Longitudinal intravital microscopy of the mouse kidney: inflammatory responses to abdominal imaging windows.

Intravital microscopy enables direct observation of cell biology and physiology at subcellular resolution in real time in living animals. Implanted windows extend the scope of intravital microscopy to processes extending for weeks or even months, such as disease progression or tumor development. However, a question that must be addressed in such studies is whether the imaging window, like any foreign body, triggers an inflammatory response, and whether that response alters the biological process under investigation. To directly evaluate this question, we conducted large-scale intravital microscopy of the kidney of LysM-EGFP mice over time after implantation of abdominal imaging windows. These studies demonstrate that windows stimulated a variety of changes consistent with a foreign body response. Within a few days of implantation, leukocytes were recruited to the window and the region between the window and kidney where, over the next 16 days, they increased in number in an expanding volume that developed a new vascular network. These changes were accompanied by a dramatic increase in glomerular albumin permeability within 2-5 days of implantation. Similar results were obtained from mice implanted with windows coated with poly(l-lysine)-graft-polyethylene glycol (PLL-g-PEG), but not from immune-deficient mice. These studies demonstrate the importance of evaluating whether implanted windows induce an inflammatory response, and whether that response impacts the processes under evaluation in longitudinal intravital microscopy studies.NEW & NOTEWORTHY Intravital microscopy studies of LysM-EGFP mice demonstrate that abdominal imaging windows placed over the kidney stimulated a variety of changes consistent with a foreign body response. Within a day of implantation, leukocytes were recruited to the window where, over the next 16 days, they increased in number in an expanding volume that developed a new vascular network. These changes were accompanied by a dramatic increase in glomerular permeability to albumin.

1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol treatment inhibits abnormal tumor growth by regulating neutrophil infiltration in a non-small cell lung carcinoma mouse model.

Excessive neutrophil infiltration into the tumor microenvironment (TME) is an important factor that contributes to tumor overgrowth and limited immunotherapy efficacy. Neutrophils activate various receptors involved in tumor progression, while suppressing the infiltration and activity of cytotoxic T cells and creating optimal conditions for tumor growth. Therefore, the appropriate control of neutrophil infiltration is an effective strategy for tumor treatment. In the present study, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) inhibited tumor overgrowth by suppressing excessive neutrophil infiltration, resulting in >74.97 % reduction in tumor size in a Lewis lung carcinoma (LLC-1) mouse model. All subjects in the positive control group died during the 90-day survival period, whereas only four subjects in the PLAG treatment group survived. PLAG had a significantly higher tumor growth inhibitory effect and survival rate than other neutrophil infiltration-targeting inhibitors (e.g., Navarixin, lymphocyte antigen 6 complex locus G6D antibody [aLy6G]). The ability of PLAG to regulate neutrophil infiltration and inhibit tumor growth depends on thioredoxin-interacting protein (TXNIP). In tumors lacking TXNIP expression, PLAG failed to control neutrophil infiltration and infiltration-related factor release, and the inhibitory effect of PLAG on tumor growth was reduced. PLAG-mediated inhibition of neutrophil infiltration enhances the efficacy of immune checkpoint inhibitors (ICIs), increasing the antitumor efficacy and survival rate by 30 %. In conclusion, PLAG could be a novel alternative to anti-tumor drugs that effectively targets excessive neutrophil infiltration into cancer tissues.

Association between body composition and chronic low back pain in Korean adults aged over 50 years: The Korea National Health and Nutrition Examination Survey (KNHANES) 2010-2011.

