DICAM in the Extracellular Vesicles from Astrocytes Attenuates Microglia Activation and Neuroinflammation.

Cross-talk between astrocytes and microglia plays an important role in neuroinflammation and central sensitization, but the manner in which glial cells interact remains less well-understood. Herein, we investigated the role of dual immunoglobulin domain-containing cell adhesion molecules (DICAM) in the glial cell interaction during neuroinflammation. DICAM knockout (KO) mice revealed enhanced nociceptive behaviors and glial cell activation of the tibia fracture with a cast immobilization model of complex regional pain syndrome (CRPS). DICAM was selectively secreted in reactive astrocytes, mainly via extracellular vesicles (EVs), and contributed to the regulation of neuroinflammation through the M2 polarization of microglia, which is dependent on the suppression of p38 MAPK signaling. In conclusion, DICAM secreted from reactive astrocytes through EVs was involved in the suppression of microglia activation and subsequent attenuation of neuroinflammation during central sensitization.

Continuous administration of mirabegron has advantages in inhibition of central sensitization compared with short-term treatment cessation in a mouse model of overactive bladder.

AIMS: There is no clear pathophysiologic evidence determining how long overactive bladder (OAB) medication should be continued. We, therefore, investigated the effect of mirabegron using cessation (CES) or continuation (CON) treatment in an OAB animal model. METHODS: Female C57BL/6 mice were divided into four groups (N = 8 each): Sham, OAB, CES, and CON groups. The OAB-like condition was induced by three times weekly intravesical instillations of KCl mixture with hyaluronidase. After the last intravesical instillation for inducing OAB, mirabegron (2 mg/kg/day) was administered in CES and CON groups for 10 and 20 days, respectively. Final experiments were carried out on 20 days from the last intravesical instillation in all groups. After cystometry, mRNA levels of bladder muscarinic, ß-adrenergic, and P2X purinergic receptors were measured to investigate bladder efferent and afferent activity. In addition, mRNA levels of CCL2 and CCR2 in L6-S1 dorsal root ganglia (DRG) were measured to assess afferent sensitization. Immunofluorescent staining of CX3CR1, GFAP, and CCR2 in the L6 spinal cord was also conducted to investigate glial activation and central sensitization. RESULTS: OAB mice showed bladder overactivity evidenced by decreased intercontraction interval (3.56 ± 0.51 vs. 5.76 ± 0.95 min in sham mice), increased non-voiding contractions (0.39 ± 0.11 vs. 0.13 ± 0.07/min in sham mice), and inefficient voiding (72.1 ± 8.6% vs. 87.1 ± 9.5% in sham mice). Increased M2, M3, ß2, ß3, P2X2 , P2X3 , P2X4 , and P2X7 levels in the bladder and increased CCL2 and CCR2 in DRG indicate bladder efferent and afferent hyperexcitability. In addition, CX3CR1, GFAP, and CCR2 in the L6 spinal cord were upregulated in OAB mice. However, the CON group exhibited reduced ß2, ß3, P2X2 , P2X3 , P2X4 , and P2X7 levels in the bladder, reduced CCL2 and CCR2 in DRG, which are markers of afferent hyperexcitability, and reduced immunoreactivities of CX3CR1, GFAP, and CCR2 in the L6 spinal cord, which are markers of the central sensitization. Moreover, the CON group showed better improvements in nonvoiding contractions (0.16 ± 0.09 vs. 0.44 ± 0.17/min) and voiding efficiency (93.9 ± 7.4% vs. 76.5 ± 13.1%) and reductions in bladder ß3 receptors and CCL2 of L6-S1 DRG, and immunoreactivities of CX3CR1 and GFAP in the L6 spinal cord compared to the CES group. CONCLUSIONS: Continuous mirabegron treatment seems to prevent central sensitization and, thus, might be desirable for long-term disease control of OAB.

Longitudinal Monitoring of Intra-Tumoural Heterogeneity Using Optical Barcoding of Patient-Derived Colorectal Tumour Models.

