COVID-19 is causing a major once-in-a-century global pandemic. The scientific and clinical community is in a race to define and develop effective preventions and treatments. The major features of ...disease are described but clinical trials have been hampered by competing interests, small scale, lack of defined patient cohorts and defined readouts. What is needed now is head-to-head comparison of existing drugs, testing of safety including in the background of predisposing chronic diseases, and the development of new and targeted preventions and treatments. This is most efficiently achieved using representative animal models of primary infection including in the background of chronic disease with validation of findings in primary human cells and tissues. We explore and discuss the diverse animal, cell and tissue models that are being used and developed and collectively recapitulate many critical aspects of disease manifestation in humans to develop and test new preventions and treatments.
Aggressive cancers often express E-cadherin in cytoplasmic vesicles rather than on the plasma membrane and this may contribute to the invasive phenotype of these tumors. Therapeutic strategies are ...not currently available that restore the anti-invasive function of E-cadherin in cancers. MDA-MB-231 cells are a frequently used model of invasive triple-negative breast cancer, and these cells express low levels of E-cadherin that is mislocalized to cytoplasmic vesicles. MDA-MB-231 cell lines stably expressing wild-type E-cadherin or E-cadherin fused to glutathione S-transferase or green fluorescent protein were used as experimental systems to probe the mechanisms responsible for cytoplasmic E-cadherin localization in invasive cancers. Although E-cadherin expression partly reduced cell invasion in vitro, E-cadherin was largely localized to the cytoplasm and did not block the invasiveness of the corresponding orthotopic xenograft tumors. Further studies indicated that the glucocorticoid dexamethasone and the highly potent class I histone deacetylase (HDAC) inhibitor largazole cooperated to induce E-cadherin localization to the plasma membrane in triple-negative breast cancers, and to suppress cellular invasion in vitro. Dexamethasone blocked the production of the cleaved form of the CDCP1 (that is, CUB domain-containing protein 1) protein (cCDCP1) previously implicated in the pro-invasive activities of CDCP1 by upregulating the serine protease inhibitor plasminogen activator inhibitor-1. E-cadherin preferentially associated with cCDCP1 compared with the full-length form. In contrast, largazole did not influence CDCP1 cleavage, but increased the association of E-cadherin with γ-catenin. This effect on E-cadherin/γ-catenin complexes was shared with the nonisoform selective HDAC inhibitors trichostatin A (TSA) and vorinostat (suberoylanilide hydroxamic acid, SAHA), although largazole upregulated endogenous E-cadherin levels more strongly than TSA. These results demonstrate that glucocorticoids and HDAC inhibitors, both of which are currently in clinical use, cooperate to suppress the invasiveness of breast cancer cells through novel, complementary mechanisms that converge on E-cadherin.
Summary Objective Hypoxia-inducible factor (HIF)-2α and the zinc-ZIP8-MTF1 axis in chondrocytes serve as catabolic regulators of osteoarthritic cartilage destruction by regulating the expression of ...catabolic factor genes. We explored possible crosstalk between these signaling pathways and its biological significance in osteoarthritis (OA). Methods Microarray analysis, various mRNA and protein assays were conducted using primary cultured mouse articular chondrocytes and experimental OA cartilage to reveal molecular mechanisms underlying the crosstalk between HIF-2α and the zinc-ZIP8-MTF1 axis. Experimental OA in mice was induced by intra-articular (IA) injection of adenovirus expressing HIF-2α (Ad- Epas1 ), ZIP8 (Ad- Zip8 ), or MTF1 (Ad- Mtf1 ) in wild-type mice or mice with cartilage-specific conditional knockout of HIF-2α ( Epas1fl/fl ; Col2a1-Cre ), ZIP8 ( Zip8fl/fl ; Col2a1-Cre ), or MTF1 ( Mtf1fl/fl ; Col2a1-Cre ). Results HIF-2α activated the zinc-ZIP8-MTF1 axis in chondrocytes by upregulating the Zn2+ transporter ZIP8, thereby increasing Zn2+ influx and activating the downstream transcription factor MTF1. The zinc-ZIP8-MTF1 axis, in turn, acted as a novel transcriptional regulator of HIF-2α. HIF-2α-induced activation of the zinc-ZIP8-MTF1 axis amplified HIF-2α regulation of OA cartilage destruction by synergistically promoting expression of matrix-degrading enzymes. Thus, HIF-2α-induced activation of the zinc-ZIP8-MTF1 axis, together with zinc-ZIP8-MTF1 regulation of HIF-2α, acted collectively to synergistically promote expression of matrix-degrading enzymes and OA cartilage destruction. Conclusion Our findings identify a reciprocal activation mechanism involving HIF-2α and the zinc-ZIP8-MTF1 axis during OA pathogenesis that amplifies catabolic signaling and cartilage destruction.