Background The relationship between overweight or obesity and low back pain (LBP) has previously been investigated. Several recent studies have focused on the relationship between other indicators of obesity, particularly indicators of fat and the risk of LBP. However, the results of body composition and LBP have been inconsistent. Methods All data for the present retrospective, cross-sectional study was extracted from the Korea National Health and Nutrition Examination Survey (KNHANES) versions V-1 and 2 conducted in 2010 and 2011 by the Korean Centers for Disease Control and Prevention. In KNHANES V-1 (2010) and V-2 (2011), those over 50 years of age completed the surveys on LBP, body weight, and body composition assessed using dual-energy X-ray absorptiometry (DXA) were included. The multivariable logistic regression analysis was used to examine the relationship between the presence of chronic LBP and body composition adjusting for confounders. Results We analyzed 3,579 persons who completed the question. In the multivariable analyses adjusting for age and sex, none of the variables, including fat mass and fat-free mass, remained positively or negatively associated with LBP. Additionally, when depression, smoking, alcohol intake, physical activity, diabetes mellitus, and fat or lean tissue mass were included in the multivariable logistic model, no significant associations were found between all measures of fat mass, fat-free mass, and LBP Conclusion This study is contrary to previous studies that concluded that there is a correlation between obesity and fat mass and LBP. LBP is not associated with increased levels of obesity and fat mass.

Visualizing reactive astrocyte-neuron interaction in Alzheimer's disease using 11C-acetate and 18F-FDG.

Reactive astrogliosis is a hallmark of Alzheimer's disease (AD). However, a clinically validated neuroimaging probe to visualize the reactive astrogliosis is yet to be discovered. Here, we show that PET imaging with 11C-acetate and 18F-fluorodeoxyglucose (18F-FDG) functionally visualizes the reactive astrocyte-mediated neuronal hypometabolism in the brains with neuroinflammation and AD. To investigate the alterations of acetate and glucose metabolism in the diseased brains and their impact on the AD pathology, we adopted multifaceted approaches including microPET imaging, autoradiography, immunohistochemistry, metabolomics, and electrophysiology. Two AD rodent models, APP/PS1 and 5xFAD transgenic mice, one adenovirus-induced rat model of reactive astrogliosis, and post-mortem human brain tissues were used in this study. We further curated a proof-of-concept human study that included 11C-acetate and 18F-FDG PET imaging analyses along with neuropsychological assessments from 11 AD patients and 10 healthy control subjects. We demonstrate that reactive astrocytes excessively absorb acetate through elevated monocarboxylate transporter-1 (MCT1) in rodent models of both reactive astrogliosis and AD. The elevated acetate uptake is associated with reactive astrogliosis and boosts the aberrant astrocytic GABA synthesis when amyloid-ß is present. The excessive astrocytic GABA subsequently suppresses neuronal activity, which could lead to glucose uptake through decreased glucose transporter-3 in the diseased brains. We further demonstrate that 11C-acetate uptake was significantly increased in the entorhinal cortex, hippocampus and temporo-parietal neocortex of the AD patients compared to the healthy controls, while 18F-FDG uptake was significantly reduced in the same regions. Additionally, we discover a strong correlation between the patients' cognitive function and the PET signals of both 11C-acetate and 18F-FDG. We demonstrate the potential value of PET imaging with 11C-acetate and 18F-FDG by visualizing reactive astrogliosis and the associated neuronal glucose hypometablosim for AD patients. Our findings further suggest that the acetate-boosted reactive astrocyte-neuron interaction could contribute to the cognitive decline in AD.

Improving anticancer effect of aPD-L1 through lowering neutrophil infiltration by PLAG in tumor implanted with MB49 mouse urothelial carcinoma.

