Suppression of smooth muscle cell inflammation by myocardin-related transcription factors involves inactivation of TANK-binding kinase 1.

Myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MRTFA, and MRTF-B/MRTFB) suppress production of pro-inflammatory cytokines and chemokines in human smooth muscle cells (SMCs) through sequestration of RelA in the NF-κB complex, but additional mechanisms are likely involved. The cGAS-STING pathway is activated by double-stranded DNA in the cytosolic compartment and acts through TANK-binding kinase 1 (TBK1) to spark inflammation. The present study tested if MRTFs suppress inflammation also by targeting cGAS-STING signaling. Interrogation of a transcriptomic dataset where myocardin was overexpressed using a panel of 56 cGAS-STING cytokines showed the panel to be repressed. Moreover, MYOCD, MRTFA, and SRF associated negatively with the panel in human arteries. RT-qPCR in human bronchial SMCs showed that all MRTFs reduced pro-inflammatory cytokines on the panel. MRTFs diminished phosphorylation of TBK1, while STING phosphorylation was marginally affected. The TBK1 inhibitor amlexanox, but not the STING inhibitor H-151, reduced the anti-inflammatory effect of MRTF-A. Co-immunoprecipitation and proximity ligation assays supported binding between MRTF-A and TBK1 in SMCs. MRTFs thus appear to suppress cellular inflammation in part by acting on the kinase TBK1. This may defend SMCs against pro-inflammatory insults in disease.

Vitamin D triggers hCAP18/LL-37 production: Implications for LL-37-induced human osteoblast cytotoxicity.

The human cathelicidin LL-37 shows activity against microorganisms, but it is also cytotoxic to host cells. The CAMP gene codes for the LL-37 precursor hCAP18 which is processed extracellularly to active LL-37. It has previously been shown that vitamin D stimulates CAMP gene activity, but less information is available demonstrating that vitamin D also can increase hCAP18/LL-37 protein production. Here, we show with RT-qPCR that a physiological concentration of vitamin D (50 nM) enhances CAMP mRNA levels by about 170 times in human THP-1 monocyte cells. Stimulation with 50 nM vitamin D increases hCAP18/LL-37 protein contents 3-4 times in THP-1 cell lysates demonstrated by both dot blot analysis and ELISA applying two different hCAP18/LL-37 antibodies. Treatment with the proteasome inhibitor MG132 enhances hCAP18/LL-37 levels, suggesting that turnover of hCAP18/LL-37 protein is regulated by the proteasome. The hCAP18/LL-37 concentration in vitamin D-stimulated THP-1 cells corresponds to 1.04 µM LL-37. Interestingly, synthetic LL-37, at this concentration, reduces viability of human osteoblast-like MG63 cells, whereas the THP-1 cells are less sensitive as demonstrated by the MTT assay. In summary, we show that vitamin D enhances hCAP18/LL-37 production, and that this effect can be of physiological/pathophysiological relevance for LL-37-induced human osteoblast toxicity.

Voluntary first responders' experiences of being dispatched to suspected out-of-hospital cardiac arrest in rural areas: an interview study.

BACKGROUND: Out-of-hospital cardiac arrest (OHCA) is a leading cause of death, and survival outcomes vary across countries and regions. To improve survival, the European Resuscitation Council Guidelines encourage the implementation of technologies like smartphone applications to alert voluntary first responders (VFRs) who are near a suspected OHCA. VFRs are of great importance in the ´chain of survival´, but there is still a lack of knowledge about their experiences; especially of those operating in rural areas. Understanding those experiences is crucial in developing appropriate interventions to train, encourage, and safeguard VFRs in their mission. Therefore, the aim of this study was to describe VFRs´ experiences of being dispatched to suspected OHCA in rural areas. METHODS: The study used an inductive design. The data were collected using individual interviews with 16 VFRs and analysed using qualitative content analysis. RESULTS: The results are presented in terms of six generic categories ''Being motivated and prepared'', ''Having strategies to undertake the mission'', ''Collaborating with others'', ''Being ethically aware'', ''Supporting the family members'', and ''Coping with the mission'', which formed the basis of the main category 'Desire to save lives and help others'. The findings showed that VFRs had a genuine desire to contribute to save lives in this rural area. Regardless of the circumstances, they were prepared to leave everything and act to the best for the victim and their family members. In theirs' missions they collaborated with others at the scene and were guided by ethics while they acted in complex circumstances. CONCLUSIONS: VFRs dispatched in rural areas express a desire to save lives. In their missions, they acted in complex situations and experienced both emotional and ethical challenges. The design, implementation, and evaluation of support interventions directed at VFRs should be prioritised, especially in rural areas, as it can contribute to more people becoming and remaining VFRs, which in turn could contribute to sustainable development.

