Glycan-Controlled Human PD-1 Variants Displaying Broad-Spectrum High Binding to PD-1 Ligands Potentiate T Cell.

Although therapeutic immunoglobulin G (IgG) antibodies that regulate the activity of immune checkpoints bring innovation to the field of immuno-oncology, they are still limited in their efficiency to infiltrate the tumor microenvironment due to their large molecular size (150 kDa) and the necessity of additional engineering works to ablate effector functions for antibodies targeting immune cells. To address these issues, the human PD-1 (hPD-1) ectodomain, a small protein moiety of 14-17 kDa, has been considered as a therapeutic agent. Here, we used bacterial display-based high-throughput directed evolution to successfully isolate glycan-controlled (aglycosylated or only single-N-linked glycosylated) human PD-1 variants exhibiting over 1000-fold increased hPD-L1 binding affinity compared to that of wild-type hPD-1. The resulting hPD-1 variants, aglycosylated JYQ12 and JYQ12-2 with a single-N-linked glycan chain, showed exceptionally high binding affinity to hPD-L1 and very high affinity to both hPD-L2 and mPD-L1. Moreover, the JYQ12-2 efficiently potentiated the proliferation of human T cells. hPD-1 variants with significantly improved binding affinities for hPD-1 ligands could be used as effective therapeutics or diagnostics that can be differentiated from large-sized IgG antibody-based molecules.

Therapeutic Effect of Losartan, an Angiotensin II Type 1 Receptor Antagonist, on CCl4-Induced Skeletal Muscle Injury.

TGF-ß1 is known to inhibit muscle regeneration after muscle injury. However, it is unknown if high systemic levels of TGF-ß can affect the muscle regeneration process. In the present study, we demonstrated the effect of a CCl4 intra-peritoneal injection and losartan (an angiotensin II type 1 receptor antagonist) on skeletal muscle (gastrocnemius muscle) injury and regeneration. Male C57BL/6 mice were grouped randomly as follows: control (n = 7), CCl4-treatment group (n = 7), and CCl4 + losartan treatment group (n = 7). After CCl4 treatment for a 16-week period, the animals were sacrificed and analyzed. The expression of dystrophin significantly decreased in the muscle tissues of the control group, as compared with that of the CCl4 + losartan group (p < 0.01). p(phospho)-Smad2/3 expression significantly increased in the muscles of the control group compared to that in the CCl4 + losartan group (p < 0.01). The expressions of Pax7, MyoD, and myogenin increased in skeletal muscles of the CCl4 + losartan group compared to the corresponding levels in the control group (p < 0.01). We hypothesize that systemically elevated TGF-ß1 as a result of CCl4-induced liver injury causes skeletal muscle injury, while losartan promotes muscle repair from injury via blockade of TGF-ß1 signaling.

Pan-cancer analysis reveals multifaceted roles of retrotransposon-fusion RNAs.

Transposon-derived transcripts are abundant in RNA sequences, yet their landscape and function, especially for fusion transcripts derived from unannotated or somatically acquired transposons, remains underexplored. Here, we developed a new bioinformatic tool to detect transposon-fusion transcripts in RNA-sequencing data and performed a pan-cancer analysis of 10,257 cancer samples across 34 cancer types as well as 3,088 normal tissue samples. We identified 52,277 cancer-specific fusions with ~30 events per cancer and hotspot loci within transposons vulnerable to fusion formation. Exonization of intronic transposons was the most prevalent genic fusions, while somatic L1 insertions constituted a small fraction of cancer-specific fusions. Source L1s and HERVs, but not Alus showed decreased DNA methylation in cancer upon fusion formation. Overall cancer-specific L1 fusions were enriched in tumor suppressors while Alu fusions were enriched in oncogenes, including recurrent Alu fusions in EZH2 predictive of patient survival. We also demonstrated that transposon-derived peptides triggered CD8+ T-cell activation to the extent comparable to EBV viruses. Our findings reveal distinct epigenetic and tumorigenic mechanisms underlying transposon fusions across different families and highlight transposons as novel therapeutic targets and the source of potent neoantigens.

Hypoxia Pretreatment Promotes Chondrocyte Differentiation of Human Adipose-Derived Stem Cells via Vascular Endothelial Growth Factor.

