Human bone marrow mesenchymal stem cells are resistant to HBV infection during differentiation into hepatocytes in vivo and in vitro

Hepatocyte-like cells induced from bone marrow mesenchymal stem cells (BMSCs) recover liver function in animal models with liver failure. Our initial findings revealed that human BMSCs improved liver function in hepatitis B patients with end stage liver disease. However, the susceptibility of BMSCs to HBV infection during induction toward hepatocytes remains unknown. We have assessed whether BMSCs-derived hepatocyte-like cells can function like liver cells and be infected by HBV. A new and efficient way to direct the differentiation of BMSCs into functional hepatocytes was developed. BMSCs obtained from hepatitis B patients were induced to differentiate into hepatocytes through exposure to HGF, FGF-4, and EGF. After 6 days of exposure, BMSCs-derived hepatocyte-like cells that expressed a subset of hepatic genes and showed hepatic functions were obtained. HBV was used to infect the differentiated cells, and subsequently these cells were assayed for the presence of HBeAg, HBsAg, and HBV DNA. BMSCs proved resistant to HBV infection, both in vitro and during differentiation into hepatocytes in vitro. This demonstrates that BMSCs are resistant to HBV infection. BMSCs are viable for transplantation and should facilitate further research exploring the in vivo HBV-resistance of the hepatocytes derived from BMSCs after transplantation, a characteristic that could form the basis for hepatocyte transplantation.     

Expression profiles of nestin in vascular smooth muscle cells in vivo and in vitro

Nestin is an intermediate filament protein expressed in neural and mesenchymal stem cells. Here, we investigated the expression of nestin in vascular smooth muscle cells (VSMCs) in vivo and in vitro. In the developing arteries, medial VSMCs were found to express nestin; its expression was prominent in embryos but was down-regulated after birth (3-6 weeks) in a region-dependent manner; its expression was abolished in the adult. Thus, the expression of nestin is specific to developing VSMCs. In primary VMSC cultures, nestin expression was induced by serum, but was independent of cell-cycle progression. Signaling analyses revealed that the serum-induced nestin expression depended on the extracellular signal-regulated kinase (ERK) and protein kinase B (PKB)(Akt) pathways, via the platelet derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. Nestin expression was closely related to the up-regulation and activation of Sp1 and Sp3. Among major serum growth factors and cytokines, PDGF-BB was the most potent inducer of nestin expression. Nestin was also up-regulated in arteries undergoing vascular remodeling following balloon injury. Its expression was particularly strong in the cells lining the lumen of the neointima, suggesting a possible correlation between nestin expression and the progression of vascular remodeling.     

Effects of freezing on biological membranes in vivo and in vitro

Membrane inactivation by freezing has been investigated using intact spinach leaves and isolated thylakoid membranes from chloroplasts of leaf cells as test material. During freezing in vitro in solutions containing neutral solute and a slight excess of inorganic salts such as NaCl, electron transport is stimulated while photophosphorylation is lost. Under more drastic freezing conditions damage increases, affecting dichlorophenolindophenol reduction, the rise in variable fluorescence, ferricyanide reduction and electron transport through Photosystem I, in that order. Semipolar compounds such as phenylalanine or phenylpyruvate exhibit a much higher membrane toxicity during freezing than inorganic salts. The profile of damage caused by this class of compounds is different from that caused by salts. Damage to membranes isolated rapidly from frost-killed leaves is similar to that produced by semipolar compounds during freezing in vitro. A few sites of damage could be identified, among them the site responsible for oxidation of water during photosynthesis. The results support the view that the sensitivity of their membranes limits the ability of cells to withstand freezing and suggest that freezing sensitivity is due to the accumulation in the cells of potentially membrane-toxic organic and norganic cell constituents.     