Response times in rural areas for emergency medical services, fire and rescue services and voluntary first responders during out-of-hospital cardiac arrests.

Aim: To increase survival in out-of-hospital cardiac arrests (OHCA), great efforts are made to improve the number of voluntary first responders (VFR). However, evidence of the potential utility of such efforts is sparse, especially in rural areas. Therefore, the aim was to describe and compare response times for emergency medical services (EMS), fire and rescue services (FRS), and VFR during OHCA in relation to population density. Methods: This observational and comparative study was based on data including positions and time stamps for VFR and response times for EMS and FRS in a region in southern Sweden. Results: In total, 285 OHCAs between 1 July 2020 and 31 December 2021 were analysed. VFR had the shortest median response time in comparison to EMS and FRS in all studied population densities. The overall median (Q1-Q3) time gain for VFR was 03:07 (01:39-05:41) minutes. A small proportion (19.2%) of alerted VFR accepted the assignments. This is most problematic in rural and sub-rural areas, where there were low numbers of alerted VFR. Also, FRS had shorter response time than EMS in all studied population densities except in urban areas. Conclusion: The differences found in median response times between rural and urban areas are worrisome from an equality perspective. More focus should be placed on recruiting VFR, especially in rural areas since VFR can potentially contribute to saving more lives. Also, since FRS has a shorter response time than EMS in rural, sub-rural, and sub-urban areas, FRS should be dispatched more frequently.

Palliative resection of the primary tumour improves survival in incurable metastatic colorectal cancer.

BACKGROUND: Studies show conflicting results on whether primary tumour resection (PTR) in metastatic colorectal cancer (mCRC) prolongs survival. The aim of this study was to analyse prognostic factors and the effects of PTR on overall survival (OS) in mCRC patients. METHODS: In this population-based cohort study, factors associated with PTR and OS were assessed in 188 mCRC patients with mCRC treated with palliative chemotherapy between 2008 and 2019. The Chi-square test and Mann-Whitney U-test were used to assess factors associated with PTR. The log-rank test was used to compare Kaplan-Meier estimates for OS. Cox regression was used to identify factors predicting OS. RESULTS: Patients undergoing PTR had significantly better performance status, fewer metastatic sites, lower CEA levels, and more often had left-sided CRC than patients not undergoing PTR. OS was longer in palliative mCRC patients undergoing PTR (P < 0.01) and PTR was an independent variable in the Cox regression analysis (P < 0.05). Median OS was 22.9 ± 1.9 months for the PTR group and 14.5 ± 1.5 months for the non-operated group. Poor performance status and liver metastases were significantly associated with poor prognosis. CONCLUSION: This study shows that PTR had a positive effect on OS and may be considered in patients suitable for surgery. PTR was offered to palliative mCRC patients with prognostic factors associated with better prognosis.

Targeted Therapy in the Palliative Setting of Colorectal Cancer-Survival and Medical Costs.

(1) Background: Targeted therapy is used alone or together with chemotherapy in metastatic colorectal cancer. The aim of this study was to assess overall survival and medical costs in a cohort of patients with metastatic colorectal cancer. (2) Methods: Demographic and clinical characteristics of 337 patients and pathological data of colorectal tumors were retrospectively collected in this population-based study. The overall survival and medical costs for patients receiving chemotherapy plus targeted therapy were compared with those for patients receiving chemotherapy only. (3) Results: Patients administered chemotherapy plus targeted therapy were less frail and had more often RAS wild-type tumors but had higher CEA levels than patients receiving chemotherapy only. No prolonged overall survival could be observed in patients receiving palliative targeted therapy. The medical costs for patients undergoing treatment with targeted therapy were significantly higher than for patients treated only with chemotherapy; they were especially higher in the group receiving targeted therapy early than late in the palliative setting. (4) Conclusions: The use of targeted therapy in metastatic colorectal cancer leads to significantly higher medical costs when used early in the palliative setting. No positive effects of the use of targeted therapy could be observed in this study; therefore, we suggest that targeted therapy be used in later lines of palliative therapy in metastatic colorectal cancer.