Geno- and phenotypic heterogeneity amongst cancer cell subpopulations are established drivers of treatment resistance and tumour recurrence. However, due to the technical difficulty associated with studying such intra-tumoural heterogeneity, this phenomenon is seldom interrogated in conventional cell culture models. Here, we employ a fluorescent lineage technique termed "optical barcoding" (OBC) to perform simultaneous longitudinal tracking of spatio-temporal fate in 64 patient-derived colorectal cancer subclones. To do so, patient-derived cancer cell lines and organoids were labelled with discrete combinations of reporter constructs, stably integrated into the genome and thus passed on from the founder cell to all its clonal descendants. This strategy enables the longitudinal monitoring of individual cell lineages based upon their unique optical barcodes. By designing a novel panel of six fluorescent proteins, the maximum theoretical subpopulation resolution of 64 discriminable subpopulations was achieved, greatly improving throughput compared with previous studies. We demonstrate that all subpopulations can be purified from complex clonal mixtures via flow cytometry, permitting the downstream isolation and analysis of any lineages of interest. Moreover, we outline an optimized imaging protocol that can be used to image optical barcodes in real-time, allowing for clonal dynamics to be resolved in live cells. In contrast with the limited intra-tumour heterogeneity observed in conventional 2D cell lines, the OBC technique was successfully used to quantify dynamic clonal expansions and contractions in 3D patient-derived organoids, which were previously demonstrated to better recapitulate the heterogeneity of their parental tumour material. In summary, we present OBC as a user-friendly, inexpensive, and high-throughput technique for monitoring intra-tumoural heterogeneity in in vitro cell culture models.

Coagulation factor-XII induces interleukin-6 by primary lung fibroblasts: a role in idiopathic pulmonary fibrosis?

The mechanisms driving idiopathic pulmonary fibrosis (IPF) remain undefined, however it is postulated that coagulation imbalances may play a role. The impact of blood-derived clotting factors, including factor XII (FXII) has not been investigated in the context of IPF. Plasma levels of FXII were measured by ELISA in patients with IPF and in age-matched healthy donors. Expression of FXII in human lung tissue was quantified using multiplex immunohistochemistry and Western blotting. Mechanistic investigation of FXII activity was assessed in vitro on primary lung fibroblasts using qPCR and specific receptor/FXII inhibition. The functional outcome of FXII on fibroblast migration was examined by high-content image analysis. Compared with 35 healthy donors, plasma levels of FXII were not higher in patients with IPF (n = 27, P > 0.05). Tissue FXII was elevated in IPF (n = 11) and increased numbers of FXII+ cells were found in IPF (n = 8) lung tissue compared with nondiseased controls (n = 6, P < 0.0001). Activated FXII induced IL6 mRNA and IL-6 protein in fibroblasts that was blocked by anti-FXII antibody, CSL312. FXII induced IL-6 production via PAR-1 and NF-κB. FXII induced migration of fibroblasts in a concentration-dependent manner. FXII is normally confined to the circulation but it leaks from damaged vessels into the lung interstitium in IPF where it 1) induces IL-6 production and 2) enhances migration of resident fibroblasts, critical events that drive chronic inflammation and therefore, contribute to fibrotic disease progression. Targeting FXII-induced fibroblastic processes in IPF may ameliorate pulmonary fibrosis.

The Cysteine-Containing Cell-Penetrating Peptide AP Enables Efficient Macromolecule Delivery to T Cells and Controls Autoimmune Encephalomyelitis.

T cells are key immune cells involved in the pathogenesis of several diseases, rendering them important therapeutic targets. Although drug delivery to T cells is the subject of continuous research, it remains challenging to deliver drugs to primary T cells. Here, we used a peptide-based drug delivery system, AP, which was previously developed as a transdermal delivery peptide, to modulate T cell function. We first identified that AP-conjugated enhanced green fluorescent protein (EGFP) was efficiently delivered to non-phagocytic human T cells. We also confirmed that a nine-amino acid sequence with one cysteine residue was the optimal sequence for protein delivery to T cells. Next, we identified the biodistribution of AP-dTomato protein in vivo after systemic administration, and transduced it to various tissues, such as the spleen, liver, intestines, and even to the brain across the blood-brain barrier. Next, to confirm AP-based T cell regulation, we synthesized the AP-conjugated cytoplasmic domain of CTLA-4, AP-ctCTLA-4 peptide. AP-ctCTLA-4 reduced IL-17A expression under Th17 differentiation conditions in vitro and ameliorated experimental autoimmune encephalomyelitis, with decreased numbers of pathogenic IL-17A+GM-CSF+ CD4 T cells. These results collectively suggest the AP peptide can be used for the successful intracellular regulation of T cell function, especially in the CNS.