Piperazine derivatives inhibit PrP/PrP propagation in vitro and in vivo

Prion diseases are fatal neurodegenerative disorders, which are not curable and no effective treatment exists so far. The major neuropathological change in diseased brains is the conversion of the normal cellular form of the prion protein PrPc^C into a disease-associated isoform PrP^Sc. PrP^Sc accumulates into multimeres and fibrillar aggregates, which leads to the formation of amyloid plaques. Increasing evidence indicates a fundamental role of PrP^Sc species and its aggregation in the pathogenesis of prion diseases, which initiates the pathological cascade and leads to neurodegeneration accompanied by spongiform changes. In search of compounds that have the potential to interfere with PrP^Sc formation and propagation, we used a cell based assay for the screening of potential aggregation inhibitors. The assay deals with a permanently prion infected cell line that was adapted for a high-throughput screening of a compound library composed of 10,000 compounds (DIVERset 2, ChemBridge).     

Antihypertensive effect of insect cells: In vitro and in vivo evaluation

In this study, we investigated the in vitro ACE inhibitory and in vivo antihypertensive effect of insect cell extracts. The IC₅₀ of three insect cell lines from different type and insect species origin: S2 (embryo, Drosophila melanogaster), Sf21 (ovary, Spodoptera frugiperda) and Bm5 (ovary, Bombyx mori), were evaluated. Most interesting results were that the IC₅₀ values ranged between 0.4 and 0.9 mg/ml, and that an extra hydrolysis with gastrointestinal enzymes did not increase the ACE inhibitory activity conspicuously. Finally, a single oral administration with a gavage of 150 mg cell extract/kg BW to spontaneous hypertensive rats (SHR) significantly decreased (p < 0.05) their systolic blood pressure (SBP) with 5-6% (9-12 mm Hg) compared to the controls at 6 h post-administration. Here the undigested and digested insect S2 cell extracts were equal in activity to lower the SBP. To the best of our knowledge, this is the first report of in vivo antihypertensive activity of insect cell extracts and this without an extra digestion requirement.     

The in vitro and in vivo apoptotic effects of Mahonia oiwakensis on human lung cancer cells

Both the root and stem bark of Mahonia species were popular folk medicines. The plant has several proven biological activities including anti-bacterial, anti-fungal, and anti-inflammatory effects. However, Mahonia has not been studied for its anticancer effects. In the present study, we made extracts from Mahonia oiwakensis (MOE), a selected species in Taiwan, and investigated their effects on various human lung cells. We found that MOE-induced apoptotic death in human A549 non-small-cell lung carcinoma (NSCLC) cells in a dose- and time-dependent manner. Treatment with the extracts also caused an increase in the sub-G1 fraction of cells, chromosome condensation, and DNA fragmentation. The mitochondrial-mediated pathway was implicated in this MOE-induced apoptosis as evidenced by the activation of the caspase cascade, cleavage of poly (ADP-ribose) polymerase (PARP), disruption of mitochondrial membrane potential, and release of cytochrome C. A higher ratio of Bax/Bcl-2 proteins and cleavage of Bid were also observed in MOE-induced cell apoptosis. In A549 tumor-xenografted nude mice, MOE also retarded in vivo proliferation (P < 0.05) and induced apoptosis in tumor cells, as shown by a decrease in Ki-67-positive staining (P < 0.05) and increased transferase-mediated dUTP nick-end labeling (TUNEL)-positive staining (P < 0.05). In conclusion, MOE inhibits the growth of human lung cancer cells in vitro and in vivo, suggesting that it may have therapeutic potential against human lung cancer.     

Low immunogenicity of allogeneic human umbilical cord blood-derived mesenchymal stem cells in vitro and in vivo

Evaluation of the immunogenicity of human mesenchymal stem cells (MSCs) in an allogeneic setting during therapy has been hampered by lack of suitable models due to technical and ethical limitations. Here, we show that allogeneic human umbilical cord blood derived-MSCs (hUCB-MSCs) maintained low immunogenicity even after immune challenge in vitro. To confirm these properties in vivo, a humanized mouse model was established by injecting isolated hUCB-derived CD34+ cells intravenously into immunocompromised NOD/SCID IL2γnull (NSG) mice. After repeated intravenous injection of human peripheral blood mononuclear cells (hPBMCs) or MRC5 cells into these mice, immunological alterations including T cell proliferation and increased IFN-γ, TNF-α, and human IgG levels, were observed. In contrast, hUCB-MSC injection did not elicit these responses. While lymphocyte infiltration in the lung and small intestine and reduced survival rates were observed after hPBMC or MRC5 transplantation, no adverse events were observed following hUCB-MSC introduction. In conclusion, our data suggest that allogeneic hUCB-MSCs have low immunogenicity in vitro and in vivo, and are therefore “immunologically safe” for use in allogeneic clinical applications.     

