An increase in apoptosis and reduction in αB-crystallin expression levels in the lens underlie the cataractogenesis of Morioka cataract (MCT) mice

We examined the morphological changes in fibers, localization of apoptotic cells, and protein expression of αB-crystallin in the lens of Morioka cataract (MCT) mice, a novel cataract model. Using a scanning electron microscope, swollen lens fibers and enlarged spaces between lens fibers were observed in the lens of 3-week-old MCT mice. At 2 weeks of age (before cataract), the single-strand DNA (ssDNA)-positive (indicating apoptosis) cell ratio of the lens epithelium was significantly higher in MCT than in wild-type ddY mice. At 2 and 4 weeks of age, αB-crystallin protein expression of the lens in MCT mice was significantly lower than that in wild-type ddY mice. These findings suggest that increase in apoptosis and reduction in αBcrystallin level are involved in the cataractogenesis of MCT mice.     

Analysis of the presence of cell proliferation-related molecules in the Tgf-β3 null mutant mouse palate reveals misexpression of EGF and Msx-1

The Tgf-β(3) null mutant mouse palate presents several cellular anomalies that lead to the appearance of cleft palate. One of them concerns the cell proliferation of both the palatal medial edge epithelium and mesenchyme. In this work, our aim was to determine whether there was any variation in the presence/distribution of several cell proliferation-related molecules that could be responsible for the cell proliferation defects observed in these palates. Our results showed no difference in the presence of EGF-R, PDGF-A, TGF-β(2), Bmp-2, and Bmp-4, and differences were minimal for FGF-10 and Shh. However, the expression of EGF and Msx-1 changed substantially. The shift of the EGF protein expression was the one that most correlated with that of cell proliferation. This molecule is regulated by TGF-β(3), and experiments blocking its activity in culture suggest that EGF misexpression in the Tgf-β(3) null mutant mouse palate plays a role in the cell proliferation defect observed.     

β-arrestin deficiency protects against pulmonary fibrosis in mice and prevents fibroblast invasion of extracellular matrix

Idiopathic pulmonary fibrosis is a progressive disease that causes unremitting extracellular matrix deposition with resulting distortion of pulmonary architecture and impaired gas exchange. β-Arrestins regulate G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors through receptor desensitization while also acting as signaling scaffolds to facilitate numerous effector pathways. Here, we examine the role of β-arrestin1 and β-arrestin2 in the pathobiology of pulmonary fibrosis. In the bleomycin-induced mouse lung fibrosis model, loss of either β-arrestin1 or β-arrestin2 resulted in protection from mortality, inhibition of matrix deposition, and protected lung function. Fibrosis was prevented despite preserved recruitment of inflammatory cells and fibroblast chemotaxis. However, isolated lung fibroblasts from bleomycin-treated β-arrestin-null mice failed to invade extracellular matrix and displayed altered expression of genes involved in matrix production and degradation. Furthermore, knockdown of β-arrestin2 in fibroblasts from patients with idiopathic pulmonary fibrosis attenuated the invasive phenotype. These data implicate β-arrestins as mediators of fibroblast invasion and the development of pulmonary fibrosis, and as a potential target for therapeutic intervention in patients with idiopathic pulmonary fibrosis.     

Changes of chondrocyte expression profiles in human MSC aggregates in the presence of PEG microspheres and TGF-β3

Biomaterial microparticles are commonly utilized as growth factor delivery vehicles to induce chondrogenic differentiation of mesenchymal stem/stromal cells (MSCs). To address whether the presence of microparticles could themselves affect differentiation of MSCs, a 3D co-aggregate system was developed containing an equal volume of human primary bone marrow-derived MSCs and non-degradable RGD-conjugated poly(ethylene glycol) microspheres (PEG-μs). Following TGF-β3 induction, differences in cell phenotype, gene expression and protein localization patterns were found when compared to MSC aggregate cultures devoid of PEG-μs. An outer fibrous layer always found in differentiated MSC aggregate cultures was not formed in the presence of PEG-μs. Type II collagen protein was synthesized by cells in both culture systems, although increased levels of the long (embryonic) procollagen isoforms were found in MSC/PEG-μs aggregates. Ubiquitous deposition of type I and type X collagen proteins was found in MSC/PEG-μs cultures while the expression patterns of these collagens was restricted to specific areas in MSC aggregates. These findings show that MSCs respond differently to TGF-β3 when in a PEG-μs environment due to effects of cell dilution, altered growth factor diffusion and/or cellular interactions with the microspheres. Although not all of the expression patterns pointed toward improved chondrogenic differentiation in the MSC/PEG-μs cultures, the surprisingly large impact of the microparticles themselves should be considered when designing drug delivery/scaffold strategies.     