Antifibrosis treatment by inhibition of VEGF, FGF, and PDGF receptors improves bladder wall remodeling and detrusor overactivity in association with modulation of C-fiber afferent activity in mice with spinal cord injury.

AIMS: Spinal cord injury (SCI) above the sacral level causes bladder dysfunction and remodeling with fibrosis. This study examined the antifibrotic effects using nintedanib, an inhibitor of vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor receptors, on detrusor overactivity (DO) and bladder fibrosis, as well as the modulation mechanisms of C-fiber afferent pathways. METHODS: Thirty female C57BL/6 mice were divided into group A (spinal intact), group B (SCI with vehicle), and group C (SCI with nintedanib). At 2 weeks after SCI, vehicle or 50 mg/kg nintedanib was administered subcutaneously for 2 weeks. Then, cystometry was conducted, followed by RT-PCR measurements of fibrosis-related molecules, muscarinic, ß-adrenergic, TRP and purinergic receptors in the bladder or L6-S1 dorsal root ganglia (DRG). Trichrome stain and Western blot analysis of transforming growth factor-beta and fibronectin were performed in the bladder. TRPV1 expression in L6 DRG was measured by immunohistochemistry. RESULTS: In cystometry, intercontraction intervals, nonvoiding contractions, voided volume, and voiding efficiency were significantly improved in group C versus group B. RT-PCR, Western blotting, and trichrome staining revealed the fibrotic changes in the bladder of group B, which was improved in group C. Increased messenger RNA levels of TRPV1, TRPA1, P2X2 , and P2X3 in DRG of group B were significantly decreased in group C. TRPV1 immunoreactivity in DRG was increased in group B, but decreased in group C. CONCLUSIONS: Nintedanib improves storage and voiding dysfunctions and bladder fibrosis in SCI mice. Also, nintedanib-induced improvement of DO is associated with reduced expression of C-fiber afferent markers, suggesting the modulation of bladder C-fiber afferent activity.

Antioxidant Analogue 6-Amino-2,4,5-Trimethylpyridin-3-ol Ameliorates Experimental Colitis in Mice.

BACKGROUND: Oxidative stress has been suggested to be a factor contributing to the disease severity of inflammatory bowel disease (IBD). BJ-1108, a derivative of 6-amino-2,4,5-trimethylpyridin-3-ol, is reported to significantly inhibit the generation of reactive oxygen species (ROS) in vitro. However, whether this molecule affects intestinal inflammation is largely unknown. We aimed to investigate the effect of BJ-1108 on dextran sulfate sodium (DSS)-induced experimental colitis in mice. METHODS: Colitis was induced in mice with DSS, and disease severity was estimated by evaluating body weight, colon length, histology, immune cell infiltration, and intestinal permeability. We examined the protective effects of BJ-1108 on barrier function using Caco-2 cells. Last, we estimated the impact of BJ-1108 on the phosphorylation of NF-kB, PI3K/AKT, and mitogen-activated protein kinases. RESULTS: Mice treated with BJ-1108 exhibited improved disease severity, as indicated by evaluations of body weight, histological scores, spleen weight, and infiltrates of T cells and macrophages. The administration of BJ-1108 inhibited the colonic mRNA expression of IL-6 and IL-1ß in vivo. Additionally, BJ-1108 limited intestinal permeability and enhanced the expression of tight junction (TJ) proteins such as claudin-1 and claudin-3 in the DSS-induced colitis model. In an in vitro model using Caco-2 cells, BJ-1108 ameliorated cytokine-induced ROS generation in a dose-dependent manner and remarkably recovered barrier dysfunction as estimated by evaluating transepithelial electrical resistance and TJ protein expression. BJ-1108 suppressed the NF-kB/ERK/PI3K pathway. CONCLUSIONS: This study demonstrated that BJ-1108 ameliorated intestinal inflammation in an experimental colitis mouse model, suggesting possible therapeutic implications for IBD.