KIT and PDGFRA Mutations and Survival of Gastrointestinal Stromal Tumor Patients Treated with Adjuvant Imatinib in a Randomized Trial.

PURPOSE: Limited data are available about the influence of KIT and PDGFRA mutations on overall survival (OS) of patients with gastrointestinal stromal tumor (GIST) treated with adjuvant imatinib. PATIENTS AND METHODS: The Scandinavian Sarcoma Group XVIII/AIO multicenter trial accrued 400 patients with a high risk for GIST recurrence after macroscopically complete surgery between February 4, 2004, and September 29, 2008. The patients received adjuvant imatinib 400 mg/day for either 1 year or 3 years based on random allocation. We analyzed using conventional sequencing KIT and PDGFRA mutations centrally from 341 (85%) patients who had localized, centrally confirmed GIST, and correlated the results with recurrence-free survival (RFS) and OS in exploratory analyses. RESULTS: During a median follow-up time of 10 years, 164 RFS events and 76 deaths occurred. Most patients were re-treated with imatinib when GIST recurred. Patients with KIT exon 11 deletion or indel mutation treated with 3 years of adjuvant imatinib survived longer than patients treated for 1 year [10-year OS 86% versus 64%, respectively; HR, 0.34; 95% confidence interval (CI), 0.15-0.72; P = 0.007], and also had longer RFS (10-year RFS 47% versus 29%; HR, 0.48; 95% CI, 0.31-0.74; P < 0.001). Patients with KIT exon 9 mutation had unfavorable OS regardless of the duration of adjuvant imatinib. CONCLUSIONS: Compared with 1 year of imatinib, 3 years of adjuvant imatinib led to 66% reduction in the estimated risk of death and a high 10-year OS rate in the subset of patients with a KIT exon 11 deletion/indel mutation.

Desquamated Epithelial Cells of Unstimulated Human Whole Saliva Express Both EGF Transcript and Protein.

Objective: The aim of this study was to investigate if desquamated oral epithelial cells (DOECs) express the epidermal growth factor (EGF) and if these cells thereby may contribute to salivary EGF contents. Background: DOECs have recently been shown to harbor the antimicrobial peptide LL-37, proposing that they may also store other biologically important salivary peptides/proteins. The EGF peptide is a growth factor which plays a critical role to maintain epithelial integrity and promote epithelial healing. The EGF is produced by salivary glands, but it is not known whether DOECs contain the EGF and thereby contribute to salivary EGF levels. Materials and Methods: DOECs were isolated from unstimulated whole saliva collected from four healthy volunteers. EGF protein expression was determined in cell lysates by dot blot and ELISA. Cellular distribution of cytokeratin, the proliferation marker Ki67, and EGF immunoreactivity were assessed by immunocytochemistry. EGF gene expression was investigated by qPCR. Expression of EGF transcript and protein in DOECs was compared to that in the human cultured keratinocyte cell line (HaCaT) cells. Results: EGF protein expression was detected in DOEC cell lysates by both dot blot and ELISA. Strong cytoplasmic EGF immunoreactivity was observed in DOECs, although some cells showed only a weak immunoreactive signal for EGF. Moreover, DOECs, besides containing EGF protein, also expressed transcript for EGF. Interestingly, ELISA analysis revealed that EGF protein contents were higher in DOECs than in HaCaT cells. ELISA analysis also disclosed that EGF concentration was about 10 times higher in whole saliva compared to DOECs. EGF transcript expression was about 50% lower in HaCaT cells stimulated with high (10%) compared to low (0.1%) concentration of fetal bovine serum, representing growth-stimulated and growth-restricted conditions, respectively, implying that growth-stimulus exerts negative feedback on EGF gene activity in HaCaT cells. Conclusion: Here, we show for the first time that DOECs express the EGF, arguing that these cells contribute to salivary EGF contents and hence may play a role in gingival epithelial repair and wound healing.

