Detection of γH2AX foci in mouse normal brain and brain tumor after boron neutron capture therapy

Aim

In this study, we investigated γH2AX foci as markers of DSBs in normal brain and brain tumor tissue in mouse after BNCT.

Background

Boron neutron capture therapy (BNCT) is a particle radiation therapy in combination of thermal neutron irradiation and boron compound that specifically accumulates in the tumor. ¹⁰B captures neutrons and produces an alpha (⁴He) particle and a recoiled lithium nucleus (⁷Li). These particles have the characteristics of extremely high linear energy transfer (LET) radiation and therefore have marked biological effects. High LET radiation causes severe DNA damage, DNA DSBs. As the high LET radiation induces complex DNA double strand breaks (DSBs), large proportions of DSBs are considered to remain unrepaired in comparison with exposure to sparsely ionizing radiation.

Materials and methods

We analyzed the number of γH2AX foci by immunohistochemistry 30 min or 24 h after neutron irradiation.

Results

In both normal brain and brain tumor, γH2AX foci induced by ¹⁰B(n,α)⁷Li reaction remained 24 h after neutron beam irradiation. In contrast, γH2AX foci produced by γ-ray irradiation at contaminated dose in BNCT disappeared 24 h after irradiation in these tissues.

Conclusion

DSBs produced by ¹⁰B(n,α)⁷Li reaction are supposed to be too complex to repair for cells in normal brain and brain tumor tissue within 24 h. These DSBs would be more difficult to repair than those by γ-ray. Excellent anti-tumor effect of BNCT may result from these unrepaired DSBs induced by ¹⁰B(n,α)⁷Li reaction.     

Honokiol Inhibits Non-Small Cell Lung Cancer Cell Migration by Targeting PGE2-Mediated Activation of β-Catenin Signaling

Lung cancer remains a leading cause of death due to its metastasis to distant organs. We have examined the effect of honokiol, a bioactive constituent from the Magnolia plant, on human non-small cell lung cancer (NSCLC) cell migration and the molecular mechanisms underlying this effect. Using an in vitro cell migration assay, we found that treatment of A549, H1299, H460 and H226 NSCLC cells with honokiol resulted in inhibition of migration of these cells in a dose-dependent manner, which was associated with a reduction in the levels of cyclooxygenase-2 (COX-2) and prostaglandin E₂ (PGE₂). Celecoxib, a COX-2 inhibitor, also inhibited cell migration. Honokiol inhibited PGE₂-enhanced migration of NSCLC cells, inhibited the activation of NF-κB/p65, an upstream regulator of COX-2, in A549 and H1299 cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, also inhibited migration of NSCLC cells. PGE₂ has been shown to activate β-catenin signaling, which contributes to cancer cell migration. Therefore, we checked the effect of honokiol on β-catenin signaling. It was observed that treatment of NSCLC cells with honokiol degraded cytosolic β-catenin, reduced nuclear accumulation of β-catenin and down-regulated matrix metalloproteinase (MMP)-2 and MMP-9, which are the down-stream targets of β-catenin and play a crucial role in cancer cell metastasis. Honokiol enhanced: (i) the levels of casein kinase-1α, glycogen synthase kinase-3β, and (ii) phosphorylation of β-catenin on critical residues Ser⁴⁵, Ser^33/37 and Thr⁴¹. These events play important roles in degradation or inactivation of β-catenin. Treatment of celecoxib also reduced nuclear accumulation of β-catenin in NSCLC cells. FH535, an inhibitor of Wnt/β-catenin pathway, inhibited PGE₂-enhanced cell migration of A549 and H1299 cells. These results indicate that honokiol inhibits non-small cell lung cancer cells migration by targeting PGE₂-mediated activation of β-catenin signaling.     

Effect of Hyperoxia on Retinoid Metabolism and Retinoid Receptor Expression in the Lungs of Newborn Mice

Background

Preterm newborns that receive oxygen therapy often develop bronchopulmonary dysplasia (BPD), which is abnormal lung development characterized by impaired alveologenesis. Oxygen-mediated injury is thought to disrupt normal lung growth and development. However, the mechanism of hyperoxia-induced BPD has not been extensively investigated. We established a neonatal mouse model to investigate the effects of normobaric hyperoxia on retinoid metabolism and retinoid receptor expression.