The role of gonadotrophins in gonocyte transformation during minipuberty.

PURPOSE: Postnatal surge of gonadotrophins, Luteinizing hormone (LH) and Follicle-Stimulating hormone (FSH) known as minipuberty, is critical for gonocyte maturation into spermatogonial stem cells (SSC) in the testis. Gonadotrophins are essential for optimum fertility in men, but very little is known how they regulate germ cells during minipuberty. This study examined whether gonadotrophins play a role on gonocyte transformation in vivo. METHODS: Testes from hypogonadal (hpg) mice and their wild type (WT) littermates (n = 6/group) were weighed, and processed in paraffin at postnatal days (D) 0, 3, 6 and 9. Mouse VASA homologue (germ cell marker), anti-Müllerian hormone (Sertoli cell marker) antibodies and DAPI (nuclei marker) were used for immunofluorescence followed by confocal imaging. Germ cells on or off basement membrane (BM) and Sertoli cells/tubule were counted using Image J and analyzed with GraphPad. RESULTS: Comparing to WT littermates, there were significantly fewer germ cells on BM/tubule (p < 0.05) in D9 hpg mice, whereas there was no significant difference for germ cells off BM/tubule and Sertoli cells/tubule between littermates. However, testicular weight was significantly reduced in D3-D9 hpg mice comparing to WT littermates. CONCLUSION: Gonadotrophin deficiency reduced D9 germ cells on BM indicating impaired gonocyte transformation into SSC. This suggests that gonadotrophins may mediate gonocyte transformation during minipuberty.

Prevalence of Multidrug-Resistant Organisms and Risk Factors for Carriage among Patients Transferred from Long-Term Care Facilities.

BACKGROUND: Patient transport between acute care hospitals and long-term care facilities (LTCFs) plays a significant role in microbial migration. The study aimed to estimate the prevalence and risk factors associated with the colonization of multidrug-resistant organisms (MDROs) among patients transferred from LTCFs. MATERIALS AND METHODS: We retrospectively reviewed medical records to examine the colonization of MDROs. All patients who were transferred from LTCFs and admitted to an acute care hospital with 800 beds in Daejeon between March 2018 and February 2019 were included in the study. We surveyed rectal cultures and nasal swabs obtained for screening vancomycin-resistant Enterococcus (VRE), carbapenem-resistant Enterobacteriaceae (CRE), and methicillin-resistant Staphylococcus aureus (MRSA) at the time of hospitalization. We conducted a multivariable logistic regression to assess the association between clinical variables and the carriage of MDROs. RESULTS: Four hundred and fifteen patients from 86 LTCFs were enrolled. A total of 31.1% (130/415) of participants carried MDROs; VRE colonization was detected in 17.1% (71/415) of participants, and MRSA colonization was shown in 19.5% (81/415) of participants. No CRE was isolated. Previous use of antibiotics within three months [odds ratio (OR) 2.28; (95% confidence interval (CI) 1.30 - 4.00), P = 0.004], use of antibiotics for longer than two weeks [OR 2.16; (95% CI 1.03 - 4.53), P = 0.040], and previous colonization of MDROs within one year [OR 2.01; (95% CI 1.15 - 3.54), P = 0.015] were independently associated with increased risk for carriage of MDROs. CONCLUSION: Our study showed that a third of patients transferred from LTCFs carried VRE or MRSA, and prior antibiotic therapy was highly associated with the carriage of MDROs, which suggested more efficient management approaches for high-risk patients.

A new method for three-dimensional immunofluorescence study of the cochlea.