LL-37 and Double-Stranded RNA Synergistically Upregulate Bronchial Epithelial TLR3 Involving Enhanced Import of Double-Stranded RNA and Downstream TLR3 Signaling.

The human host defense peptide LL-37 influences double-stranded RNA signaling, but this process is not well understood. Here, we investigate synergistic actions of LL-37 and synthetic double-stranded RNA (poly I:C) on toll-like receptor 3 (TLR3) expression and signaling, and examine underlying mechanisms. In bronchial epithelial BEAS-2B cells, LL-37 potentiated poly I:C-induced TLR3 mRNA and protein expression demonstrated by qPCR and Western blot, respectively. Interestingly, these effects were associated with increased uptake of rhodamine-tagged poly I:C visualized by immunocytochemistry. The LL-37/poly I:C-induced upregulation of TLR3 mRNA expression was prevented by the endosomal acidification inhibitor chloroquine, indicating involvement of downstream TLR3 signaling. The glucocorticoid dexamethasone reduced LL-37/poly I:C-induced TLR3 expression on both mRNA and protein levels, and this effect was associated with increased IκBα protein expression, suggesting that dexamethasone acts via attenuation of NF-κB activity. We conclude that LL-37 potentiates poly I:C-induced upregulation of TLR3 through a mechanism that may involve enhanced import of poly I:C and that LL-37/poly I:C-induced TLR3 expression is associated with downstream TLR3 signaling and sensitive to inhibition of NF-κB activity.

The antimicrobial peptide LL-37 triggers release of apoptosis-inducing factor and shows direct effects on mitochondria.

The human antimicrobial peptide LL-37 permeabilizes the plasma membrane of host cells, but LL-37-induced direct effects on mitochondrial membrane permeability and function has not been reported. Here, we demonstrate that LL-37 is rapidly (within 20 min) internalized by human osteoblast-like MG63 cells, and that the peptide co-localizes with MitoTracker arguing for accumulation in mitochondria. Subcellular fractionation and Western blot disclose that stimulation with LL-37 (8 µM) for 2 h triggers release of the mitochondrial protein apoptosis-inducing factor (AIF) to the cytosol, whereas LL-37 causes no release of cytochrome C oxidase subunit IV of the inner mitochondrial membrane, suggesting that LL-37 affects mitochondrial membrane permeability in a specific manner. Next, we investigated release of AIF and cytochrome C from isolated mitochondria by measuring immunoreactivity by dot blot. The media of mitochondria treated with LL-37 (8 µM) for 2 h contained 50% more AIF and three times more cytochrome C than that of control mitochondria, showing that LL-37 promotes release of both AIF and cytochrome C. Moreover, in vesicles reflecting mitochondrial membrane lipid composition, LL-37 stimulates membrane permeabilization and release of tracer molecules. We conclude that LL-37 is rapidly internalized by MG63 cells and accumulates in mitochondria, and that the peptide triggers release of pro-apoptotic AIF and directly affects mitochondrial membrane structural properties.

Fibrinogen-like protein 2 in gastrointestinal stromal tumour.

Gastrointestinal stromal tumour (GIST), the most common sarcoma of the gastrointestinal tract, can be treated effectively with tyrosine kinase inhibitors, such as imatinib. Cancer immune therapy has limited efficacy, and little is known about the immune suppressive factors in GISTs. Fibrinogen-like protein 2 (FGL2) is expressed either as a membrane-associated protein or as a secreted soluble protein that has immune suppressive functions. We found that GISTs expressed FGL2 mRNA highly compared to other types of cancer in a large human cancer transcriptome database. GIST expressed FGL2 frequently also when studied using immunohistochemistry in two large clinical series, where 333 (78%) out of the 425 GISTs were FGL2 positive. The interstitial cells of Cajal, from which GISTs may originate, expressed FGL2. FGL2 expression was associated with small GIST size, low mitotic counts and low tumour-infiltrating lymphocyte (TIL) counts. Patients whose GIST expressed FGL2 had better recurrence-free survival than patients whose GIST lacked expression. Imatinib upregulated FGL2 in GIST cell lines, and the patients with FGL2-negative GIST appeared to benefit most from long duration of adjuvant imatinib. We conclude that GISTs express FGL2 frequently and that FGL2 expression is associated with low TIL counts and favourable survival outcomes.