Methods

Newborn mice were exposed to hyperoxic or normoxic conditions for 15 days. The concentration of retinol and retinyl palmitate in the lung was measured by HPLC to gauge retinoid metabolism. Retinoid receptor mRNA levels were assessed by real-time PCR. Proliferation and retinoid receptor expression in A549 cells were assessed in the presence and absence of exogenous vitamin A.

Results

Hyperoxia significantly reduced the body and lung weight of neonatal mice. Hyperoxia also downregulated expression of RARα, RARγ, and RXRγ in the lungs of neonatal mice. In vitro, hyperoxia inhibited proliferation and expression of retinoid receptors in A549 cells.

Conclusion

Hyperoxia disrupted retinoid receptor expression in neonatal mice.     

Characterization of Stimulus-Secretion Coupling in the Human Pancreatic EndoC-βH1 Beta Cell Line

Aims/Hypothesis

Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.

Methods

Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay), gene expression (Gene Chip array), metabolite levels (GC/MS), respiration (Seahorse XF24 Extracellular Flux Analyzer), glucose utilization (radiometric), lactate release (enzymatic colorimetric), ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca²⁺ responses (microfluorometry) were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.

Results

Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.

Conclusions/Interpretation

Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca²⁺ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the advantage of carrying the human genome, allowing studies of human genetic variants, epigenetics and regulatory RNA molecules.     

Regulation of Autophagy Via PERK-eIF2α Effectively Relieve the Radiation Myelitis Induced by Iodine-125

Radiation myelitis is the most serious complication in clinical radiotherapy for spinal metastases. We previously showed that ¹²⁵I brachytherapy induced apoptosis of spinal cord neurons accompanied by autophagy. In this study, we further investigated the mechanism by which ¹²⁵I radiation triggered autophagy in neural cells. We found that autophagy induced by ¹²⁵I radiation was involved in endoplasmic reticulum (ER) stress and mainly dependent on PERK-eIF2α pathway. The expressions of LC3II, ATG12 and PI3K were significantly suppressed in PERK knockout neural cells. Meanwhile, the expressions of phosphorylated-Akt s473 and caspase3/8 all significantly increased in neural cells transfected with a PERK siRNA and which enhanced apoptosis of neurons after ¹²⁵I radiation. The results were consistent with that by MTT and Annexin-FITC/PT staining. In annimal model of banna pigs with radiation myelitis caused by ¹²⁵I brachytherapy, we have successfully decreased PERK expression by intrathecal administration of the lentivirus vector. The apoptosis rate was significantly higher than that in control group and which deteriorated radiation myelitis of banna pigs. Thus, autophagy caused by ¹²⁵I radiation was mainly as an attempt of cell survival at an early stage, but it would be a self-destructive process and promoted the process of apoptosis and necrosis radiated by ¹²⁵I for more than 72 hours. The study would be useful and helpful to maximize efficiency of radiation therapy in clinical therapy.     

Left Atrial Appendage Closure Guided by Integrated Echocardiography and Fluoroscopy Imaging Reduces Radiation Exposure

Aims

To investigate whether percutaneous left atrial appendage (LAA) closure guided by automated real-time integration of 2D-/3D-transesophageal echocardiography (TEE) and fluoroscopy imaging results in decreased radiation exposure.

Methods and Results

In this open-label single-center study LAA closure (Amplatzer^TM Cardiac Plug) was performed in 34 consecutive patients (8 women; 73.1±8.5 years) with (n = 17, EN+) or without (n = 17, EN-) integrated echocardiography/fluoroscopy imaging guidance (EchoNavigator^® [EN]; Philips Healthcare). There were no significant differences in baseline characteristics between both groups. Successful LAA closure was documented in all patients. Radiation dose was reduced in the EN+ group about 52% (EN+: 48.5±30.7 vs. EN-: 93.9±64.4 Gy/cm²; p = 0.01). Corresponding to the radiation dose fluoroscopy time was reduced (EN+: 16.7±7 vs. EN-: 24.0±11.4 min; p = 0.035). These advantages were not at the cost of increased procedure time (89.6±28.8 vs. 90.1±30.2 min; p = 0.96) or periprocedural complications. Contrast media amount was comparable between both groups (172.3±92.7 vs. 197.5±127.8 ml; p = 0.53). During short-term follow-up of at least 3 months (mean: 8.1±5.9 months) no device-related events occurred.