Brugia pahangi: Feeding and nutrient uptake in vitro and in vivo

Incubation in vitro of adult Brugia pahangi in an apparatus which permitted the separate exposure of the anterior, middle, or posterior region of the worms to medium-containing radioactively labeled d-glucose, l-leucine, and adenosine has provided evidence that these materials are taken up in physiologically significant amounts by a transcuticular route. No evidence for an oral ingestion of materials has been obtained from worms in vitro, but in vivo an oral uptake of Trypan blue has been demonstrated. The ultrastructure and cytochemical staining reactions for nonspecific esterase, acid phosphatase (EC-3.1.3.2), and leucine naphthylamidase of the gut and body wall are described.     

Activities in vitro and in vivo of enzymes of benzodiazepine alkaloid biosynthesis during development of Penicillium cyclopium

In the hyphae of Penicillium cyclopium the in vitro measurable activities of 3 enzymes of alkaloid biosynthesis are induced endogenously during     

Inhibition of c-Jun N-terminal kinase increases apoE expression in vitro and in vivo

Apolipoprotein E (apoE), a ligand for the low-density lipoprotein receptor family, has been implicated in modulating glial inflammatory responses and the risk of neurodegeneration associated with Alzheimer’s disease. Glial cells activated by lipopolysaccharide (LPS) have decreased apoE levels and we recently showed that apoE itself can modulate the inflammatory response by reducing c-Jun N-terminal kinase (JNK) activation. Reduced JNK phosphorylation is vital to overcome the LPS-induced decrease in apoE expression, suggesting that JNK inhibition may be an effective way to increase apoE protein and protract its anti-inflammatory properties. This study investigates the impact of JNK inhibition on apoE production using two JNK inhibitors. Our work in primary glia and in vivo in mice injected with JNK inhibitor demonstrates that inhibition of JNK may be an effective way to increase apoE expression.     

In vitro and in vivo digestion of octenyl succinic starch

This study aimed to understand effects of octenyl succinic anhydride (OSA) modification of normal corn (NCS) and high-amylose corn (HA7) starch on their enzymatic hydrolysis rates. After modification with 3% and 10% OSA, resistant starch (RS) contents of the cooked OS-NCS increased from 0.8% of the control starch to 6.8% and 13.2% (Englyst Method), respectively, whereas that of the cooked OS-HA7 decreased from 24.1% to 23.7% and 20.9%, respectively. When the cooked NCS, HA7 and OS (10%)-HA7 were used to prepare diets for rats at 55% (w/w) starch, RS contents of the diets were 1.1%, 13.2% and 14.6%, respectively. After feeding to the rats, 20.2–31.1% of the starch in the OS (10%)-HA7-diet was not utilized in vivo and was found in rat feces, which was substantially larger than that of the HA7-diet (≤4.9%) and NCS-diet (≤0.2%). The body weights of the rats, however, remained similar between different groups.     

Formaldehyde induces hyperphosphorylation and polymerization of Tau protein both in vitro and in vivo

Background

Chronic formaldehyde exposure leads to memory impairment and abnormal elevation of endogenous formaldehyde has been found in the brains of Alzheimer's disease (AD) patients. Hyperphosphorylated Tau protein with subsequent aggregates as neurofibrillary tangles (NFTs) is one of the typical pathological characteristics in AD brains. The mechanism underlying abnormally elevated concentrations of endogenous formaldehyde that induce Tau hyperphosphorylation is unknown.

Methods

N2a cells and mice were treated with formaldehyde for different time points, then Western blotting and immunocytochemistry were utilized to determine the phosphorylation and polymerization of Tau protein. HPLC was used to detect the concentration of formaldehyde in cell media.

