochondria permeability transition pore decreased or remained unchanged. Furthermore, the expression of caspase 3 was enhanced in aged ileum with increased DNA fragmentation, while nuclear translocation of apoptosis-inducing factor and endonuclease G were decreased with aging. In conclusion, our findings indicate that the mitochondrial translocation of Bax by increased oxidative stress may result in cell death through caspase-dependent apoptosis in aged ileum, thereby leading to a decrease in intestinal mucosal thickness during aging.

Helicobacter pylori accelerates hepatic fibrosis by sensitizing transforming growth factor-ß1-induced inflammatory signaling.

Our earlier report has shown that Helicobacter pylori promoted hepatic fibrosis in a murine model. Herein, in order to elucidate the mechanism by which H. pylori accelerate liver fibrosis, the authors investigated the changes in expression levels of mitogen-activated protein kinases (MAPKs), p53-related proteins, antioxidants, and proinflammatory cytokines in liver samples. H. pylori infection enhanced CCl4-induced MAP kinase activation and p53 signaling pathway as well as Bax- and proliferating-cell nuclear antigen expressions, whereas H. pylori alone induced neither of these expressions nor hepatic fibrosis. Moreover, mRNA expressions of inflammatory cytokines, glutathione peroxidase expression, and the proliferative index were strongly augmented in livers of the H. pylori with CCl4 treatment group compared with those of the CCl4-alone treatment group, whereas there was no difference in apoptotic index between the two groups. Interestingly, H. pylori treatment increased the number of α-fetoprotein-expressing hepatocytes independently of CCl4 intoxication. In vitro analyses, using an immortalized rat hepatic stellate cell (HSC) line, revealed that H. pylori lysates increased the proliferation of HSCs, which was boosted by the addition of transforming growth factor-beta1 (TGF-ß1). Furthermore, the treatment of H. pylori lysates promoted the translocation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) into the nucleus based on an increase in the degradation of NF-κB inhibitor alpha, in the presence of TGF-ß1, as did H2O2 treatment. In conclusion, H. pylori infection along with an elevated TGF-ß1 may accelerate hepatic fibrosis through increased TGF-ß1-induced pro-inflammatory signaling pathways in HSCs. Moreover, H. pylori infection might increase the risk of TGF-ß1-mediated tumorigenesis by disturbing the balance between apoptosis and proliferation of hepatocytes.

In vitro inhibition of Helicobacter pylori growth and of adherence of cagA-positive strains to gastric epithelial cells by Lactobacillus paraplantarum KNUC25 isolated from kimchi.

Lactobacillus paraplantarum KNUC25 strain was isolated from overfermented kimchi, a Korean traditional food. The strain had a broad antimicrobial activity spectrum, from gram-positive to gram-negative bacteria. The aim of this study was to evaluate the activity of L. paraplantarum KNUC25 against Helicobacter pylori strains. Judged by a disc agar diffusion method, the anti-H. pylori activity existed in the cell-free supernatants (CFSs) of KNUC25. The mean diameters of growth inhibition by 10, 30, and 60 microL of a 15-fold concentrated CFS per disc were 11.2, 17.7, and 23.7 mm, respectively. The neutralized CFS lost its anti-H. pylori activity, suggesting that acidic pH in CFS may be responsible for the anti-H. pylori activity. Adherence was determined by urease activity of H. pylori adhered to gastric epithelial cell line AGS cells after co-incubation of AGS cells with CFS and H. pylori strain ATCC43504 (s1m1vacA/cagA(+)), ATCC51932 (s2m2vacA/cagA(-)), or SS1 (s2m2vacA/cagA(+)) in vitro followed by three washes by means of centrifugation with saline. Adherence of ATCC43504 or SS1 to AGS cells was reduced by about 70% after a 30-minute incubation with 30 microL of a 15-fold concentrated KNUC25 CFS, whereas that of ATCC51932 to AGS cells was not. The results show KNUC25 CFS is effective in inhibiting the growth of H. pylori, which is related to pH and the adherence of cagA-positive H. pylori to gastric cells.

Multiple perianal infundibular follicular cysts in a dog.