Visualisation of cochlear histopathology in three-dimensions has been long desired in the field of hearing research. This paper outlines a technique that has made this possible and shows a research application in the field of hearing protection after cochlear implantation. The technique utilises robust immunofluorescent labelling followed by effective tissue clearing and fast image acquisition using Light Sheet Microscopy. We can access the health of individual components by immunofluorescent detection of proteins such as myosin VIIa to look at cochlear hair cells, NaKATPase alpha 3 to look at spiral ganglion neurons, and IBA1 to look at macrophages within a single cochlea, whilst maintaining the integrity of fine membranous structures and keeping the cochlear implant in place. This allows the tissue response to cochlear implantation to be studied in detail, including the immune reaction to the implant and the impact on the structure and health of neural components such as hair cells. This technique reduces time and labour required for sectioning of cochleae and can allow visualisation of cellular detail. Use of image analysis software allows conversion of high-resolution image stacks into three-dimensional interactive data sets so volumes and numbers of surfaces can be measured. Immunofluorescent whole cochlea labelling and Light Sheet Microscopy have the capacity to be applied to many questions in hearing research of both the cochlea and vestibular system.

Role of Plasmodium falciparum Protein GEXP07 in Maurer's Cleft Morphology, Knob Architecture, and P. falciparum EMP1 Trafficking.

The malaria parasite Plasmodium falciparum traffics the virulence protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of infected red blood cells (RBCs) via membranous organelles, known as the Maurer's clefts. We developed a method for efficient enrichment of Maurer's clefts and profiled the protein composition of this trafficking organelle. We identified 13 previously uncharacterized or poorly characterized Maurer's cleft proteins. We generated transfectants expressing green fluorescent protein (GFP) fusions of 7 proteins and confirmed their Maurer's cleft location. Using co-immunoprecipitation and mass spectrometry, we generated an interaction map of proteins at the Maurer's clefts. We identified two key clusters that may function in the loading and unloading of PfEMP1 into and out of the Maurer's clefts. We focus on a putative PfEMP1 loading complex that includes the protein GEXP07/CX3CL1-binding protein 2 (CBP2). Disruption of GEXP07 causes Maurer's cleft fragmentation, aberrant knobs, ablation of PfEMP1 surface expression, and loss of the PfEMP1-mediated adhesion. ΔGEXP07 parasites have a growth advantage compared to wild-type parasites, and the infected RBCs are more deformable and more osmotically fragile.IMPORTANCE The trafficking of the virulence antigen PfEMP1 and its presentation at the knob structures at the surface of parasite-infected RBCs are central to severe adhesion-related pathologies such as cerebral and placental malaria. This work adds to our understanding of how PfEMP1 is trafficked to the RBC membrane by defining the protein-protein interaction networks that function at the Maurer's clefts controlling PfEMP1 loading and unloading. We characterize a protein needed for virulence protein trafficking and provide new insights into the mechanisms for host cell remodeling, parasite survival within the host, and virulence.

Biogenesis of the Spacious Coxiella-Containing Vacuole Depends on Host Transcription Factors TFEB and TFE3.

Coxiella burnetii is an obligate intracellular bacterial pathogen that replicates inside the lysosome-derived Coxiella-containing vacuole (CCV). To establish this unique niche, C. burnetii requires the Dot/Icm type IV secretion system (T4SS) to translocate a cohort of effector proteins into the host cell, which modulate multiple cellular processes. To characterize the host-pathogen interactions that occur during C. burnetii infection, stable-isotope labeling by amino acids in cell culture (SILAC)-based proteomics was used to identify changes in the host proteome during infection of a human-derived macrophage cell line. These data revealed that the abundances of many proteins involved in host cell autophagy and lysosome biogenesis were increased in infected cells. Thus, the role of the host transcription factors TFEB and TFE3, which regulate the expression of a network of genes involved in autophagy and lysosomal biogenesis, were examined in the context of C. burnetii infection. During infection with C. burnetii, both TFEB and TFE3 were activated, as demonstrated by the transport of these proteins from the cytoplasm into the nucleus. The nuclear translocation of these transcription factors was shown to be dependent on the T4SS, as a Dot/Icm mutant showed reduced nuclear translocation of TFEB and TFE3. This was supported by the observation that blocking bacterial translation with chloramphenicol resulted in the movement of TFEB and TFE3 back into the cytoplasm. Silencing of the TFEB and TFE3 genes, alone or in combination, significantly reduced the size of the CCV, which indicates that these host transcription factors facilitate the expansion and maintenance of the organelle that supports C. burnetii intracellular replication.