Conclusions

Automated real-time integration of echocardiography and fluoroscopy can be incorporated into procedural work-flow of percutaneous left atrial appendage closure without prolonging procedure time. This approach results in a relevant reduction of radiation exposure.

Trial Registration

ClinicalTrials.gov NCT01262508     

Bystander effects induced by direct and scattered radiation generated during penetration of medium inside a water phantom

Background

The biological effects of ionizing radiation have long been thought to results from direct targeting of the nucleus leading to DNA damage. Over the years, a number of non-targeted or epigenetic effects of radiation exposure have been reported where genetic damage occurs in cells that are not directly irradiated but respond to signals transmitted from irradiated cells, a phenomenon termed the “bystander effects”.

Aim

We compared the direct and bystander responses of human A 549, BEAS-2-B and NHDF cell lines exposed to both photon (6 MV) and electron (22 MeV) radiation inside a water phantom. The cultures were directly irradiated or exposed to scattered radiation 4 cm outside the field. In parallel, non-irradiated cells (termed bystander cells) were incubated in ICM (irradiation conditioned medium) collected from another pool of irradiated cells (termed donor cells).

Materials and methods

In directly irradiated cells as well as ICM-treated cells, the frequency of micronuclei and condensation of chromatin characteristic for the apoptotic process were estimated using the cytokinesis-block micronucleus test.

Results

In all tested cell lines, radiation induced apoptosis and formation of micronuclei. A549 and BEAS-2B cells cultured in ICM showed increased levels of micronuclei and apoptosis, whereas normal human fibroblasts (NHDF line) were resistant to bystander response. In A549 and BEAS-2B cells placed outside the radiation field and exposed to scattered radiation the formation of micronuclei and induction of apoptosis were similar to that after ICM-treatment.

Conclusion

Results suggest that the genetic damage in cells exposed to scattered radiation is caused by factors released by irradiated cells into the medium rather than by DNA damage induced directly by X rays. It seems that bystander effects may have important clinical implications for health risk after low level radiation exposure of cells lying outside the radiation field during clinical treatment.     

Increased γ-H2A.X Intensity in Response to Chronic Medium-Dose-Rate γ-Ray Irradiation

Background

The molecular mechanisms of DNA repair following chronic medium-dose-rate (MDR) γ-ray-induced damage remain largely unknown.

Methodology/Principal Findings

We used a cell function imager to quantitatively measure the fluorescence intensity of γ-H2A.X foci in MDR (0.015 Gy/h and 0.06 Gy/h) or high-dose-rate (HDR) (54 Gy/h) γ-ray irradiated embryonic fibroblasts derived from DNA-dependent protein kinase mutated mice (scid/scid mouse embryonic fibroblasts (scid/scid MEFs)). The obtained results are as follows: (1) Automatic measurement of the intensity of radiation-induced γ-H2A.X foci by the cell function imager provides more accurate results compared to manual counting of γ-H2A.X foci. (2) In high-dose-rate (HDR) irradiation, γ-H2A.X foci with high fluorescence intensity were observed at 1 h after irradiation in both scid/scid and wild-type MEFs. These foci were gradually reduced through de-phosphorylation at 24 h or 72 h after irradiation. Furthermore, the fluorescence intensity at 24 h increased to a significantly greater extent in scid/scid MEFs than in wild-type MEFs in the G₁ phase, although no significant difference was observed in G₂/M-phase MEFs, suggesting that DNA-PKcs might be associated with non-homologous-end-joining-dependent DNA repair in the G₁ phase following HDR γ-ray irradiation. (3) The intensity of γ-H2A.X foci for continuous MDR (0.06 Gy/h and 0.015 Gy/h) irradiation increased significantly and in a dose-dependent fashion. Furthermore, unlike HDR-irradiated scid/scid MEFs, the intensity of γ-H2A.X foci in MDR-irradiated scid/scid MEFs showed no significant increase in the G₁ phase at 24 h, indicating that DNA repair systems using proteins other than DNA-PKcs might induce cell functioning that are subjected to MDR γ-ray irradiation.

Conclusions

Our results indicate that the mechanism of phosphorylation or de-phosphorylation of γ-H2A.X foci induced by chronic MDR γ-ray irradiation might be different from those induced by HDR γ-ray irradiation.     