BACKGROUND: The PD-L1 antibody is an immune checkpoint inhibitor (ICI) attracting attention. The third-generation anticancer drug has been proven to be very effective due to fewer side effects and higher tumor-specific reactions than conventional anticancer drugs. However, as tumors produce additional resistance in the host immune system, the effectiveness of ICI is gradually weakening. Therefore, it is very important to develop a combination therapy that increases the anticancer effect of ICI by removing anticancer resistance factors present around the tumor. METHODS: The syngeneic model was used (n = 6) to investigate the enhanced anti-tumor effect of PD-L1 antibody with the addition of PLAG. MB49 murine urothelial cancer cells were implanted into the C57BL/6 mice subcutaneously. PLAG at different dosages (50/100 mpk) was daily administered orally for another 4 weeks with or without 5 mpk PD-L1 antibody (10F.9G2). PD-L1 antibody was delivered via IP injection once a week. RESULTS: The aPD-L1 monotherapy group inhibited tumor growth of 56% compared to the positive group, while the PLAG and aPD-L1 co-treatment inhibited by 89%. PLAG treatment effectively reduced neutrophils infiltrating localized in tumor and converted to a tumor microenvironment with anti-tumor effective T-cells. PLAG increased tumor infiltration of CD8 positive cytotoxic T-cell populations while effectively inhibiting the infiltration of neoplastic T-cells such as CD4/FoxP3. Eventually, neutrophil-induced tumor ICI resistance was resolved by restoring the neutrophil-to-lymphocyte ratio to the normal range. In addition, regulation of cytokine and chemokine factors that inhibit neutrophil infiltration and increase the killing activity of cytotoxic T cells was observed in the tumors of mice treated with PLAG + aPD-L1. CONCLUSIONS: PLAG effectively turned the tumor-promoting microenvironment into a tumor-suppressing microenvironment. As a molecule that increases the anti-tumor effectiveness of aPD-L1, PLAG has the potential to be an essential and effective ICI co-therapeutic agent.

PLAG co-treatment increases the anticancer effect of Adriamycin and cyclophosphamide in a triple-negative breast cancer xenograft mouse model.

Chemotherapy induces tumor cell death and inhibits tumor progression, but the accompanying immune responses in the surrounding dying tissue cause significant inflammation. These responses, such as excessive neutrophil infiltration into tumor tissue, are the main causes of resistance to anticancer treatment. The development of drugs that reduce neutrophil infiltration into tumors is necessary to increase the anticancer effect of chemotherapy. Here, we show that the antitumor effect of the chemotherapy AC regimen (Adriamycin and cyclophosphamide) was increased by 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) cotreatment in the MDA-MB-231 triple-negative breast cancer xenograft mouse model. Tumor growth was inhibited up to 56% in mice treated with AC and inhibited up to 94% in mice cotreated with AC and PLAG. Side effects of chemotherapy, such as a reduction in body weight, were alleviated in mice cotreated with AC and PLAG. Excessive neutrophil infiltration caused by the AC regimen was successfully cleared in mice cotreated with AC and PLAG. We conclude that PLAG inhibits excessive neutrophil infiltration that aids tumor growth. Reduced neutrophils and increased lymphocytes in PLAG-treated mice can maximize the antitumor effect of the AC regimen and inhibit tumor growth.

Suppression of tumor progression by thioredoxin-interacting protein-dependent adenosine 2B receptor degradation in a PLAG-treated Lewis lung carcinoma-1 model of non-small cell lung cancer.

Extracellular adenosine in the tumor microenvironment plays a vital role in cancer development. Specifically, activation of adenosine receptors affects tumor cell growth and adenosine release. We examined the anti-tumor efficacy of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) in animal models, revealing the role of PLAG in inhibiting tumor progression by promoting the degradation of adenosine 2B receptors (A2BRs) in tumors. PLAG induced the expression of thioredoxin-interacting protein (TXNIP), a type of α-arrestin that accelerates A2BR internalization by interacting with A2BR complexes containing ß-arrestin. Engulfed receptors bound to TXNIP were rapidly degraded after E3 ligase recruitment and ubiquitination, resulting in early termination of intracellular signals that promote tumor overgrowth. However, in control cancer cells, A2BRs bound to protein phosphatase 2A and were returned to the cell membrane instead of being degraded, resulting in continuous receptor-mediated signaling by pathways including the Raf-Erk axis, which promotes tumor proliferation. A TXNIP-silenced cell-implanted mouse model and TXNIP knockout (KO) mice were used to verify that PLAG-mediated suppression of tumor progression is dependent on TXNIP expression. Increased tumor growth was observed in TXNIP-silenced cell-implanted mice, and the anti-tumor effects of PLAG, including delayed tumor overgrowth, were greatly reduced. However, the anti-tumor effects of PLAG were observed in cancer cell-implanted TXNIP-KO mice, which indicates that PLAG produces anti-tumor effects by enhancing TXNIP expression in tumor cells. These essential functions of PLAG, including delaying tumor growth via A2BR degradation, suggest innovative directions for anticancer drug development.