Cell Type Dependent Suppression of Inflammatory Mediators by Myocardin Related Transcription Factors.

Myocardin related transcription factors (MRTFs: MYOCD/myocardin, MRTF-A, and MRTF-B) play a key role in smooth muscle cell differentiation by activating contractile genes. In atherosclerosis, MRTF levels change, and most notable is a fall of MYOCD. Previous work described anti-inflammatory properties of MRTF-A and MYOCD, occurring through RelA binding, suggesting that MYOCD reduction could contribute to vascular inflammation. Recent studies have muddled this picture showing that MRTFs may show both anti- and pro-inflammatory properties, but the basis of these discrepancies remain unclear. Moreover, the impact of MRTFs on inflammatory signaling pathways in tissues relevant to human arterial disease is uncertain. The current work aimed to address these issues. RNA-sequencing after forced expression of myocardin in human coronary artery smooth muscle cells (hCASMCs) showed reduction of pro-inflammatory transcripts, including CCL2, CXCL8, IL6, and IL1B. Side-by-side comparison of MYOCD, MRTF-A, and MRTF-B in hCASMCs, showed that the anti-inflammatory impact was shared among MRTFs. Correlation analyses using human arterial transcriptomic datasets revealed negative correlations between MYOCD, MRTFA, and SRF, on the one hand, and the inflammatory transcripts, on the other. A pro-inflammatory drive from lipopolysaccharide, did not change the size of the suppressive effect of MRTF-A in hCASMCs on either mRNA or protein levels. To examine cell type-dependence, we compared the anti-inflammatory impact in hCASMCs, with that in human bladder SMCs, in endothelial cells, and in monocytes (THP-1 cells). Surprisingly, little anti-inflammatory activity was seen in endothelial cells and monocytes, and in bladder SMCs, MRTF-A was pro-inflammatory. CXCL8, IL6, and IL1B were increased by the MRTF-SRF inhibitor CCG-1423 and by MRTF-A silencing in hCASMCs, but depolymerization of actin, known to inhibit MRTF activity, had no stimulatory effect, an exception being IL1B. Co-immunoprecipitation supported binding of MRTF-A to RelA, supporting sequestration of this important pro-inflammatory mediator as a mechanism. Dexamethasone treatment and silencing of RelA (by 76 ± 1%) however only eliminated a fraction of the MRTF-A effect (≈25%), suggesting mechanisms beyond RelA binding. Indeed, SRF silencing suggested that MRTF-A suppression of IL1B and CXCL8 depends on SRF. This work thus supports an anti-inflammatory impact of MRTF-SRF signaling in hCASMCs and in intact human arteries, but not in several other cell types.

Exogenous LL-37 but not homogenates of desquamated oral epithelial cells shows activity against Streptococcus mutans.

OBJECTIVE: The antimicrobial peptide hCAP18/LL-37 is detected in desquamated epithelial cells of human whole saliva, but the functional importance of this pool of hCAP18/LL-37 is not understood. Here, we assess the impact of homogenates of desquamated oral epithelial cells and exogenous, synthetic LL-37 on two oral bacteria: S. mutans and S. gordonii. MATERIAL AND METHODS: Desquamated epithelial cells of unstimulated whole saliva were isolated and cellular and extracellular levels of hCAP18/LL-37 analyzed by ELISA. Bacterial viability was determined by BacLight Live/Dead staining and confocal laser scanning microscopy. RESULTS: Desquamated oral epithelial cells harboured hCAP18/LL-37, and they spontaneously released/leaked the peptide to their medium. Exogenous, synthetic LL-37 showed cytotoxic activity against S. mutans but not S gordonii, suggesting that LL-37 acts differentially on these two types of oral bacteria. Homogenates of desquamated oral epithelial cells had no effect on S. mutans viability. Treatment with exogenous, synthetic LL-37 (8 and 10 µM) reduced S. mutans viability, whereas lower concentrations (0.1 and 1 µM) of the peptide lacked effect. CONCLUSIONS: Desquamated oral epithelial cells contain hCAP18/LL-37, but their cellular levels of hCAP18/LL-37 are too low to affect S. mutans viability, whereas exogenous, synthetic LL-37 has a strong effect on these bacteria.