Deletion of the D domain of the human parainfluenza virus type 3 (HPIV3) PD protein results in decreased viral RNA synthesis and beta interferon (IFN-β) expression

Parvoviridae is a family of small non-enveloped viruses and divided into two subfamilies. The family members infect a wide range of organisms from insects to humans and some of the members (e.g., nonpathogenic adeno-associated viruses) are effective gene therapy delivery vectors. We detailed the synonymous codon usage pattern of Parvoviridae family from the available 58 sequenced genomes through multivariate statistical methods. Our results revealed that nine viruses showed some degree of strong codon bias, and the others possessed a general weak trend of codon bias. ENc-plot and neutrality plot results showed that selective pressure dominated over mutation in shapes coding sequence’s composition. The overall GC content and GC content at the third synonymous codon position were the principal determinants behind the variations within the codon usage patterns, as they both significantly correlated with the first axis of correspondence analysis. In addition, gene length had no direct influence on the codon usage pattern. Densovirinae subfamily and Parvovirinae subfamily possessed nine identical preferred codons, though most of the two subfamilies codon usage frequencies were significantly different. The result of cluster analysis based on synonymous codon usage was discordant with that of taxonomic classification. Adeno-associated viruses formed a separated clade far from other Parvoviridae members in the dendrogram. Thus, we concluded that natural selection rather than mutation pressure accounts for the main factor that affects the codon bias in Parvoviridae family.     

Astragaloside effect on TGF-β1, SMAD2/3, and α-SMA expression in the kidney tissues of diabetic KKAy mice

Numerous cytokines participate in the occurrence and development of inflammation and renal interstitial fibrosis. Previous studies confirmed that TGF-β1 overexpressed in diabetic nephropathy. As a downstream signal protein of TGF-β1 family, SMAD has an important role in the process of α-SMA mediated renal interstitial fibrosis. This study aimed to study astragaloside effect on TGF-β1, SMAD2/3, and α-SMA expression in the kidney tissue of diabetic KKAy mice, to reveal its potential impact on renal interstitial fibrosis. 20 type II diabetic KKAy mice were randomly equally divided into model group and astragaloside group, while 10 male C57BL/6J mice were selected as the control. Astragaloside at 40 mg/(kg•d) was given when the KKAy mice fed with high-fat diet to 14 weeks old. The mice were killed at 24 weeks old and the kidney tissue samples were collected. Pathology morphological changes were observed. TGF-β1, SMAD2/3, and α-SMA expression levels were determined by immunohistochemistry. Compared with control, mice kidney in model group appeared obvious fibrosis and up-regulated blood glucose level, TGF-β1, SMAD2/3, and α-SMA expression (P < 0.05). Mice in astragaloside group exhibited alleviated renal interstitial fibrosis compared with the model. Its blood glucose level, TGF-β1, SMAD2/3, and α-SMA expression levels were significantly lower than the model group (P < 0.05). Astragaloside can delay the renal fibrosis process in diabetic mice by influencing the TGF-β/SMADS signaling pathway and down-regulating TGF-β1, SMAD2/3, and α-SMA expression.     

Construction of the recombinant expression vector for CD80-IgG fusion gene and its expression in Chinese hamster ovary cells

Itiswellestablishedthatoptimalactivationof na veTcellsrequiressignalingthroughtheT cell receptoraswellasthroughothercostimulatorypathways.Na veTcellswillbecomenonrespons ive,anergicorapoptoticifthetumorantigenispr esentedtothemwithoutthecostimulatorysignal,whichmakestumorcellsescapefromtheimmuno surveillance.Oneofthemostimportantcostimula torysignalsofthiskindcanbeprovidedbythein teractionofB7.1(CD80)onantigen presenting cells(APC)withCD28onTcells.Acutemyelogenousleukemia(AML)cellsarenot…     

Upregulated expression and function of the α4β1 integrin in multiple myeloma cells resistant to bortezomib