PLAG alleviates cisplatin-induced cachexia in lung cancer implanted mice.

Chemotherapy-induced cachexia has been a significant challenge to the successful treatment of cancer patients. Chemotherapy leads to loss of muscle, loss of appetite, and excessive weight loss, which makes these necessary treatments intolerable for most patients. Therefore, it is necessary to alleviate cachexia to successfully treat cancer patients. In this study, tumor-implanted mouse models administered cisplatin showed rapid weight loss and reduced feeding rate by the second week of treatment, and 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) effectively alleviated cisplatin-induced cachexia. In mice treated with cisplatin on a sacrificial day after 6 weeks, the weight of the two major leg muscles (quadriceps femoris and gastrocnemius) were reduced by up to 70%, but this muscle reduction was successfully prevented in the PLAG co-treatment group. The distribution and size of muscle fibers that appear in small units in cisplatin-treated mice were restored to normal levels by PLAG co-treatment. Furthermore, myostatin expression levels were upregulated by cisplatin, whereas myostatin decreased to normal levels with muscle recovery in the PLAG co-treated group. Tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), which are commonly expressed in cachexia, were significantly increased in cisplatin-treated mice but were reduced to normal levels in PLAG co-treated mice. Glucose absorption, an indicator of muscle tissue activity, decreased with cisplatin treatment and recovered to normal levels with PLAG co-treatment. Overall, PLAG effectively alleviated cisplatin-induced cachexia symptoms and reduced tumor growth in tumor-implanted mice. These findings suggest PLAG may be a promising drug to alleviate cachexia in cancer patients receiving chemotherapy.

High-Throughput Magnetic Actuation Platform for Evaluating the Effect of Mechanical Force on 3D Tumor Microenvironment.

Accurately replicating and analyzing cellular responses to mechanical cues is vital for exploring metastatic disease progression. However, many of the existing in vitro platforms for applying mechanical stimulation seed cells on synthetic substrates. To better recapitulate physiological conditions, a novel actuating platform is developed with the ability to apply tensile strain on cells at various amplitudes and frequencies in a high-throughput multi-well culture plate using a physiologically-relevant substrate. Suspending fibrillar fibronectin across the body of the magnetic actuator provides a matrix representative of early metastasis for 3D cell culture that is not reliant on a synthetic substrate. This platform enables the culturing and analysis of various cell types in an environment that mimics the dynamic stretching of lung tissue during normal respiration. Metabolic activity, YAP activation, and morphology of breast cancer cells are analyzed within one week of cyclic stretching or static culture. Further, matrix degradation is significantly reduced in breast cancer cell lines with metastatic potential after actuation. These new findings demonstrate a clear suppressive cellular response due to cyclic stretching that has implications for a mechanical role in the dormancy and reactivation of disseminated breast cancer cells to macrometastases.

In Vitro Magnetic Techniques for Investigating Cancer Progression.

Worldwide, there are currently around 18.1 million new cancer cases and 9.6 million cancer deaths yearly. Although cancer diagnosis and treatment has improved greatly in the past several decades, a complete understanding of the complex interactions between cancer cells and the tumor microenvironment during primary tumor growth and metastatic expansion is still lacking. Several aspects of the metastatic cascade require in vitro investigation. This is because in vitro work allows for a reduced number of variables and an ability to gather real-time data of cell responses to precise stimuli, decoupling the complex environment surrounding in vivo experimentation. Breakthroughs in our understanding of cancer biology and mechanics through in vitro assays can lead to better-designed ex vivo precision medicine platforms and clinical therapeutics. Multiple techniques have been developed to imitate cancer cells in their primary or metastatic environments, such as spheroids in suspension, microfluidic systems, 3D bioprinting, and hydrogel embedding. Recently, magnetic-based in vitro platforms have been developed to improve the reproducibility of the cell geometries created, precisely move magnetized cell aggregates or fabricated scaffolding, and incorporate static or dynamic loading into the cell or its culture environment. Here, we will review the latest magnetic techniques utilized in these in vitro environments to improve our understanding of cancer cell interactions throughout the various stages of the metastatic cascade.