Oxygen and Hydrogen Stable Isotope Ratios of Bulk Needles Reveal the Geographic Origin of Norway Spruce in the European Alps

Background

Tracking timber is necessary in order to prevent illegal logging and protect local timber production, but there is as yet no suitable analytical traceability method. Stable isotope ratios in plants are known to reflect geographical variations. In this study we analysed four stable isotope ratios in order to develop a model able to identify the geographic origin of Norway spruce in the European Alps.

Methodology and Principal Findings

δ¹⁸O, δ²H, δ¹³C and δ¹⁵N were measured in bulk needles of Picea abies sampled in 20 sites in and around the European Alps. Environmental and spatial variables were found to be related to the measured isotope ratios. An ordinary least squares regression was used to identify the most important factor in stable isotope variability in bulk needles. Spatial autocorrelation was tested for all isotope ratios by means of Moran’s I. δ¹⁸O, δ²H and δ¹⁵N values differed significantly between sites. Distance from the coast had the greatest influence on δ²H, while latitude and longitude were strongly related to δ¹⁸O. δ¹³C values did not appear to have any relationship with geographical position, while δ¹⁵N values were influenced by distance from the motorway. The regression model improved the explanatory power of the spatial and environmental variables. Positive spatial autocorrelations were found for δ¹⁸O and δ²H values.

Conclusions

The δ ¹⁸O, δ²H and δ¹⁵N values in P. abies bulk needles are a suitable proxy to identify geographic origin as they vary according to geographical position. Although the regression model showed the explanatory variables to have significant power and stability, we conclude that our model might be improved by multivariate spatial interpolation of the δ ¹⁸O and δ²H values.     

Non-small cell lung carcinoma cell motility, rac activation and metastatic dissemination are mediated by protein kinase C epsilon

Background

Protein kinase C (PKC) ε, a key signaling transducer implicated in mitogenesis, survival, and cancer progression, is overexpressed in human primary non-small cell lung cancer (NSCLC). The role of PKCε in lung cancer metastasis has not yet been established.

Principal Findings

Here we show that RNAi-mediated knockdown of PKCε in H358, H1299, H322, and A549 NSCLC impairs activation of the small GTPase Rac1 in response to phorbol 12-myristate 13-acetate (PMA), serum, or epidermal growth factor (EGF). PKCε depletion markedly impaired the ability of NSCLC cells to form membrane ruffles and migrate. Similar results were observed by pharmacological inhibition of PKCε with εV1-2, a specific PKCε inhibitor. PKCε was also required for invasiveness of NSCLC cells and modulated the secretion of extracellular matrix proteases and protease inhibitors. Finally, we found that PKCε-depleted NSCLC cells fail to disseminate to lungs in a mouse model of metastasis.

Conclusions

Our results implicate PKCε as a key mediator of Rac signaling and motility of lung cancer cells, highlighting its potential as a therapeutic target.     

An Exploratory Study on 99mTc-RGD-BBN Peptide Scintimammography in the Assessment of Breast Malignant Lesions Compared to 99mTc-3P4-RGD2

Purpose

This study aimed to explore the diagnostic performance of single photon emission computed tomography / computerized tomography (SPECT/CT) using a new radiotracer ^99mTc-RGD-BBN for breast malignant tumor compared with ^99mTc-3P4-RGD₂.

Methods

6 female patients with breast malignant tumors diagnosed by fine needle aspiration cytology biopsy (FNAB) who were scheduled to undergo surgery were included in the study. ^99mTc-3P4-RGD₂ and ^99mTc-RGD-BBN were performed with single photon emission computed tomography (SPECT) at 1 hour after intravenous injection of 299 ± 30 MBq and 293 ± 32 MBq of radiotracers respectively at separate day. The results were evaluated by the Tumor to non-Tumor ratios (T/NT). ^99mTc-RGD-BBN and ^99mTc-3P4-RGD₂ SPECT/CT images were interpreted independently by 3 experienced nuclear medicine physicians using a 3-point scale system. All of the samples were analyzed immunohistochemically to evaluate the integrin αvβ3 and gastrin-releasing peptide receptor (GRPR) expression. The safety, biodistribution and radiation dosimetry of ^99mTc-RGD-BBN were also evaluated in the healthy volunteers.

Results

No serious adverse events were reported in any of the patients during the study. The effective radiation dose entirely conformed to the relevant standards. A total of 6 palpable malignant lesions were detected using ^99mTc-RGD-BBN SPECT/CT with clear uptake. All malignant lesions were also detected using ^99mTc-3P4-RGD₂ SPECT/CT. The results showed that five malignant lesions were with clear uptake and the other one with barely an uptake. 4 malignant cases were found with both αvβ3 and GRPR expression, 1 case with only GRPR positive expression (integrin αvβ3 negative) and 1 case with only integrin αvβ3 positive expression (GRPR negative).