Mechanisms involved in regulation of periodontal ligament cell production of pro-inflammatory cytokines: Implications in periodontitis.

It is well recognized that human periodontal ligament cells (PDL cells) may represent local immune cells of the periodontal tissues. However, it is unclear whether they represent "true" immune cells, since they can produce pro-inflammatory cytokines not only after stimulation with bacterial lipopolysaccharides but also in response to other stimuli such as mechanical stress. Stimulation with bacterial lipopolysaccharides strongly enhances PDL cell production of pro-inflammatory cytokines through activation of toll-like receptors and NF-κB signaling. Less information is available regarding putative modulators of cytokine production and their mechanisms of action in PDL cells. The anti-inflammatory glucocorticoid dexamethasone reduces lipopolysaccharide-induced PDL cell production of cytokines. Recent observations show that vitamin D and the antimicrobial peptide LL-37 antagonize lipopolysaccharide-stimulated PDL cell production of pro-inflammatory cytokines. Secretory leukocyte protease inhibitor is endogenously expressed by PDL cells, and this protein negatively regulates PDL cell-evoked cytokine production. More information and knowledge about the regulation of PDL cell production of cytokines may clarify the role of PDL cells in oral innate immunity and their importance in periodontitis.

LL-37-induced caspase-independent apoptosis is associated with plasma membrane permeabilization in human osteoblast-like cells.

The host defense peptide LL-37 is active against both gram-positive and gram-negative bacteria, but it has also been shown to reduce human host cell viability. However, the mechanisms behind LL-37-induced human host cell cytotoxicity are not yet fully understood. Here, we assess if LL-37-evoked attenuation of human osteoblast-like MG63 cell viability is associated with apoptosis, and if the underlying mechanism may involve LL-37-induced plasma membrane permeabilization. MG63 cell viability and plasma membrane permeabilization were investigated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and by measuring lactate dehydrogenase (LDH) release, respectively. Apoptosis was assessed by the terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) assay and Annexin V flow cytometry, and caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage were determined by Western blot. LL-37 (4 and 10 µM) reduced both cell number and cell viability, and these effects were associated with a pro-apoptotic effect demonstrated by positive TUNEL staining and Annexin V flow cytometry. LL-37-induced apoptosis was not coupled to either caspase-3 or PARP cleavage, suggesting that LL-37 causes caspase-independent apoptosis in MG63 cells. Both LL-37 and the well-known plasma membrane permeabilizer Triton X-100 reduced cell viability and stimulated LDH release. Triton X-100-treated cells showed positive TUNEL staining, and the detergent accumulated cells in late apoptosis/necrosis. Similar to LL-37, Triton X-100 caused no PARP cleavage. We conclude that LL-37 promotes caspase-independent apoptosis, and that this effect seems coupled to plasma membrane permeabilization in human MG63 cells.

Survival Outcomes Associated With 3 Years vs 1 Year of Adjuvant Imatinib for Patients With High-Risk Gastrointestinal Stromal Tumors: An Analysis of a Randomized Clinical Trial After 10-Year Follow-up.