The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment promotes MM cell retention, survival, and resistance to different anti-MM agents, including proteasome inhibitors (PIs) such as bortezomib (BTZ). The α4β1 integrin is a main adhesion receptor mediating MM cell–stroma interactions and MM cell survival, and its expression and function are downregulated by BTZ, leading to inhibition of cell adhesion-mediated drug resistance (CAM-DR) and MM cell apoptosis. Whether decreased α4β1 expression and activity are maintained or recovered upon development of resistance to BTZ represents an important question, as a potential rescue of α4β1 function could boost MM cell survival and disease progression. Using BTZ-resistant MM cells, we found that they not only rescue their α4β1 expression, but its levels were higher than in parental cells. Increased α4β1 expression in resistant cells correlated with enhanced α4β1-mediated cell lodging in the BM, and with disease progression. BTZ-resistant MM cells displayed enhanced NF-κB pathway activation relative to parental counterparts, which contributed to upregulated α4 expression and to α4β1-dependent MM cell adhesion. These data emphasize the upregulation of α4β1 expression and function as a key event during resistance to BTZ in MM, which might indirectly contribute to stabilize this resistance, as stronger MM cell attachment to BM stroma will regain CAM-DR and MM cell growth and survival. Finally, we found a strong correlation between high ITGB1 (integrin β1) expression in MM and poor progression-free survival (PFS) and overall survival (OS) during treatment of MM patients with BTZ and IMIDs, and combination of high ITGB1 levels and presence of the high-risk genetic factor amp1q causes low PFS and OS. These results unravel a novel prognostic value for ITGB1 in myeloma. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Risk of genetic and epigenetic alteration in children conceived following ART: Is it time to return to nature whenever possible?

Assisted reproductive technology may influence epigenetic signature as the procedures coincide with the extensive epigenetic modification occurring from fertilization to embryo implantation. However, it is still unclear to what extent ART alters the embryo epigenome. In vivo fertilization occurs in the fallopian tube, where a specific and natural environment enables the embryo's healthy development. During this dynamic period, major waves of epigenetic reprogramming, crucial for the normal fate of the embryo, take place. Over the past decade, concerns relating to the raised incidence of epigenetic anomalies and imprinting following ART have been raised by several authors. Epigenetic reprogramming is particularly susceptible to environmental conditions during the periconceptional period; therefore, unphysiological conditions, including ovarian stimulation, in vitro fertilization, embryo culture, cryopreservation of gametes and embryos, parental lifestyle, and underlying infertility, have the potential to contribute to epigenetic dysregulation independently or collectively. This review critically appraises the evidence relating to the association between ART and genetic and epigenetic modifications that may be transmitted to the offspring.     

Evaluation of the expression of TLR-2, Dectin-1 and TNF-α level in invasive aspergillosis in cancer mice

Aspergillus fumigatus (A. fumigatus) is an opportunistic fungal pathogen that can cause life-threatening invasive fungal disease (invasive aspergillosis (IA)) in immuno-compromised individuals. In this study, we explored whether the innate resistance to IA in cancer mice has resulted in altered expression of TLR-2 and Dectin-1 in macrophages (flow cytometry) and production of tumour necrosis factor alpha (TNF-α; ELISA) as well as overall mortality. The data demonstrated significant increases in Dectin-1 and TLR-2 on peritoneal macrophages and mortality in cancer mice intravenously infected with A. fumigatus. TNF-α levels were not significantly increased in this group. Probably, IA causes some disorganization in inflammatory responses. We hypothesize that concomitance of IA and cancer may change the micro-environment for local or systemic immune responses. Other complementary studies are required to support our hypothesis.     

Oxymatrine attenuates bleomycin-induced pulmonary fibrosis in mice via the inhibition of inducible nitric oxide synthase expression and the TGF-β/Smad signaling pathway