Results

Under formaldehyde stress, Tau became hyperphosphorylated, not only in the cytoplasm, but also in the nucleus of neuroblastoma (N2a) cells, and mouse brains. Polymers of cellular phospho-Tau were also detected. Significant accumulation of glycogen synthase kinase-3β (GSK-3β) in the nucleus of N2a and mouse brain cells, and elevation of its phosphorylation at Y216, was observed under formaldehyde stress. Formaldehyde-induced Tau hyperphosphorylation was blocked in the presence of LiCl and CT99021, inhibitors of GSK-3β, and by RNAi interference.

Conclusions

Formaldehyde, which may cause age-related memory loss, can act as a factor triggering Tau hyperphosphorylation via GSK-3β catalysis and induces polymerization of Tau.

General significance

Investigation of formaldehyde-induced Tau hyperphosphorylation may provide novel insights into mechanisms underlying tauopathies.     

The miR-182/SORT1 axis regulates vascular smooth muscle cell calcification in vitro and in vivo

Arterial calcification is a common feature of cardiovascular disease. Sortilin is involved in the development of atherosclerosis, but the specific mechanism is unclear. In this study, we established calcification models in vivo and in vitro by using vitamin D₃ and β-glycerophosphate, respectively. In vivo, the expression of SORT1 was up-regulated and the expression of miR-182 was down-regulated in calcified arterial tissues. Meanwhile there was a negative correlation between SORT1 expression and miR-182 levels. In vitro, downregulating SORT1 expression using shRNA inhibited β-glycerophosphoric induced vascular smooth muscle cells (VSMCs) calcification. Moreover, reduced sortilin levels followed transfection of miR-182 mimics, whereas there was a significant increase in sortilin levels after transfection of miR-182 inhibitors. A luciferase reporter assay confirmed that SORT1 is the direct target of miR-182. Our study suggests that SORT1 plays a vital role in the development of arterial calcification and is regulated by miR-182.     

Antibacterial performance of nanoscaled visible-light responsive platinum-containing titania photocatalyst in vitro and in vivo

Background

Traditional antibacterial photocatalysts are primarily induced by ultraviolet light to elicit antibacterial reactive oxygen species. New generation visible-light responsive photocatalysts were discovered, offering greater opportunity to use photocatalysts as disinfectants in our living environment. Recently, we found that visible-light responsive platinum-containing titania (TiO₂–Pt) exerted high performance antibacterial property against soil-borne pathogens even in soil highly contaminated water. However, its physical and photocatalytic properties, and the application in vivo have not been well-characterized.

Methods

Transmission electron microscopy, energy dispersive spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction, ultraviolet–visible absorption spectrum and the removal rate of nitrogen oxides were therefore analyzed. The antibacterial performance under in vitro and in vivo conditions was evaluated.

Results

The apparent quantum efficiency for visible light illuminated TiO₂–Pt is relatively higher than several other titania photocatalysts. The killing effect achieved approximately 2 log reductions of pathogenic bacteria in vitro. Illumination of injected TiO₂–Pt successfully ameliorated the subcutaneous infection in mice.

Conclusions

This is the first demonstration of in vivo antibacterial use of TiO₂–Pt nanoparticles. When compared to nanoparticles of some other visible-light responsive photocatalysts, TiO₂–Pt nanoparticles induced less adverse effects such as exacerbated platelet clearance and hepatic cytotoxicity in vivo.

General significance

These findings suggest that the TiO₂–Pt may have potential application on the development of an antibacterial material in both in vitro and in vivo settings.     