Importance: Adjuvant imatinib is associated with improved recurrence-free survival (RFS) when administered after surgery to patients with operable gastrointestinal stromal tumor (GIST), but its influence on overall survival (OS) has remained uncertain. Objective: To evaluate the effect of adjuvant imatinib on OS of patients who have a high estimated risk for GIST recurrence after macroscopically complete surgery. Design, Setting, and Participants: In this open-label, randomized (1:1), multicenter phase 3 clinical trial conducted in Finland, Germany, Norway, and Sweden, 400 patients who had undergone macroscopically complete surgery for GIST with a high estimated risk for recurrence according to the modified National Institutes of Health Consensus Criteria were enrolled between February 2004 and September 2008. Data for this follow-up analysis were analyzed from September to November, 2019. Interventions: Imatinib 400 mg/d administered orally for either 12 months or 36 months after surgery. Main Outcomes And Measures: The primary end point was RFS; the secondary objectives included OS and treatment safety. Results: The intention-to-treat cohort consisted of 397 patients (12-month group, 199; 36-month group, 198; 201 men and 196 women; median [IQR] age, 62 (51-69) years and 60 (51-67) years, during a median follow-up time of 119 months after the date of randomization, 194 RFS events and 96 OS events were recorded in the intention-to-treat population. Five-year and 10-year RFS was 71.4% and 52.5%, respectively, in the 36-month group and 53.0% and 41.8% in the 12-month group (hazard ratio [HR], 0.66; 95% CI, 0.49-0.87; P = .003). In the 36-month group, 5-year OS and 10-year OS rates were 92.0% and 79.0%, respectively, and in the 12-month group 85.5% and 65.3% (HR, 0.55; 95% CI, 0.37-0.83; P = .004). The results were similar in the efficacy population, from which 15 patients who did not have GIST in central pathology review and 24 patients who had intra-abdominal metastases removed at surgery were excluded (36-month group, 10-year OS 81.6%; 12-month group, 66.8%; HR, 0.50; 95% CI, 0.32-0.80; P = .003). No new safety signals were detected. Conclusions and Relevance: Three years of adjuvant imatinib is superior in efficacy compared with 1 year of imatinib. Approximately 50% of deaths may be avoided during the first 10 years of follow-up after surgery with longer adjuvant imatinib treatment. Trial Registration: ClinicalTrials.gov Identifier: NCT00116935.

What can we learn about functional importance of human antimicrobial peptide LL-37 in the oral environment from severe congenital neutropenia (Kostmann disease)?

The human antimicrobial peptide LL-37 is produced by neutrophils and epithelial cells, and the peptide can be detected in plasma as well as saliva. LL-37 is active against both gram-positive and gram-negative bacteria including oral pathogens such as Porphyromonas gingivalis and Streptococcus mutans. Besides its antimicrobial properties, LL-37 modulates the innate immune system, and furthermore, it also affects host cell viability. Although, both structural and functional properties of LL-37 have been extensively investigated, its physiological/pathophysiological importance in-vivo is not completely understood. In this review, Kostmann disease (morbus Kostmann) is highlighted since it may represent a LL-37 knockdown model which can provide new important information and insights about the functional role of LL-37 in the human in-vivo setting. Patients with Kostmann disease suffer from neutropenia, and although they are treated with recombinant granulocyte colony-stimulating factor (G-CSF) to normalize their levels of neutrophils, they lack or have very low levels of LL-37 in plasma, saliva and neutrophils. Interestingly, these patients suffer from severe periodontal disease, linking LL-37-deficiency to oral infections. Thus, LL-37 seems to play an important pathophysiological role in the oral environment antagonizing oral pathogens and thereby prevents oral infections.

Human host defense peptide LL-37 facilitates double-stranded RNA pro-inflammatory signaling through up-regulation of TLR3 expression in vascular smooth muscle cells.

OBJECTIVE: The importance of human host defense peptide LL-37 in vascular innate immunity is not understood. Here, we assess the impact of LL-37 on double-stranded RNA (dsRNA) signaling in human vascular smooth muscle cells. MATERIALS AND METHODS: Cellular import of LL-37 and synthetic dsRNA (poly I:C) were investigated by immunocytochemistry and fluorescence imaging. Transcript and protein expression were determined by qPCR, ELISA and Western blot. Knockdown of TLR3 was performed by siRNA. RESULTS: LL-37 was rapidly internalized, suggesting that it has intracellular actions. Co-stimulation with poly I:C and LL-37 enhanced pro-inflammatory IL-6 and MCP-1 transcripts several fold compared to treatment with poly I:C or LL-37 alone. Poly I:C increased IL-6 and MCP-1 protein production, and this effect was potentiated by LL-37. LL-37-induced stimulation of poly I:C signaling was not associated with enhanced import of poly I:C. Treatment with poly I:C and LL-37 in combination increased expression of dsRNA receptor TLR3 compared to stimulation with poly I:C or LL-37 alone. In TLR3 knockdown cells, treatment with poly I:C and LL-37 in combination had no effect on IL-6 and MCP-1 expression, showing loss of function. CONCLUSIONS: LL-37 potentiates dsRNA-induced cytokine production through up-regulation of TLR3 expression representing a novel pro-inflammatory mechanism.