Oxymatrine (OM) is an alkaloid extracted from the Chinese herb Sophora flavescens Ait. with a variety of pharmacological activities. The aim of this study was to investigate the preventive effects of OM on bleomycin (BLM)-induced pulmonary fibrosis (PF) and to further explore the underlying mechanisms. C57BL/6 mice were randomly assigned to five groups: the saline sham group; the BLM group, in which mice were endotracheally instilled with BLM (3.0?mg/kg); and the BLM plus OM groups, in which OM was given to mice daily (10, 20 or 40?mg/kg) one day after BLM instillation for 21?days. The bronchoalveolar lavage fluid (BALF) and lung tissues were collected at 15 and 22?days post BLM administration, respectively. Lung tissues were stained with hematoxylin and eosin (H&E) for histological evaluation. Levels of tumor necrosis factor (TNF)-α, interleukin-6 (IL-6) and nitric oxide (NO) in mouse BALF were measured, as well as myeloperoxidase (MPO) activity and malondialdehyde (MDA) content in lung homogenates. The inducible nitric oxide synthase (iNOS) expression in the lung tissues was determined by immunohistochemical staining, quantitative real-time PCR and western blot analysis. Moreover, the expression of transforming growth factor (TGF)-β1, Smad2, Smad3, p-Smad2 and p-Smad3 were also detected. We found that OM improved BLM-induced lung pathological changes, inhibited MPO activity and reduced MDA levels in a dose-dependent manner. OM also dose-dependently inhibited the release of TNF-α and IL-6, and decreased the expression of iNOS in lung tissues and thus prevented NO release in response to BLM challenge. In addition, OM decreased the expression of TGF-β1, p-Smad2 and p-Smad3, which are all important members of the TGF-β/Smad signaling pathway. Our study provides evidence that OM significantly ameliorated BLM-induced PF in mice via the inhibition of iNOS expression and the TGF-β/Smad pathway.     

Lead exposure increases levels of β-amyloid in the brain and CSF and inhibits LRP1 expression in APP transgenic mice

Lead (Pb) is an environmental factor suspected of contributing to neurodegenerative diseases such as Alzheimer's disease (AD). In AD, it has been postulated that increased production and/or decreased metabolism/clearance of β-amyloid (Aβ) may lead to amyloid plaque deposition as well as a cascade of other neuropathological changes. It has been suggested that Pb exposure may be associated with AD-like pathology and severe memory deficits in humans. Therefore, we investigated whether Pb exposure could induce Aβ accumulation in the brain. In this study, we demonstrated that acute Pb treatments lead to increased levels of Aβ in the cerebrospinal fluid (CSF) and brain tissues. Interestingly, Pb treatments did not affect Aβ production in brain neurons. Furthermore, Pb treatments significantly decreased LRP1 protein expression in the choroid plexus (CP). Our results suggest disrupted LRP1-mediated transport of Aβ in this region may be responsible for the Aβ accumulation in brain.     

Pulmonary epithelial expression of human α1-antitrypsin in transgenic mice results in delivery of α1-antitrypsin protein to the interstitium

α₁-Antitrypsin (α₁AT) therapy is used as a treatment for α₁AT deficiency. It has also been proposed as a therapy for cigarette smoke-induced emphysema, although the efficacy of such therapy is as yet unproven. Moreover, the optimal route of delivery of α₁AT to the lung interstitium, the crucial locus of action, is unknown. We created transgenic mice with expression of the human α₁AT gene directed by a human surfactant protein C (SpC) promoter fragment or a rat Clara cell 10-kDa protein (CC10) promoter fragment in order to examine the ability of pulmonary epithelial cell expression of α₁AT to deliver protein to the interstitium, and to produce a model that would allow studies on the efficacy of α₁AT in preventing lung damage after cigarette smoke exposure. Four transgenic lines were studied. In situ hybridization and light microscopic immunohistochemistry showed that two CC10 driven lines expressed human α₁AT in type II alveolar cells and airway epithelial cells; α₁AT expression was seen in the alveolar parenchyma in two SpC driven lines, and in small airway epithelium in one of the SpC lines. Electron microscopic immunochemistry showed the presence of the human α₁AT protein in the interstitium in all lines. Mean levels of human protein varied from 0.37 to 2.9 μg/g lung protein and serum levels from 0.72 to 1.3 μg/ml, compared to normal human serum α₁AT levels of 2–5 mg/ml. We conclude that transgene-mediated expression of α₁AT in pulmonary epithelial cells results in diffuse expression of the transgene in the alveolar parenchyma and reproducibly leads to transfer of protein to the interstitium. The present model is, however, limited by low levels of protein production; limited protein production may be a problem in other forms of gene therapy in which relatively large amounts of extracellular protein are needed in the lung for a therapeutic effect. Received: 5 August 1998 / Accepted: 25 January 1999     