This case report describes a 7-year-old male cocker spaniel dog with multiple perianal infundibular follicular cysts. Clinically the dog had moderate anal sacculitis, peri-anal pruritus causing it to 'scoot' and lick the area. On examination of the perianal area, there were over 100 firm, well circumscribed papules, ranged from 0.2 to 0.5 cm in diameter with a central pore, and were found in the perianal region. Alopecia was present in the perianal region. The skin tissue in the perianal region resected surgically was submitted for histological examination. Microscopically, the tissue revealed multiple dilated cysts filled with keratins and the papules corresponded to infundibular follicular cysts. The affected dog showed moderate anal sacculitis. Anal sacculitis commonly causes repeated scooting or licking the area around the anus. Therefore, the multiple follicular cysts in the present case appear to be primarily a sequela to chronic external trauma to the perianal area, probably in response to anal sacculitis. To the best of the authors' knowledge, the present report is the first documented case of multiple perianal infundibular follicular cysts in a dog.

Vitamin C deficiency attenuates liver fibrosis by way of up-regulated peroxisome proliferator-activated receptor-gamma expression in senescence marker protein 30 knockout mice.

UNLABELLED: Senescence marker protein 30 (SMP30), an important aging marker molecule that is highly expressed in the liver, has been known to protect hepatocytes from apoptosis by the synthesis of vitamin C. To explore the function of SMP30 in liver fibrosis, the effect of SMP30 deficiency on liver fibrosis was investigated in SMP30 knockout (KO) mice. Moreover, the in vivo results were further confirmed by way of hepatic stellate cell (HSC) isolation. We demonstrated that carbon tetrachloride (CCl(4))-induced liver fibrosis and the nuclear translocation of p-Smad2/3, the immediate downstream of transforming growth factor beta (TGF-beta), were significantly inhibited in the liver of SMP30 KO mice compared with wildtype (WT) mice. We also confirmed that both WT and SMP30 KO HSCs did not express SMP30. Finally, we further confirmed that up-regulation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) caused by a lack of vitamin C was the pivotal factor in the mechanisms for attenuated liver fibrosis of SMP30 KO mice, and feeding with vitamin C restored CCl(4)-induced liver fibrosis in SMP30 KO mice. CONCLUSION: Vitamin C deficiency by SMP30 depletion attenuated liver fibrosis by way of up-regulated PPAR-gamma expression in SMP30 KO mice. Our results provide, for the first time, the possible mechanisms underlying inhibition of HSC activation associated with vitamin C and PPAR-gamma up-regulation in liver fibrosis of SMP30 KO mice.

Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage.

Recently, reactive oxygen species (ROS) have been studied as a regulator of differentiation into specific cell types in embryonic stem cells (ESCs). However, ROS role in human ESCs (hESCs) is unknown because mouse ESCs have been used mainly for most studies. Herein we suggest that ROS generation may play a critical role in differentiation of hESCs; ROS enhances differentiation of hESCs into bi-potent mesendodermal cell lineage via ROS-involved signaling pathways. In ROS-inducing conditions, expression of pluripotency markers (Oct4, Tra 1-60, Nanog, and Sox2) of hESCs was decreased, while expression of mesodermal and endodermal markers was increased. Moreover, these differentiation events of hESCs in ROS-inducing conditions were decreased by free radical scavenger treatment. hESC-derived embryoid bodies (EBs) also showed similar differentiation patterns by ROS induction. In ROS-related signaling pathway, some of the MAPKs family members in hESCs were also affected by ROS induction. p38 MAPK and AKT (protein kinases B, PKB) were inactivated significantly by buthionine sulfoximine (BSO) treatment. JNK and ERK phosphorylation levels were increased at early time of BSO treatment but not at late time point. Moreover, MAPKs family-specific inhibitors could prevent the mesendodermal differentiation of hESCs by ROS induction. Our results demonstrate that stemness and differentiation of hESCs can be regulated by environmental factors such as ROS.

Subcutaneous leiomyosarcoma in an adrenomedullin heterozygous mouse.

The present study reports a case of a 5-month-old female adrenomedullin (AM) heterozygous (+/-) mouse that presented a mass of leiomyosarcoma found in the right shoulder girdle region. The neoplastic mass extended to the sternal region and showed hemorrhages, congestion and necrotic foci. The excised tumor with a diameter of 2.5cm was firm, ill-demarcated and had focally infiltrated the surrounding muscles. The cut surface was homogeneously whitish with multi-focal reddish lesions. Microscopically, the tumor composed of variable fascicles of spindle-shaped cells with pleomorphic and cigar-shaped nuclei. The nuclei were round and elongated. Metastasis of tumor cell to skeletal muscle was frequently observed. Immunohistochemically, desmin, vimentin and alpha-smooth muscle actin (alpha-SMA) were demonstrated in neoplastic cells but tumor cells were negative for cytokeratin (CK) and S-100. Based on gross finding, microscopical examination and immunohistochemistry, the present case was diagnosed as a subcutaneous leiomyosarcoma.