Pretreatment Tumor DNA Sequencing of KIT and PDGFRA in Endosonography-Guided Biopsies Optimizes the Preoperative Management of Gastrointestinal Stromal Tumors.

BACKGROUND: Neoadjuvant tyrosine kinase inhibitor (TKI) therapy increases the chance of organ-preserving, radical resection in selected patients with gastrointestinal stromal tumors (GISTs). We aimed to evaluate systematic, immediate DNA sequencing of KIT and PDGFRA in pretreatment GIST tissue to guide neoadjuvant TKI therapy and optimize preoperative tumor response. METHODS: All patients who were candidates for neoadjuvant therapy of a suspected GIST [the study cohort (SC)] were prospectively included from January 2014 to March 2018. Patients were subjected to pretreatment endosonography-guided fine-needle biopsy (EUS-FNB) or transabdominal ultrasound-guided needle biopsy (TUS-NB), followed by immediate tumor DNA sequencing (< 2 weeks). A historic (2006-2013) reference cohort (RC) underwent work-up without sequencing before neoadjuvant imatinib (n = 42). The rate of optimal neoadjuvant therapy (TherapyOPTIMAL) was calculated, and the induced tumor size reduction (Tumor RegressionMAX, %) was evaluated by computed tomography (CT) scan. RESULTS: The success rate of pretreatment tumor DNA sequencing in the SC (n = 81) was 77/81 (95%) [EUS-FNB 71/74 (96%); TUS-NB 6/7 (86%)], with mutations localized in KIT (n = 58), PDGFRA (n = 18), or neither gene, wild type (n = 5). In patients with a final indication for neoadjuvant therapy, the TherapyOPTIMAL was higher in the SC compared with the RC [61/63 (97%) versus 33/42 (79%), p = 0.006], leading to a significantly higher Tumor RegressionMAX in patients treated with TKI (27% vs. 19%, p = 0.015). CONCLUSIONS: Pretreatment endosonography-guided biopsy sampling followed by immediate tumor DNA sequencing of KIT and PDGFRA is highly accurate and valuable in guiding neoadjuvant TKI therapy in GIST. This approach minimizes maltreatment with inappropriate regimens and leads to improved tumor size reduction before surgery.

Antimicrobial peptide LL-37 and its pro-form, hCAP18, in desquamated epithelial cells of human whole saliva.

The antimicrobial peptide LL-37 is active against oral bacteria and has been demonstrated to be present in human saliva, but its distribution in different fractions of saliva is not known. LL-37 is formed from its intracellular pro-form, hCAP18, in an extracellular enzymatic reaction catalyzed by proteinase 3 and kallikrein 5. Here, we prepared cell-containing and cell-free fractions of unstimulated human whole saliva by centrifugation after depolymerization of mucins with dithiothreitol, and measured the levels of hCAP18/LL-37 in these fractions using ELISA. Cellular expression of hCAP18/LL-37 was determined by western blotting and immunocytochemistry. The ELISA analyses demonstrated that both cells and cell-free saliva contained hCAP18/LL-37. Western blot analysis of cell-pellet homogenates showed a strong band corresponding to hCAP18 at the correct molecular weight and a weak band corresponding to LL-37. Phase-contrast and light microscopy revealed that the cells consisted of desquamated epithelial cells. These cells expressed cytoplasmic immunoreactivity for hCAP18/LL-37. The peripheral part of the cytoplasm, corresponding to the plasma membrane, was particularly rich in hCAP18/LL-37 immunoreactivity. No immunoreactivity was observed after omission of the primary antibody. We conclude that desquamated epithelial cells of human whole saliva contain antimicrobial hCAP18/LL-37, suggesting that these cells may take part in the innate immune system by harboring and releasing these peptides.