Although intracranial atherosclerotic disease is often encountered during endovascular treatment for acute vertebrobasilar occlusions, its clinical implication is not well-known. We aimed to evaluate ...whether intracranial atherosclerotic disease influences the clinical outcomes following endovascular treatment of acute vertebrobasilar occlusive stroke.
Fifty-one patients with acute vertebrobasilar occlusive stroke were included. The onset-to-groin puncture time was ≤12 hours, and aspiration- or stent-based thrombectomy was used as the primary treatment method. Following primary endovascular treatment, intracranial atherosclerotic disease (IAD group) was angiographically diagnosed when a fixed focal stenosis was observed at the occlusion site, whereas embolism (embolic group) was diagnosed if no stenosis was observed. Clinical and treatment variables were compared in both groups, and IAD was evaluated as a prognostic factor for clinical outcomes.
The baseline NIHSS score tended to be lower (14 versus 22,
= .097) in the IAD group (
= 19) than in the embolic group (
= 32). The procedural time was longer in the IAD group (96 versus 61 minutes,
= .002), despite similar rates of TICI 2b-3 (89.5% versus 87.5%,
= 1.000). The NIHSS score at 7 days was higher (21 versus 8,
= .060) and poor outcomes (mRS 4-6 at 3 months) were more frequent in the IAD group (73.7% versus 43.8%,
= .038). IAD (odds ratio, 5.469; 95% CI, 1.09-27.58;
= .040) was independently associated with poor outcomes.
An arterial occlusion related to IAD was associated with a longer procedural time and poorer clinical outcome. Further studies are warranted to elucidate the appropriate endovascular strategy.
Microglia are the resident immune cells in the central nervous system and key players against pathogens and injury. However, persistent microglial activation often exacerbates pathological damage and ...has been implicated in many neurological diseases. Despite their pivotal physiological and pathophysiological roles, how the survival and death of activated microglia is regulated remains poorly understood. We report here that microglia activated through Toll-like receptors (TLRs) undergo RIP1/RIP3-dependent programmed necrosis (necroptosis) when exposed to the pan caspase inhibitor zVAD-fmk. Although zVAD-fmk and the caspase-8 inhibitor IETD-fmk had no effect on unstimulated primary microglia, they markedly sensitized microglia to TLR1/2,3,4,7/8 ligands or TNF treatment, triggering programmed necrosis that was completely blocked by R1P1 kinase inhibitor necrostatin-1. Interestingly, necroptosis induced by TLR ligands and zVAD was restricted to microglial cells and was not observed in astrocytes, neurons or oligodendrocytes even though they are known to express certain TLRs. Deletion of genes encoding TNF or TNFR1 failed to prevent lipopolysaccharide- and poly(I:C)-induced microglial necroptosis, unveiling a TNF-independent programmed necrosis pathway in TLR3- and TLR4-activated microglia. Microglia from mice lacking functional TRIF were fully protected against TLR3/4 activation and zVAD-fmk-induced necrosis, and genetic deletion of rip3 also prevented microglia necroptosis. Activation of c-jun N-terminal kinase and generation of specific reactive oxygen species were downstream signaling events required for microglial cell death execution. Taken together, this study reveals a robust RIP3-dependent necroptosis signaling pathway in TLR-activated microglia upon caspase blockade and suggests that TLR signaling and programmed cell death pathways are closely linked in microglia, which could contribute to neuropathology and neuroinflammation when dysregulated.