Frequent aberrant expression of the human ether à go-go (hEAG1) potassium channel in head and neck cancer: pathobiological mechanisms and clinical implications

Compelling evidence indicates that the human ether-à-go-go voltage-gated potassium channels (hEAG1) may represent new valuable membrane therapeutic targets and diagnostic/prognostic biomarkers in various cancers. This study is the first to investigate the expression of hEAG1 potassium channel subunit in both primary tumors and HNSCC-derived cell lines to ascertain its clinical and biological role in tumor progression. Our findings demonstrate that hEAG1 is frequently aberrantly expressed in a high percentage of primary tumors (83?%, 45/54 cases) and HNSCC-derived cell lines (83?%, 10/12 cell lines). hEAG1 expression increased during HNSCC progression and was more frequent in advanced tumors. Strikingly, hEAG1 expression was also detected in a notable proportion (39?%, 17/44 cases) of patient-matched normal adjacent mucosa, whereas no expression was detected in normal epithelia from non-oncologic patients without exposure to tobacco carcinogens. In an attempt to identify the underlying mechanisms of aberrant hEAG1 expression in HNSCC, we found that hEAG1 gene copy gain occurred at a low frequency (15?%, 13/88 cases) in primary tumors but was not observed in early stages of HNSCC tumorigenesis. Furthermore, this study provides original evidence supporting the involvement of histone acetylation (i.e., H3Ac and H4K16Ac activating marks) in the regulation of hEAG1 expression in HNSCC. In addition, functional studies in HNSCC cells further revealed that hEAG1 expression is a biologically relevant feature that promotes cell proliferation and invasion, although independently of its ion-conducting function. Our findings strongly support the notion that hEAG1 may represent a promising candidate as tumor marker and membrane therapeutic target for HNSCC treatment.     

Imbalance in the ratio of CpG and polyG contributes to impaired interferon-α expression

The secretion of interferon-α (IFN-α) is impaired during hepatitis B virus (HBV) infection. DNA sequences purified from distinct viruses, for example, HBV versus members of Herpesviridae, have been shown to differ in their IFN-α signaling properties. The present study found that DNA from HBV inhibited, while DNA from members of Herpesviridae induced, the expression of IFN-α. Furthermore, stimulatory cytosine-phosphate-guanosine (CpG) sequences derived from these DNA viruses could induce the secretion of IFN-α, while inhibitory guanosine-rich oligodeoxynucleoti (polyG) oligonucleotide sequences derived from these DNA viruses could inhibit CpG-induced IFN-α secretion. Using a computational analysis of genomic DNA sequences, the discrimination between the genomes of HBV and those of other DNA viruses that can also cause inflammation of the liver is based on different frequencies of the CpG and polyG motifs. The underrepresentation of stimulatory CpG motifs and overrepresentation of inhibitory polyG motifs were documented in HBV genomes, whereas the DNA from other viral genomes displayed the opposite trend. Moreover, it was demonstrated that HBV could suppress the activation of IFN-α via its own DNA through the high proportion of polyG motifs. To our knowledge, this is the first demonstration of a specific role for polyG motifs in the inhibition of the IFN-α response following DNA virus infection.     

Response of ERβ and aromatase expression in the monkey hippocampal formation to ovariectomy and menopause

Changes in the expression of estrogen-related substances in monkeys’ brains at the menopausal transition, when estrogen deficit starts to occur, have not yet been examined thoroughly. In the present study, we immunohistochemically investigated the expression levels of estrogen receptor beta (ERβ) and aromatase (local estrogen synthesizing enzyme) in the hippocampal formation of premenopausal, menopausal, and ovariectomized premenopausal monkeys. In all monkeys tested, ERβ immunoreactivity was observed in interneurons located in the subiculum and the Ammon's horn, and most of these ERβ-immunoreactive neurons coexpressed a GABAergic neuron marker, parvalbumin. In the menopausal monkeys who exhibited a decline in estrogen concentration, hippocampal ERβ was highly upregulated, while aromatase expression was not markedly changed. By contrast, aromatase in the ovariectomized monkeys was significantly upregulated, while ERβ expression was not changed. In the brains of ovariectomized and menopausal monkeys, depletion of ovary-derived estrogen brought about different reactions which may be attributed to the senescence of brain aging.