BACKGROUND: Human adipose tissue-derived stem cells (ADSCs) are attractive multipotent stem cell sources with therapeutic potential in various fields requiring repair and regeneration, such as acute and chronically damaged tissues. ADSC is suitable for cell-based therapy, but its use has been hampered due to poor survival after administration. Potential therapeutic use of ADSC requires mass production of cells through in vitro expansion. Many studies have consistently observed the tendency of senescence by mesenchymal stem cell (MSC) proliferation upon expansion. Hypoxia has been reported to improve stem cell proliferation and survival. METHODS: We investigated the effects of hypoxia pretreatment on ADCS proliferation, migration capacity, differentiation potential and cytokine production. We also analyzed the effects of vascular endothelial growth factor (VEGF) on osteogenic and chondrogenic differentiation of ADSCs by hypoxia pretreatment. RESULTS: Hypoxia pretreatment increased the proliferation of ADSCs by increasing VEGF levels. Interestingly, hypoxia pretreatment significantly increased chondrogenic differentiation but decreased osteogenic differentiation compared to normoxia. The osteogenic differentiation of ADSC was decreased by the addition of VEGF but increased by the depletion of VEGF. We have shown that hypoxia pretreatment increases the chondrogenic differentiation of ADSCs while reducing osteogenic differentiation in a VEGF-dependent manner. CONCLUSION: These results show that hypoxia pretreatment can provide useful information for studies that require selective inhibition of osteogenic differentiation, such as cartilage regeneration.

Helicobacter pylori promotes hepatic fibrosis in the animal model.

Helicobacter pylori infection has been reported to be very common in patients with chronic liver diseases, including cirrhosis. To elucidate the pathological effect of H. pylori infection on the progression of hepatic fibrosis, C57BL/6 mice and Sprague-Dawley rats were orally inoculated with H. pylori, and hepatic fibrosis was induced with carbon tetrachloride (CCl(4)) administration. We observed the histopathological changes and the presence of H. pylori genes by PCR in the liver. Significant increase in the fibrotic score as well as in serum alanine aminotransferase and aspartate aminotransferase levels was shown in the CCl(4)+H. pylori group compared with that in the CCl(4)-treated group. Compared with the CCl(4)-treated group, alpha-smooth muscle actin and transforming growth factor-beta1 were enhanced; however, senescence marker protein-30, a multifunctional protein protecting hepatocytes against oxidative stress and apoptosis, was suppressed in the CCl(4)+H. pylori group. The 16S rRNA (400 bp) was demonstrated by PCR for H. pylori genes from genomic DNA extracted from the liver, and H. pylori-infected mice showed 93.8% (15 of 16) seropositivity by contrast with seronegativity in all H. pylori-noninfected mice. In addition, immunohistochemical study against H. pylori showed positive antigen fragments in the liver of the infected groups. Consequently, our data suggest that H. pylori infection could be an important contributing infectious factor to the development of liver cirrhosis.

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.

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.

TiO2 particles induce ER stress and apoptosis in human hepatoma cells, HepG2, in a particle size-dependent manner.

The cytotoxicity of TiO2 nanoparticles are well-known, but the particle size-dependent induction of ER stress and apoptosis by TiO2 in hepatocytes has not been elucidated clearly. In the present study, we investigated whether a fine TiO2 particle and two types of TiO2 nanoparticles induce ER stress and apoptosis differently in HepG2 cells. A particle size-dependent decrease in cell viability was observed after exposure to the TiO2 particles. The levels of ER stress-related proteins (BiP, CHOP, ATF6α, and p-PERK) were increased with decreasing particle size. TiO2 particles induced ER stress-mediated apoptosis in a particle size-dependent manner as seen by a decrease in the expression of Bcl-2, and increases in the expression of Bax, caspase-12, and cleaved caspase-3. These results indicated that the cytotoxicity produced by TiO2 particles was related to particle size, with smaller TiO2 nanoparticles producing greater toxic effects involving ER stress and apoptosis in the HepG2 cells.

Hydrogel-incorporating unit in a well: 3D cell culture for high-throughput analysis.

The microfluidic 3D cell culture system has been an attractive model because it mimics the tissue and disease model, thereby expanding our ability to control the local cellular microenvironment. However, these systems still have limited value as quantitative assay tools due to the difficulties associated with the manipulation and maintenance of microfluidic cells, and their lack of compatibility with the high-throughput screening (HTS) analysis system. In this study, we suggest a microchannel-free, 3D cell culture system that has a hydrogel-incorporating unit integrated with a multi-well plate (24- to 96-well plate), which can provide better reproducibility in biological experiments. This plate was devised considering the design constraints imposed by various cell biology applications as well as by high-throughput analysis where the physical dimensions of the micro-features in the hydrogel-incorporating units were altered. We also demonstrated that the developed plate is potentially applicable to a variety of quantitative biochemical assays for qRT-PCR, Western blotting, and microplate-reader-based assays, such as ELISA, viability assay, and high content-screening (HCS) as well as the co-culture for biological studies. Human neural progenitor cells (hNPCs) that produce pathogenic Aß species for modeling Alzheimer's disease (AD) were three-dimensionally cultured, and the efficacy of the inhibitors of Aß production was assessed by ELISA in order to demonstrate the performance of this plate.