Development and Validation of a Novel Warfarin Dosing Algorithm for Korean Patients With VKORC1 1173C.

BACKGROUND: Differences in the performance of suggested warfarin dosing algorithms among different ethnicities and genotypes have been reported; this necessitates the development of an algorithm with enhanced performance for specific population groups. Previous warfarin dosing algorithms underestimated warfarin doses in VKORC1 1173C carriers. We aimed to develop and validate a new warfarin dosing algorithm for Korean patients with VKORC1 1173C. METHODS: A total of 109 patients carrying VKORC1 1173CT (N=105) or 1173CC (N=4) were included in this study. Multiple regression analysis was performed to deduce a new dosing algorithm. Following literature searches for genotype-guided warfarin dosing algorithms, 21 algorithms were selected and evaluated using the correlation coefficient (ρ) of actual dose and estimated dose, mean error, and root mean square error. RESULTS: The developed algorithm is as follows: maintenance dose (mg/week)=exp [3.223-0.009×(age)+0.577×(body surface area [BSA])+0.178×(sex)-0.481×(CYP2C9 genotype)+0.227×(VKORC1 genotype)]. Integrated variables explained 44% of the variance in the maintenance dose. The predicted and actual doses showed moderate correlation (ρ=0.641) with the best performance with a mean error of -1.30 mg/week. The proportion of underestimated groups was 17%, which was lower than with the other algorithms. CONCLUSIONS: This is the first study to develop and validate a warfarin dosing algorithm based on data from VKORC1 1173C carriers; it showed superior predictive performance compared with previously published algorithms.

Does the apoptosis pathway play a critical role in gonocyte transformation?

PURPOSE: Undescended Testes (UDT) are prevalent in 2%-5% of male infants and cause malignancy and infertility. During germ cell development, abnormal gonocytes usually undergo apoptosis. This process is believed to involve BAX (Bcl-2 Associated X) protein in clearing abnormal gonocytes which may fail in UDT, resulting in persisting gonocytes causing seminomas later in life. We aim to investigate the role of BAX in gonocyte apoptosis. MATERIALS AND METHODS: BAXKO (BAX-knockout) mice were back-crossed to OG2 mice (Oct4-promoter driving enhanced green fluorescent protein-eGFP) to produce BAXOG2 mice. Testes (wildtype-BAX+/+, heterozygous-BAX+/- and homozygous-BAX-/- mice, n = 6/group) on postnatal days 1, 3, 6, 9 were fixed and embedded in OCT for frozen sectioning. Sections were labeled with Anti-Müllerian Hormone (Sertoli cell marker), Mouse Vasa Homolog (germ cell marker) and DAPI (nucleus marker) and imaged using confocal microscopy. Oct4-GFP+ve germ cells, germ cells on/off the basement membrane and Sertoli cells were counted using ImageJ followed by data analysis with GraphPad. RESULTS: BAX-/-OG2 mice had significantly higher number of germ cells/tubule comparing to BAX+/+OG2 on day 9. There were Oct4-GFP+ve gonocyte-like germ cells that persisted in the center of the tubules in BAX-/-OG2 even after the completion of gonocyte transformation. This suggests that abnormal gonocytes in BAX-/-OG2 mice failed to undergo apoptosis and are allowed to persist. CONCLUSION: This study demonstrated that apoptosis is important in regulating germ cell migration and differentiation during gonocyte transformation in neonatal mice. In addition, inhibition of apoptosis results in persisting neonatal gonocytes which might become seminomas in patients with UDT.

Degrading products of chondroitin sulfate can induce hypertrophy-like changes and MMP-13/ADAMTS5 production in chondrocytes.