Conclusion

^99mTc-RGD-BBN is a safe agent for detecting breast cancer. ^99mTc-RGD-BBN may have the potential to make up for the deficiency of ^99mTc-3P4-RGD₂ in the detection of breast cancer with only GRPR positive expression (integrin α_vβ₃ negative). The preliminary application of ^99mTc-RGD-BBN has demonstrated its powerful potential in breast cancer diagnosis and therapy.     

Clara cell 10-kDa protein gene transfection inhibits NF-κB activity in airway epithelial cells

Background

Clara cell 10-kDa protein (CC10) is a multifunctional protein with anti-inflammatory and immunomodulatory effects. Induction of CC10 expression by gene transfection may possess potential therapeutic effect. Nuclear factor κB (NF-κB) plays a key role in the inflammatory processes of airway diseases.

Method/Results

To investigate potential therapeutic effect of CC10 gene transfection in controlling airway inflammation and the underlying intracellular mechanisms, in this study, we constructed CC10 plasmid and transfected it into bronchial epithelial cell line BEAS-2B cells and CC10 knockout mice. In BEAS-2B cells, CC10''s effect on interleukin (IL)-1β induced IL-8 expression was explored by means of RT-PCR and ELISA and its effect on NF-κB classical signaling pathway was studied by luciferase reporter, western blot, and immunoprecipitation assay. The effect of endogenous CC10 on IL-1β evoked IL-8 expression was studied by means of nasal explant culture. In mice, CC10''s effect on IL-1β induced IL-8 and nuclear p65 expression was examined by immunohistochemistry. First, we found that the CC10 gene transfer could inhibit IL-1β induced IL-8 expression in BEAS-2B cells. Furthermore, we found that CC10 repressed IL-1β induced NF-κB activation by inhibiting the phosphorylation of IκB-α but not IκB kinase-α/β in BEAS-2B cells. Nevertheless, we did not observe a direct interaction between CC10 and p65 subunit in BEAS-2B cells. In nasal explant culture, we found that IL-1β induced IL-8 expression was inversely correlated with CC10 levels in human sinonasal mucosa. In vivo study revealed that CC10 gene transfer could attenuate the increase of IL-8 and nuclear p65 staining in nasal epithelial cells in CC10 knockout mice evoked by IL-1β administration.

Conclusion

These results indicate that CC10 gene transfer may inhibit airway inflammation through suppressing the activation of NF-κB, which may provide us a new consideration in the therapy of airway inflammation.     

In Vivo Differences between Two Optical Isomers of Radioiodinated o-iodo-trans-decalinvesamicol for Use as a Radioligand for the Vesicular Acetylcholine Transporter

Purpose

To develop a superior VAChT imaging probe for SPECT, radiolabeled (-)-OIDV and (+)-OIDV were isolated and investigated for differences in their binding affinity and selectivity to VAChT, as well as their in vivo activities.

Procedures

Radioiodinated o-iodo-trans-decalinvesamicol ([¹²⁵I]OIDV) has a high binding affinity for vesicular acetylcholine transporter (VAChT) both in vitro and in vivo. Racemic [¹²⁵I]OIDV was separated into its two optical isomers (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV by HPLC. To investigate VAChT binding affinity (Ki) of two OIDV isomers, in vitro binding assays were performed. In vivo biodistribution study of each [¹²⁵I]OIDV isomer in blood, brain regions and major organs of rats was performed at 2,30 and 60 min post-injection. In vivo blocking study were performed to reveal the binding selectivity of two [¹²⁵I]OIDV isomers to VAChT in vivo. Ex vivo autoradiography were performed to reveal the regional brain distribution of two [¹²⁵I]OIDV isomers and (-)-[¹²³I]OIDV for SPECT at 60 min postinjection.