Inhibition of skin carcinogenesis by suppression of NF-κB dependent ITGAV and TIMP-1 expression in IL-32γ overexpressed condition.

BACKGROUND: Interleukin-32 (IL-32) has been associated with various diseases. Previous studies have shown that IL-32 inhibited the development of several tumors. However, the role of IL-32γ, an isotype of IL-32, in skin carcinogenesis remains unknown. METHODS: We compared 7,12-Dimethylbenz[a]anthracene/12-O-Tetradecanoylphorbol-13-acetate (DMBA/TPA)-induced skin carcinogenesis in wild type (WT) and IL-32γ-overexpressing mice to evaluate the role of IL-32γ. We also analyzed cancer stemness and NF-κB signaling in skin cancer cell lines with or without IL-32γ expression by western blotting, quantitative real-time PCR and immunohistochemistry analysis. RESULTS: Carcinogen-induced tumor incidence in IL-32γ mice was significantly reduced in comparison to that in WT mice. Infiltration of inflammatory cells and the expression levels of pro-inflammatory mediators were decreased in the skin tumor tissues of IL-32γ mice compared with WT mice. Using a genome-wide association study analysis, we found that IL-32 was associated with integrin αV (ITGAV) and tissue inhibitor of metalloproteinase-1 (TIMP-1), which are critical factor for skin carcinogenesis. Reduced expression of ITGAV and TIMP-1 were identified in DMBA/TPA-induced skin tissues of IL-32γ mice compared to that in WT mice. NF-κB activity was also reduced in DMBA/TPA-induced skin tissues of IL-32γ mice. IL-32γ decreased cancer cell sphere formation and expression of stem cell markers, and increased chemotherapy-induced cancer cell death. IL-32γ also downregulated expression of ITGAV and TIMP-1, accompanied with the inhibition of NF-κB activity. In addition, IL-32γ expression with NF-κB inhibitor treatment further reduced skin inflammation, epidermal hyperplasia, and cancer cell sphere formation and downregulated expression levels of ITGAV and TIMP-1. CONCLUSIONS: These findings indicated that IL-32γ suppressed skin carcinogenesis through the inhibition of both stemness and the inflammatory tumor microenvironment by the downregulation of TIMP-1 and ITGAV via inactivation of NF-κB signaling.

IL-32 gamma reduces lung tumor development through upregulation of TIMP-3 overexpression and hypomethylation.

The low expression of tissue inhibitor of metalloproteinase 3 (TIMP-3) is important in inflammatory responses. Therefore, inhibition of TIMP-3 may promote tumor development. Our study showed that expression of TIMP-3 was elevated in lL-32γ mice lung tissues. In this study, we investigated whether IL-32γ mice inhibited lung tumor development through overexpression of TIMP-3 and its methylation. To explore the possible underlying mechanism, lung cancer cells were transfected with IL-32γ cDNA plasmid. A marked increase in TIMP-3 expression was caused by promoter methylation. Mechanistic studies indicated that TIMP-3 overexpression reduced NF-κB activity, which led to cell growth inhibition in IL-32γ transfected lung cancer cells. We also showed that IL-32γ inhibits expression of DNA (cytosine-5-)-methyltransferase 1 (DNMT1). Moreover, IL-32γ inhibits the binding of DNMT1 to TIMP-3 promoter, but this effect was reversed by the treatment of DNA methyltransferase inhibitor (5-Aza-CdR) and NF-κB inhibitor (PS1145), suggesting that a marked increase in TIMP-3 expression was caused by inhibition of promoter hypermethylation via decreased DNMT1 expression through the NF-κB pathway. In an in vivo carcinogen induced lung tumor model, tumor growth was inhibited in IL-32γ overexpressed mice with elevated TIMP-3 expression and hypomethylation accompanied with reduced NF-κB activity. Moreover, in the lung cancer patient tissue, the expression of IL-32 and TIMP-3 was dramatically decreased at a grade-dependent manner compared to normal lung tissue. In summary, IL-32γ may increase TIMP-3 expression via hypomethylation through inactivation of NF-κB activity, and thereby reduce lung tumor growth.

Differentiation potential and profile of nuclear receptor expression during expanded culture of human adipose tissue-derived stem cells reveals PPARγ as an important regulator of Oct4 expression.