Adenovirus-induced Reactive Astrogliosis Exacerbates the Pathology of Parkinson's Disease.

Parkinson's disease (PD) is the most prevalent neurodegenerative motor disorder. While PD has been attributed to dopaminergic neuronal death in substantia nigra pars compacta (SNpc), accumulating lines of evidence have suggested that reactive astrogliosis is critically involved in PD pathology. These pathological changes are associated with α-synuclein aggregation, which is more prone to be induced by an A53T mutation. Therefore, the overexpression of A53T-mutated α-synuclein (A53T-α-syn) has been utilized as a popular animal model of PD. However, this animal model only shows marginal-to-moderate extents of reactive astrogliosis and astrocytic α-synuclein accumulation, while these phenomena are prominent in human PD brains. Here we show that Adeno-GFAP-GFP virus injection into SNpc causes severe reactive astrogliosis and exacerbates the A53T-α-syn-mediated PD pathology. In particular, we demonstrate that AAV-CMV-A53T-α-syn injection, when combined with Adeno-GFAP-GFP, causes more significant loss of dopaminergic neuronal tyrosine hydroxylase level and gain of astrocytic GFAP and GABA levels. Moreover, the combination of AAV-CMV-A53T-α-syn and Adeno-GFAP-GFP causes an extensive astrocytic α-syn expression, just as in human PD brains. These results are in marked contrast to previous reports that AAV-CMV-A53T-α-syn alone causes α-syn expression mostly in neurons but rarely in astrocytes. Furthermore, the combination causes a severe PD-like motor dysfunction as assessed by rotarod and cylinder tests within three weeks from the virus injection, whereas Adeno-GFAP-GFP alone or AAV-CMV-A53T-α-syn alone does not. Our findings implicate that inducing reactive astrogliosis exacerbates PD-like pathologies and propose the virus combination as an advanced strategy for developing a new animal model of PD.

Association Between Electronic Cigarette Use and Levels of High-Sensitivity C-Reactive Protein and Uric Acid.

The present study investigated whether electronic cigarette use, which is becoming increasingly common, was related to systemic inflammation that may lead to cardiovascular disease, similar to conventional cigarette smoking. The study included 1208 men (19-65 years old) who participated in the 7th Korean National Health and Nutrition Examination Survey (2016). The participants were categorized as electronic cigarette users, conventional cigarette users, and nonsmokers. Serum high-sensitivity C-reactive protein was used as an inflammatory index, and uric acid level was used as a metabolic indicator. After adjusting for confounding factors, electronic cigarette use was significantly associated with elevated serum high-sensitivity C-reactive protein levels (ß = 1.326, P = .002), uric acid levels (ß = 0.400, P = .042), and hyperuricemia (uric acid level of >7 mg/mL; odds ratio = 2.67, 95% confidence interval = 1.27-5.58). These findings suggest that electronic cigarette use may be associated with systemic inflammation markers, similar to conventional cigarette use.

Novel Method for S1 Transforaminal Epidural Steroid Injection.