Reconstruction of functional endometrium-like tissue in vitro and in vivo using cell sheet engineering

Uterus is a female specific reproductive organ and plays critical roles in allowing embryo to grow. Therefore, the endometrial disorders lead to female infertility. Hence, the regeneration of endometrium allowing fertilized ovum to implant might be valuable in the field of fertility treatment. Recently, cell sheet engineering using a temperature-responsive culture dish has advanced in regenerative medicine. With this technology, endometrial cells were harvested as a contiguous cell sheet by reducing temperature. Firstly, mouse endometrial cell sheets were re-cultured for 3 days to evaluate the function. Histological analyses revealed that endometrial epithelial cell-specific cytokeratin 18 and female-specific hormone receptors, estrogen receptor β and progesterone receptor, were expressed. Furthermore, endometrial epithelial cells constructed epithelial layer at the apical side. Then, endometrial cell sheets from green-fluorescent-protein rat cells were transplanted onto the buttock muscle of nude rat for evaluating the function in vivo. Histological analyses showed that endometrial cell sheets reconstructed endometrium-like tissue, which was found to form uterus-specific endometrial glands having hormonal receptor to estrogen. In this study, endometrial cell sheets were speculated to contribute to the regeneration of functional endometrium as a new therapy.     

An easy-to-use FRET protein substrate to detect calpain cleavage in vitro and in vivo

Calpain-1 and -2 are Ca^2 +-activated intracellular cysteine proteases that regulate a wide range of cellular functions through the cleavage of their protein substrates. Unlike degradative proteases, calpains make limited, transformative cleavages, typically in accessible sequences linking discrete subdomains, to irreversibly alter substrate functions. The biological roles of calpain and their interplay with calcium signaling are of significant biomedical interest as biomarkers and potential therapeutic targets in a growing number of diseases including Alzheimer's, cancer and fibrosis. Unfortunately, many of the colorimetric and fluorimetric assays that have been developed to study calpain activity suffer from low sensitivity and/or poor calpain specificity. To address the need for a highly sensitive and calpain-specific substrate suitable for in vitro and in vivo calpain activity analysis, we have developed a protein FRET probe. We inserted the optimized calpain cleavage sequence PLFAAR between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) and modulated its flanking sequences for optimal calpain cleavage. We demonstrate greater sensitivity and calpain-specificity of an optimal 16-residue PLFAAR-based FRET substrate compared to a standard α-spectrin-based probe. The 16-residue PLFAAR protein FRET substrate is not significantly cleaved by trypsin, chymotrypsin, cathepsin-L or caspase-3, and is highly sensitive to both calpain-1 and -2. After transfection of the substrate gene into breast cancer cells the PLFAAR protein FRET product was cut in lysed wild-type cells but not in those with a calpain knock-out phenotype. Blockage of substrate cleavage in the lysates by endogenous and exogenous calpastatin was observed, and was overcome by adding extra calpain.     

Differential integrin expression in pre-implantation embryos developing under in vivo and in vitro conditions

Implantation failure is a major problem in human assisted reproduction, which persists regardless the optimization of endometrial receptivity and selection of genetically and morphologically healthy embryos. Since embryo-endometrium interaction depends on cell junctional, cell adhesion and cell-substratum adhesion molecules, the present study inquired whether in vitro growing murine embryos display similar to the in vivo growing embryos patterns of adhesion molecules. To this extend aVb3 expression and distribution in zygotes and 2-cell stage embryos were studied. The results demonstrated that only the in vivo growing embryos displayed specifically polarized aVb3 distribution, indicating their potential successful interaction with endometrium. Based on previous studies showing that L-carnitine (L-Cn) could affect embryonic development, it was demonstrated that the addition of L-Cn to the culture medium, could lead the in vitro growing embryos to acquire aVb3 expression and distribution similar to the in vivo growing embryos. Visualization of the effect of L-Cn using third harmonic generation imaging showed decreased lipid droplet levels in 2-cell-stage embryos, observation that correlates with an active energetic state of the growing embryos. Thus, the application of L-Cn to the culture medium could assist pre-implantation-state embryos to acquire aVb3 expression and distribution similar to the in vivo developing conditions.     

Ultrastructure of the median neurosecretory cells of Manduca sexta in vivo and in vitro

Ultrastructural comparisons of median neurosecretory cells of Manduca sexta were made by using fresh and cultured specimens. Examination of the endomembrane system, the abundance and electron density of neurosecretory granules, and the condition and abundance of mitochondria showed that brains cultured for 4 days were similar to those taken directly from the insect. After 10 days, the neurosecretory cells had broken down but the neurosecretory granules were still present.