Abstract Objective Mesenchymal stem cells (MSCs) have been studied in regenerative medicine because of their unique immunologic characteristics. However, before clinical application in humans, animal ...models are needed to confirm their safety and efficacy. To date, appropriate methods and sources to obtain mouse MSCs have not been identified. Therefore, we investigated MSCs isolated from 3 strains of mice and 3 sources for the development of MSCs in a mouse model. Materials and Methods Male BALB/c, C3H and C57BL/6 mice were used to isolate MSCs from various tissues including bone marrow (BM), compact bone, and adipose tissue. The MSCs were maintained in StemXVivo medium. Immunophenotypes of the MSCs were analyzed by FACS and their growth potential estimated by the number of colony-forming unit fibroblasts. Results All MSCs that were isolated from BM, compact bone, and adipose tissue showed plastic-adherent, fibroblastic-like morphologic characteristics regardless of the mouse strain or cell source. However, culture of BM MSCs was less successful than the other tissue types. The FACS phenotype analysis revealed that the MSCs were positive for CD29, CD44, CD105, and Sca-1, but negative for CD34, TER-119, CD45, and CD11b. According to the results of the characterization, the adipose tissue MSCs showed higher growth potential than did other MSCs. Conclusion The results of this study showed that culture of adipose tissue and compact bone-MSCs was easier than BM MSCs. Based on the results of immunophenotype and growth potential, C57BL/6 AT-MSCs might be a suitable source to establish a mouse model of MSCs.
The disappearing and forming behavior of B2 ordered phase in high Si steels with Si-levels between 5 and 6.5% (by weight, unless specified otherwise) was investigated by observing TEM and measuring ...electrical resistivity and micro- and nano-hardness. The critical cooling rate for suppressing the formation of B2 ordered phase increased exponentially with Si-content. The ordered phase coarsening was observed to occur actively above 800
°C, where atoms have enough mobility. The removal of solidification segregation is necessary in order to reduce the amount of B2 ordered phase in the as-cast or hot-rolled state, to lower the annealing temperature for the dissolution of B2 ordered phase, and to shorten the annealing time. Micro-Vickers hardness depended only on Si-content even under various heat treatment conditions, and the nano-hardness had a small difference between B2 and A2 phases. In the high Si steels, the short-range order in A2 disordered phase was likely to cause a comparable hardness as that of B2 ordered phase.
Minimally invasive treatment has become a mainstay management strategy for early gastric cancer (EGC). Full-thickness incision of the gastric wall using natural orifice transluminal endoscopic ...surgery (NOTES) has been reported but is not easily applicable in clinical settings at present. The aim of the current study was to assess the feasibility of hybrid NOTES, which consists of endoscopic full-thickness gastric resection and a laparoscopic lymphadenectomy.
This was a prospective, pilot study at a single tertiary care referral center. A total of 14 patients with EGC located above the lower third of the stomach underwent hybrid NOTES. Clinically, the patients had contraindications to exclusive treatment using endoscopic submucosal dissection (ESD). The main outcome measure was technical success of hybrid NOTES.
All cases were resected en bloc with negative surgical margins. Histologically, four cases were mucosal cancers, and 10 cases were submucosal cancers. The median tumor size was 26 mm (range 12 - 90 mm). Lymphatic vessel invasion was found in four cases without lymph node metastasis (LNM). The median number of obtained lymph nodes was 18 (range 7 - 67). LNM was discovered in one case of undifferentiated submucosal cancer without lymphovascular invasion. Hybrid NOTES was conducted without intraoperative or postoperative adverse events in nine cases. The median operating time and estimated blood loss of successful cases were 143 minutes (range 110 - 253 minutes) and 16 mL (range 5 - 30 mL), respectively. The median hospital stay was 6 days (range 4 - 10 days). Five cases were converted to a subtotal gastrectomy for various reasons.
Hybrid NOTES could be a bridge between endoscopic resection and laparoscopic surgery and may prevent extensive gastrectomy in patients with EGC.
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