Results

VAChT binding affinity (Ki) of (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV was 22.1 nM and 79.0 nM, respectively. At 2 min post-injection, accumulation of (-)-[¹²⁵I]OIDV was the same as that of (+)-[¹²⁵I]OIDV. However, (+)-[¹²⁵I]OIDV clearance from the brain was faster than (-)-[¹²⁵I]OIDV. At 30 min post-injection, accumulation of (-)-[¹²⁵I]OIDV (0.62 ± 0.10%ID/g) was higher than (+)-[¹²⁵I]OIDV (0.46 ± 0.07%ID/g) in the cortex. Inhibition of OIDV binding showed that (-)-[¹²⁵I]OIDV was selectively accumulated in regions known to express VAChT in the rat brain, and ex vivo autoradiography further confirmed these results showing similar accumulation of (-)-[¹²⁵I]OIDV in these regions. Furthermore, (-)-[¹²³I]OIDV for SPECT showed the same regional brain distribution as (-)-[¹²⁵I]OIDV.

Conclusion

These results suggest that radioiodinated (-)-OIDV may be a potentially useful tool for studying presynaptic cholinergic neurons in the brain.     

Activation of Dll4/Notch Signaling and Hypoxia-Inducible Factor-1 Alpha Facilitates Lymphangiogenesis in Lacrimal Glands in Dry Eye

Purpose

By using hypoxia-inducible factor-1 alpha conditional knockout (HIF-1α CKO) mice and a dry eye (DE) mouse model, we aimed to determine the role played by delta-like ligand 4 (Dll4)/Notch signaling and HIF-1α in the lymphangiogenesis of lacrimal glands (LGs).

Methods

C57BL/6 mice were housed in a controlled-environment chamber for DE induction. During DE induction, the expression level of Dll4/Notch signaling and lymphangiogenesis in LGs was measured by quantitative RT-PCR, immunoblot, and immunofluorescence staining. Next, lymphangiogenesis was measured after Dll4/Notch signal inhibition by anti-Dll4 antibody or γ-secretase inhibitor. Using HIF-1α CKO mice, the expression of Dll4/Notch signaling and lymphangiogenesis in LGs of DE-induced HIF-1α CKO mice were assessed. Additionally, the infiltration of CD45⁺ cells in LGs was assessed by immunohistochemical (IHC) staining and flow cytometry for each condition.

Results

DE significantly upregulated Dll4/Notch and lymphangiogenesis in LGs. Inhibition of Dll4/Notch significantly suppressed lymphangiogenesis in LGs. Compared to wild-type (WT) mice, DE induced HIF-1α CKO mice showed markedly low levels of Dll4/Notch and lymphangiogenesis. Inhibition of lymphangiogenesis by Dll4/Notch suppression resulted in increased CD45⁺ cell infiltration in LGs. Likewise, CD45⁺ cells infiltrated more in the LGs of HIF-1α CKO DE mice than in non-DE HIF-1α CKO mice.

Conclusions

Dll4/Notch signaling and HIF-1α are closely related to lymphangiogenesis in DE-induced LGs. Lymphangiogenesis stimulated by Dll4/Notch and HIF-1α may play a role in protecting LGs from DE-induced inflammation by aiding the clearance of immune cells from LGs.     

Norepinephrine and Epinephrine Enhanced the Infectivity of Enterovirus 71

Background

Enterovirus 71 (EV71) infections may be associated with neurological complications, including brainstem encephalitis (BE). Severe EV71 BE may be complicated with autonomic nervous system (ANS) dysregulation and/or pulmonary edema (PE). ANS dysregulation is related to the overactivation of the sympathetic nervous system, which results from catecholamine release.

Objective

The aims of this study were to explore the effects of catecholamines on severe EV71 infection and to investigate the changes in the percentages of EV71-infected cells, virus titer, and cytokine production on the involvement of catecholamines.

Study Design

Plasma levels of norepinephrine (NE) and epinephrine (EP) in EV71-infected patients were measured using an enzyme-linked immunoassay. The expression of adrenergic receptors (ADRs) on RD, A549, SK-N-SH, THP-1, Jurkat and human peripheral blood mononuclear cells (hPBMCs) were detected using flow cytometry. The percentages of EV71-infected cells, virus titer, and cytokine production were investigated after treatment with NE and EP.