BACKGROUND: S1 transforaminal epidural steroid injection (S1-TFESI) results in positive clinical outcomes for the treatment of pain associated with the S1 nerve root. S1-TFESI via the transforaminal approach is commonly performed under fluoroscopic guidance. Ultrasound guidance is an alternative to mitigate radiation exposure. However, performing spinal procedures under ultrasound guidance has some limitations in confirming the position of the needle tip and vascular uptake. New techniques are therefore needed to make ultrasound and fluoroscopy complementary. Our objective was to describe a novel technique for S1-TFESI and confirm its reproducibility. METHODS: Records of patients with S1 radiculopathy were reviewed retrospectively; those treated using the new S1-TFESI technique were selected. Initially, ultrasound was used to distinguish anatomy of the sacral foramen and guide initial placement of the needle entry point. Fluoroscopy was subsequently used to confirm needle tip position and vascular injection. The number of times the needle required reinsertion was recorded, and ultrasound and C-arm images were stored. RESULTS: Sixty-seven S1-TFESIs were performed in 56 patients. All injections exhibited epidural spread of contrast media, not only to the S1 nerve. The cephalad angle was 16.25 ± 6.75° (range, 5-27°), the oblique angle was 2.48 ± 2.62° (range, 0-7°), and the mean number of attempts was 1.24 ± 1.25. CONCLUSIONS: The new technique, involving the use of ultrasound to guide initial placement of the needle entry point, followed by confirmatory imaging and any needed adjustment with the use of fluoroscopy, can be a technique to complement the shortcomings of using ultrasound or fluoroscopy alone.

Remote real-time monitoring of subsurface landfill gas migration.

The cost of monitoring greenhouse gas emissions from landfill sites is of major concern for regulatory authorities. The current monitoring procedure is recognised as labour intensive, requiring agency inspectors to physically travel to perimeter borehole wells in rough terrain and manually measure gas concentration levels with expensive hand-held instrumentation. In this article we present a cost-effective and efficient system for remotely monitoring landfill subsurface migration of methane and carbon dioxide concentration levels. Based purely on an autonomous sensing architecture, the proposed sensing platform was capable of performing complex analytical measurements in situ and successfully communicating the data remotely to a cloud database. A web tool was developed to present the sensed data to relevant stakeholders. We report our experiences in deploying such an approach in the field over a period of approximately 16 months.

MMHD [(S,E)-2-methyl-1-(2-methylthiazol-4-yl) hexa-1,5-dien-ol], a novel synthetic compound derived from epothilone, suppresses nuclear factor-kappaB-mediated cytokine expression in lipopolysaccharide-stimulated BV-2 microglia.

The effects of MMHD [(S,E)-2-methyl-1-(2-methylthiazol-4-yl) hexa-1,5-dien-ol], a novel synthetic compound derived from epothilone, was investigated for its effects on the expression of proinflammatory mediators in lipopolysaccharide-stimulated BV-2 microglia. MMHD attenuated the expressions of inducible nitric oxide synthase and cyclooxygenase-2 mRNA and protein without affecting cell viability. Moreover, MMHD suppressed nuclear factor-kappaB (NF-kappaB) activation via the translocation of p65 into the nucleus. These results indicate that MMHD exerts anti-inflammatory properties by suppressing the transcription of proinflammatory cytokine genes through the NF-kappaB signaling pathway.

Cancer stem cell markers CD133 and CD24 correlate with invasiveness and differentiation in colorectal adenocarcinoma.

AIM: To verify that CD markers are available for detecting cancer stem cell populations and to evaluate their clinical significance in colon cancer. METHODS: Immunohistochemistry for CD133, CD24 and CD44 was performed on the tissue microarray of 523 colorectal adenocarcinomas. Medical records were reviewed and clinicopathological analysis was performed. RESULTS: In colorectal adenocarcinoma, 128 of 523 cases (24.5%) were positive and 395 cases (75.5%) were negative for CD133 expression. Two hundred and sixty-four of 523 cases (50.5%) were positive and 259 cases (49.5%) were negative for CD24 expression. Five hundred and two of 523 cases (96%) were negative and 21 cases (4%) were positive for CD44 expression. Upon clinicopathological analysis, CD133 expression was present more in male patients (P = 0.002) and in advanced T stage cancer (P = 0.024). Correlation between CD24 expression and clinicopathological factors was seen in the degree of differentiation (P = 0.006). Correlation between CD44 expression and clinicopathological factors was seen in the tumor size (P = 0.001). Survival was not significantly related to CD133, CD24 and CD44 expression. CONCLUSION: CD markers were related to invasiveness and differentiation of colorectal adenocarcinoma. However, CD expression was not closely related to survival.