Salivary mucocele with osseous metaplasia in a dog.

A 4-year-old, male, dachshund was referred to a certain local veterinary hospital because of a soft and fluctuant swelling in the left upper cervical region. The swelling was surgically removed and appeared to be filled with bloody mucus. Grossly, the swelling was identified as salivary mucocele and showed small multifocal whitish ossified tissue on its surface. Microscopically, the wall of salivary mucocele appeared as granulation tissue surrounding mucin, which was composed of loose edematous and vascularized connective tissue containing chronic inflammatory cells such as lymphocytes, plasma cells and macrophages. Characteristically, present case had ossifying components formed by metaplastic spindle cells in the wall of salivary mucocele. Therefore, the present case was diagnosed as salivary mucocele with osseous metaplasia in a dog.

Potent neutralization of vacuolating cytotoxin (VacA) of Helicobacter pylori by immunoglobulins against the soluble recombinant VacA.

BACKGROUND: The recombinant vacuolating cytotoxin (rVacA) of Helicobacter pylori that retains native conformational epitopes was evaluated as a vaccine antigen for anti-H. pylori treatment. METHODS: s1m1 vacA gene fraction encoding the mature VacA protein was expressed as a soluble protein in E. coli at low temperature. The efficacy of anti-rVacA antibody against VacA or H. pylori was assessed in vitro using AGS cells and in vivo using a murine model. RESULTS: The rabbit antisera against rVacA completely neutralized the vacuolating activity and partially inhibited the cell death induced by VacA in AGS cells. Oral immunization of C57BL/6 mice with rVacA plus CpG-oligodeoxynucleotide (ODN) as an ajuvant stimulated specific anti-VacA antibody and mucosal immune responses which correlated with decreased systemic immune responses and gastric urease activities (p>0.05). CONCLUSION: The rVacA antigen possessing conformational epitopes may have potential as a vaccine component and may be useful in serological and histopathological analysis.

Two different types of malignant fibrous histiocytomas from pet dogs.

We describe 2 cases of malignant fibrous histiocytomas (MFHs) that spontaneously developed in young pet dogs. To classify these tumors, we applied a panel of antibodies (vimentin, desmin, alpha-SMA, and ED1) and Azan staining for collagen. The MFHs were most consistent with osteoclastlike giant and inflammatory cell types. The first case had positive staining for ED1 and vimentin, and given the osteoclast-like giant cells, calcification sites accompanying peripheral giant cell infiltrates. The latter case, the inflammatory cell type, exhibited a storiform-pleomorphic variant of neoplastic cells, including an ossifying matrix. MFHs are among the most highly aggressive tumors occurring in soft tissue sarcomas in elderly dogs; however, MFHs have been poorly studied from a diagnostic point of view. Herein, we describe the histologic and immunohistologic features of MFHs in detail, thus classifying the subtypes of these tumors.

Discoid lupus erythematosus (DLE) in a Spitz dog.

A 3-year-old, female Spitz, was presented due to lack of response to therapies with a 6-month history of skin lesions characterized by diffuse erythema and scaling on the dorsal trunk. Physical examination revealed the dog was active and healthy. Skin culture isolated no fungus. Histological examination of skin biopsy specimens revealed interface dermatitis with hydropic degeneration of the basal layers, predominant plasmacytic perivascular accumulation in the dermis, and intensive plasma cell-rich interface mural folliculitis. Moderate CD3-positive lymphocytes infiltrated the superficial dermis. This report may provide unique information of canine discoid lupus erythematosus in an unusual breed with atypical cutaneous lesions.

Macrophages, myofibroblasts and mast cells in a rat liver infected with Capillaria hepatica.

We trapped a rat (Rattus norvegicus) infected with Capillaria hepatica. At necropsy, grossly yellowish-white nodules (2-3 mm in diameter) were noted to be scattered on the liver's surface. Microscopically, granulomatous and fibrotic nodules that contained the eggs and/or adult worms of Capillaria hepatica were detected in the liver. Septal fibrosis was diffusely formed throughout the liver. There were a number of ED1-positive macrophages located in the sinusoids of the pseudolobules. On the double staining, myofibroblasts and mast cells were generally observed within the fibrous septa with the mast cells in close proximity to the myofibroblasts. We suggest that the interactions between macrophages, myofibroblasts and mast cells play a role in the septal fibrosis observed in rats infected by Capillaria hepatica.