Chondroitin sulfate (CS) is the most abundant glycosaminoglycan (GAG) in articular cartilage and the loss of CS-GAG occurs early in OA. As a major component of perichondral matrix interacting directly with chondrocytes, the active turnover of CS can affect to break the homeostasis of chondrocytes. Here we employ CS-based 3-dimensional (3D) hydrogel scaffold system to investigate how the degradation products of CS affect the catabolic phenotype of chondrocytes. The breakdown of CS-based ECM by the chondroitinase ABC (ChABC) resulted in a hypertrophy-like morphologic change in chondrocytes, which was accompanied by catabolic phenotypes, including increased MMP-13 and ADAMTS5 expression, nitric oxide (NO) production and oxidative stress. The inhibition of Toll-like receptor 2 (TLR2) or TLR4 with OxPAPC (TLR2 and TLR4 dual inhibitor) and LPS-RS (TLR4-MD2 inhibitor) ameliorated these catabolic phenotypes of chondrocytes by CS-ECM degradation, suggesting a role of CS breakdown products as damage-associated molecular patterns (DAMPs). As downstream signals of TLRs, MAP kinases, NF-kB, NO and STAT3-related signals were responsible for the catabolic phenotypes of chondrocytes associated with ECM degradation. NO in turn reinforced the activation of MAP kinases as well as NFkB signaling pathway. Thus, these results propose that the breakdown product of CS-GAG can recapitulate the catabolic phenotypes of OA.

Lipid order and charge protect killer T cells from accidental death.

Killer T cells (cytotoxic T lymphocytes, CTLs) maintain immune homoeostasis by eliminating virus-infected and cancerous cells. CTLs achieve this by forming an immunological synapse with their targets and secreting a pore-forming protein (perforin) and pro-apoptotic serine proteases (granzymes) into the synaptic cleft. Although the CTL and the target cell are both exposed to perforin within the synapse, only the target cell membrane is disrupted, while the CTL is invariably spared. How CTLs escape unscathed remains a mystery. Here, we report that CTLs achieve this via two protective properties of their plasma membrane within the synapse: high lipid order repels perforin and, in addition, exposed phosphatidylserine sequesters and inactivates perforin. The resulting resistance of CTLs to perforin explains their ability to kill target cells in rapid succession and to survive these encounters. Furthermore, these mechanisms imply an unsuspected role for plasma membrane organization in protecting cells from immune attack.

Polydeoxyribonucleotide injection in the treatment of patients with carpal tunnel syndrome: Retrospective preliminary study.

BACKGROUND: Corticosteroid injection is beneficial in treating carpal tunnel syndrome (CTS) due to its anti-inflammatory effects. However, its side effects limit widespread usage. Recently, several studies have found that polydeoxyribonucleotide offers anti-inflammatory capabilities with fewer side effects, making it an ideal alternative. Nevertheless, there has been no study on its effectiveness in patients with CTS. Therefore, we evaluate the effectiveness of polydeoxyribonucleotide in patients with CTS. Based on the criteria, 30 patients with CTS who received two-consecutive polydeoxyribonucleotide injections (with a week interval) were initially included. METHOD: Patients with CTS were investigated retrospectively. To evaluate the effectiveness of polydeoxyribonucleotide in patients with CTS, numeric rating scale (NRS), cross-sectional area (CSA) of the median nerve, and severity and functional status scores of CTS based on the Boston Carpal Tunnel Syndrome Questionnaire (BCTQ) were assessed. RESULTS: There was a significant improvement in the NRS, CSA, and functional and severity scores of BCTQ after two-consecutive polydeoxyribonucleotide injections (P < .05). CONCLUSION: In conclusion, although more research is needed to evaluate the effectiveness of polydeoxyribonucleotide in patients with CTS, the findings here suggest that polydeoxyribonucleotide may be a viable alternative to corticosteroids in patients with CTS.

DICAM Attenuates Experimental Colitis via Stabilizing Junctional Complex in Mucosal Barrier.