Potential therapeutic use of human adipose tissue-derived stem cells (hADSCs) requires the production of large cell numbers by in vitro expansion. However, long-term in vitro culture is associated with reduced stem cell characteristics and differentiation capability. We investigated the proliferation rate and expression of p16(INK4a) mRNA, surface stem cell markers, and stem cell transcription factors. The proliferation rate decreased significantly as passages increased, and the expression of p16(INK4a) mRNA significantly increased. FACS analysis of CD73, CD90, and CD105 expression showed no significant difference among examined passages; however, the mRNA expression levels of pluripotent markers, Oct4 and Nanog, were significantly decreased at higher passages. At passages 12 and 20, there was decreased differentiation capability into insulin-producing cells, evidenced by significantly decreased expression of insulin and related ß cell markers. Adipogenic and osteogenic differentiation was also decreased at higher passages. We then analyzed the transcriptional expression profiles of 48 nuclear receptors at four different passages. We found that the expression of peroxisome proliferator-activated receptor γ (PPARγ) and thyroid hormone receptor TRß was significantly decreased at higher passages. Treatment with PPARγ activators or overexpression of PPARγ in hADSCs at passage 20 could recover Oct4 expression levels and increase Oct4 promoter activity. PPARγ inactivation by GW9662 inhibited the troglitazone-induced Oct4 mRNA expression. Furthermore, PPARγ overexpression in hADSC at passage 20 improved the differentiation potential to insulin-producing cells. In conclusion, we demonstrated that hADSCs undergo characteristic changes and reduction of differentiation capability during expanded culture in vitro, and revealed the role of PPARγ as one potential factor in the regulation of Oct4 expression during in vitro aging of hADSCs.

Ascorbic acid deficiency accelerates aging of hepatic stellate cells with up-regulation of PPARγ.

Senescent cells have been observed in certain aged or damaged tissues. However, the information about the effects of aging on liver cells is limited. In the present study, we have examined age-related histological changes in the livers of senescence marker protein knockout (SMP30-/-) mice, which are considered as a murine aging model due to the more sensitive response to apoptotic reagents and due to their shorter life span. In livers of old SMP30-/- mice, numerous hepatic stellate cells (HSCs) were hypertrophic and contained abundant microvesicular lipid droplets in cytoplasm. We have found that the expression of peroxisome proliferators-activated receptor γ (PPARγ), which is a protein related to lipid metabolism and HSC quiescence, was increased in hypertrophic HSCs by aging and vitamin C (VC) deficiency, whereas these phenomena were dramatically reduced by antioxidant treatment. Therefore, these prominent phenotypic changes can be considered as aging markers in the livers of animals which are subjected to antioxidant property evaluation.

The protective effect of ENA Actimineral resource A on CCl4-induced liver injury in rats.

ENA Actimineral Resource A (ENA-A) is alkaline water that is composed of refined edible cuttlefish bone and two different species of seaweed, Phymatolithon calcareum and Lithothamnion corallioides. In the present study, ENA-A was investigated as an antioxidant to protect against CCl(4)-induced oxidative stress and hepatotoxicity in rats. Liver injury was induced by either subacute or chronic CCl(4) administration, and the rats had free access to tap water mixed with 0% (control group) or 10% (v/v) ENA-A for 5 or 8 weeks. The results of histological examination and measurement of antioxidant activity showed that the reactive oxygen species production, lipid peroxidation, induction of CYP2E1 were decreased and the antioxidant activity, including glutathione and catalase production, was increased in the ENA-A groups as compared with the control group. On 2-DE gel analysis of the proteomes, 13 differentially expressed proteins were obtained in the ENA-A groups as compared with the control group. Antioxidant proteins, including glutathione S-transferase, kelch-like ECH-associated protein 1, and peroxiredoxin 1, were increased with hepatocyte nuclear factor 3-beta and serum albumin precursor, and kininogen precursor decreased more in the ENA-A groups than compared to the control group. In conclusion, our results suggest that ENA-A does indeed have some protective capabilities against CCl(4)-induced liver injury through its antioxidant function.

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.

Primary biliary cirrhosis, similar to that in human beings, in a male C57BL/6 mouse infected with Helicobacter pylori.

We report a case of primary biliary cirrhosis (PBC) that occurred in a 24-month-old male C57BL/6 mouse infected with Helicobacter pylori (H. pylori). Microscopically, the portal tract in the liver showed nonsuppurative destructive cholangitis with variable cytologic distortion of the epithelial cells and peribiliary lymphoplasmacytic infiltration. Immunohistochemistry using alpha-smooth muscle actin demonstrated fibrous bands associating with the wall of vasculature. The level of serum antivacuolating toxin IgG in this mouse showed the highest value (optical density=2.1470) of the H. pylori-infected group (n=13) (optical density=1.7168+/-0.1759, mean+/-SD). Spontaneously developed PBC-like lesions in C57BL/6 mice have been reported by several authors. However, this case strikingly resembles human PBC with its characterized histological features. Therefore, we propose that the increase in vacuolating toxin caused by H. pylori infection may be related to the development of PBC by molecular mimicry.