Results

The plasma levels of NE and EP were significantly higher in EV71-infected patients with ANS dysregulation and PE than in controls. Both α_1A- and β₂-ADRs were expressed on A549, RD, SK-N-SH, HL-60, THP-1, Jurkat cells and hPBMCs. NE treatment elevated the percentages of EV71-infected cells to 62.9% and 22.7% in THP-1 and Jurkat cells, respectively. Via treatment with EP, the percentages of EV71-infected cells were increased to 64.6% and 26.9% in THP-1 and Jurkat cells. The percentage of EV71-infected cells increased upon NE or EP treatment while the α- and β-blockers reduced the percentages of EV71-infected cells with NE or EP treatment. At least two-fold increase in virus titer was observed in EV71-infected A549, SK-N-SH and hPBMCs after treatment with NE or EP. IL-6 production was enhanced in EV71-infected hPBMCs at a concentration of 10² pg/mL NE.

Conclusion

The plasma levels of NE and EP elevated in EV71-infected patients with ANS dysregulation and PE. Both NE and EP enhanced the percentages of infected cells and virus titers in EV71 infection in vitro. NE and EP may play a role in the pathogenesis of EV71 BE complicated with ANS dysregulation and PE.     

Dynamic diagnosis of familial prion diseases supports the β2-α2 loop as a universal interference target

Background

Mutations in the cellular prion protein associated to familial prion disorders severely increase the likelihood of its misfolding into pathogenic conformers. Despite their postulation as incompatible elements with the native fold, these mutations rarely modify the native state structure. However they variably have impact on the thermodynamic stability and metabolism of PrP^C and on the properties of PrP^Sc aggregates. To investigate whether the pathogenic mutations affect the dynamic properties of the HuPrP(125-229) α-fold and find possible common patterns of effects that could help in prophylaxis we performed a dynamic diagnosis of ten point substitutions.

Methodology/Principal Findings

Using all-atom molecular dynamics simulations and novel analytical tools we have explored the effect of D178N, V180I, T183A, T188K, E196K, F198S, E200K, R208H, V210I and E211Q mutations on the dynamics of HuPrP(125-228) α-fold. We have found that while preserving the native state, all mutations produce dynamic changes which perturb the coordination of the α2-α3 hairpin to the rest of the molecule and cause the reorganization of the patches for intermolecular recognition, as the disappearance of those for conversion inhibitors and the emergence of an interaction site at the β2-α2 loop region.

Conclusions/Significance

Our results suggest that pathogenic mutations share a common pattern of dynamical alterations that converge to the conversion of the β2-α2 loop into an interacting region that can be used as target for interference treatments in genetic diseases.     

In vivo Molecular Imaging and Radionuclide (131I) Therapy of Human Nasopharyngeal Carcinoma Cells Transfected with a Lentivirus Expressing Sodium Iodide Symporter

Introduction

Despite recent improvements in the survival rates for nasopharyngeal carcinoma (NPC), novel treatment strategies are required to improve distant metastasis-free survival. The sodium iodine symporter (NIS) gene has been applied for in vivo imaging and cancer therapy. In this study, we examined the potential of NIS gene therapy as a therapeutic approach in NPC by performing non-invasive imaging using ¹²⁵I and ¹³¹I therapy in vivo.

Methods

We constructed a lentiviral vector expressing NIS and enhanced green fluorescent protein (EGFP) under the control of the human elongation factor-1α (EF1α) promoter, and stably transfected the vector into CNE-2Z NPC cells to create CNE-2Z-NIS cells. CNE-2Z and CNE-2Z-NIS tumor xenografts were established in nude mice; ¹²⁵I uptake, accumulation and efflux were measured using micro-SPECT/CT imaging; the therapeutic effects of treatment with ¹³¹I were assessed over 25 days by measuring tumor volume and immunohistochemical staining of the excised tumors.

Results

qPCR, immunofluorescence and Western blotting confirmed that CNE-2Z-NIS cells expressed high levels of NIS mRNA and protein. CNE-2Z-NIS cells and xenografts took up and accumulated significantly more ¹²⁵I than CNE-2Z cells and xenografts. In vitro, ¹³¹I significantly reduced the clonogenic survival of CNE-2Z-NIS cells. In vivo, ¹³¹I effectively inhibited the growth of CNE-2Z-NIS xenografts. At the end of ¹³¹I therapy, CNE-2Z-NIS xenograft tumor cells expressed higher levels of NIS and caspase-3 and lower levels of Ki-67.

Conclusion

Lentiviruses effectively delivered and mediated long-lasting expression of NIS in CNE-2Z cells which enabled uptake and accumulation of radioisotopes and provided a significant therapeutic effect in an in vivo model of NPC. NIS-mediated radioiodine treatment merits further investigation as a potentially effective, low toxicity therapeutic strategy for NPC.