BACKGROUND: Adhesion molecules maintain the intestinal barrier function that is crucial to prevent intestinal inflammation. Dual immunoglobulin domain-containing adhesion molecule (DICAM) has been recently identified and known for the involvement in cell-cell adhesion through homophilic interaction and heterophilic interaction with integrin αVß3. We tested whether the change of DICAM expression affects the severity of colonic inflammation. METHODS: Colitis was induced with oral administration of 2.5% dextran sulfate sodium (DSS) in 8-week-old male mice for 5 days. The function of DICAM under inflammatory condition was investigated using loss-of-function and gain-of-function models such as DICAM-deficient mice and adenoviral transduction of DICAM into Caco-2 colonic epithelial cells. RESULTS: DICAM increased in parallel with the degree of inflammation after 5-day administration of DSS and decreased with the resolution of inflammation. DICAM was expressed in the epithelial junctional complex and colocalized with ZO-1. Treatment with TNF-α or IFN-γ in Caco-2 cells significantly increased DICAM in protein and RNA level. The DICAM knockout mice showed more severe DSS-induced colitis compared with WT littermates. Adenoviral transduction of DICAM into Caco-2 cells significantly attenuated the inflammation-mediated decrease of adhesion molecules, including ZO-1 and occludin. Furthermore, Caco-2 cells with DICAM overexpression maintained intestinal barrier function under IFN-γ treatment as estimated by transepithelial electrical resistance. CONCLUSION: Our study demonstrates that DICAM which is increased in an inflammatory condition has a protective role in experimental colitis by stabilizing the integrity of junctional complex in the intestinal mucosal barrier.

Targeted Graphene Oxide Networks: Cytotoxicity and Synergy with Anticancer Agents.

An effective strategy to inhibit endocytosis in cancer cells is presented where modified net-type graphene oxide (GO) sheets, bound with multiple cell surface receptors, are introduced and synthesized as novel anticancer agents. The results suggest that the binding connects GO sheets with neighboring lipid rafts, neutralizes endocytosis, and causes metabolic deprivation. As a result, tumor cell survival and proliferation are reduced. Live cell confocal microscopy imaging reveals that GO-PEGFA (folate-PEGylated GO) (PEG, polyethylene glycol) is internalized by tumor cells, while GO-PEGRGD (tripeptide Arg-Gly-Asp PEGylated GO) associates with the external cell membrane (not internalized). In vitro exposure of tumor cells to GO-PEGFA or GO-PEGRGD reduces the cell viability by 35%, compared to 50% reduction using methotrexate (100 µM). The combination of modified GO sheets with methotrexate or doxorubicin shows a greater toxicity (80% reduction in cell viability) than the individual agents. The proposed setup demonstrates a significant synergy in limiting tumor cell growth.

Surgical stress response and promotion of metastasis in colorectal cancer: a complex and heterogeneous process.

Surgery remains the curative treatment modality for colorectal cancer in all stages, including stage IV with resectable liver metastasis. There is emerging evidence that the stress response caused by surgery as well as other perioperative therapies such as anesthesia and analgesia may promote growth of pre-existing micro-metastasis or potentially initiate tumor dissemination. Therapeutically targeting the perioperative period may therefore reduce the effect that surgical treatments have in promoting metastases, for example by combining ß-adrenergic receptor antagonists and cyclooxygenase-2 (COX-2) inhibitors in the perioperative setting. In this paper, we highlight some of the mechanisms that may underlie surgery-related metastatic development in colorectal cancer. These include direct tumor spillage at the time of surgery, suppression of the anti-tumor immune response, direct stimulatory effects on tumor cells, and activation of the coagulation system. We summarize in more detail results that support a role for catecholamines as major drivers of the pro-metastatic effect induced by the surgical stress response, predominantly through activation of ß-adrenergic signaling. Additionally, we argue that an improved understanding of surgical stress-induced dissemination, and more specifically whether it impacts on the level and nature of heterogeneity within residual tumor cells, would contribute to the successful clinical targeting of this process. Finally, we provide a proof-of-concept demonstration that ex-vivo analyses of colorectal cancer patient-derived samples using RGB-labeling technology can provide important insights into the heterogeneous sensitivity of tumor cells to stress signals. This suggests that intra-tumor heterogeneity is likely to influence the efficacy of perioperative ß-adrenergic receptor and COX-2 inhibition, and that ex-vivo characterization of heterogeneous stress response in tumor samples can synergize with other models to optimize perioperative treatments and further improve outcome in colorectal and other solid cancers.