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.

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.

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.

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.

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.

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.

Odontoblast-like MDPC-23 cells produce pro-inflammatory IL-6 in response to lipoteichoic acid and express the antimicrobial peptide CRAMP.

Objective: Odontoblasts are thought to be involved in innate immunity but their precise role in this process is not fully understood. Here, we assess effects of lipopolysaccharide (LPS) and lipoteichoic acid (LTA), produced by Gram-negative and Gram-positive bacteria, respectively, on matrix metalloproteinase-8 (MMP-8), interleukin-6 (IL-6) and cathelin-related antimicrobial peptide (CRAMP) expression in odontoblast-like MDPC-23 cells.Material and methods: Gene activity and protein production was determined by quantitative real-time RT-PCR and ELISA, respectively. Cellular expression of CRAMP was determined by immunocytochemistry.Results: Stimulation with LTA (5 and 25 µg/ml) but not LPS (1 and 5 µg/ml) for 24 h enhanced IL-6 mRNA expression. The LTA-induced up-regulation of IL-6 mRNA levels was associated with increased IL-6 protein levels. Stimulation with either LPS or LTA for 24 h lacked effect on both MMP-8 transcript and protein expression. Immunocytochemistry disclosed that MDPC-23 cells expressed immunoreactivity for CRAMP. MDPC-23 cells showed mRNA expression for CRAMP, but stimulation with either LPS or LTA did not modulate CRAMP transcript expression.Conclusions: We show that MDPC-23 cells possess immune-like cell properties such as LTA-induced IL-6 production and expression of the antimicrobial peptide CRAMP, suggesting that odontoblasts may modulate innate immunity via these mechanisms.

Polyamines and microbiota in bicuspid and tricuspid aortic valve aortopathy.

Polyamines are small aliphatic cationic molecules synthesized via a highly regulated pathway and involved in general molecular and cellular phenomena. Both mammalian cells and microorganisms synthesize polyamines, and both sources may contribute to the presence of polyamines in the circulation. The dominant location for microorganisms within the body is the gut. Accordingly, the gut microbiota probably synthesizes most of the polyamines in the circulation in addition to those produced by the mammalian host cells. Polyamines are mandatory for cellular growth and proliferation. Established evidence suggests that the polyamine spermidine prolongs lifespan and improves cardiovascular health in animal models and humans through both local mechanisms, involving improved cardiomyocyte function, and systemic mechanisms, including increased NO bioavailability and reduced systemic inflammation. Higher levels of polyamines have been detected in non-dilated aorta of patients affected by bicuspid aortic valve congenital malformation, an aortopathy associated with an increased risk for thoracic ascending aorta aneurysm. In this review, we discuss metabolism of polyamines and their potential effects on vascular smooth muscle and endothelial cell function in vascular pathology of the thoracic ascending aorta associated with bicuspid or tricuspid aortic valve.

Beneficial effects of spermidine on cardiovascular health and longevity suggest a cell type-specific import of polyamines by cardiomyocytes.

Recent and exciting in vivo studies show that supplementation with the polyamine spermidine (Spd) is cardioprotective and prolongs lifespan in both mice and humans. The mechanisms behind Spd-induced cardioprotection are supposed to involve Spd-evoked stimulation of autophagy, mitophagy and mitochondrial respiration and improved the mechano-elastical function of cardiomyocytes. Although cellular uptake of Spd was not characterized, these results suggest that Spd is imported by the cardiomyocytes and acts intracellularly. In the light of these new and thrilling data, we discuss in the present review cellular polyamine import with a special focus on mechanisms that may be relevant for Spd uptake by electrically excitable cells such as cardiomyocytes.

The host defense peptide LL-37 is internalized by human periodontal ligament cells and prevents LPS-induced MCP-1 production.

OBJECTIVE: The human host defense peptide LL-37 both shows antimicrobial effects and modulates host cell properties. Here, we assess the effects of synthesized LL-37 on lipopolysaccharide (LPS)-induced inflammation in human periodontal ligament (PDL) cells and investigates underlying mechanisms. BACKGROUND: LL-37 has been detected in the periodontal tissues, but its functional importance for PDL cell innate immune responses is not known. METHODS: Human PDL cells were obtained from premolars extracted on orthodontic indications. Cellular pro-inflammatory monocyte chemoattractant protein-1 (MCP-1) mRNA expression was determined using quantitative real-time RT-PCR. MCP-1 protein production was assessed by western blot and ELISA. Internalization of LL-37 by PDL cells was visualized by immunocytochemistry. Nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-κB) activity was assessed by western blot of phosphorylated p65, phosphorylated p105, and IκBα proteins. Binding of LL-37 to PDL cell DNA was determined by isolation and purification of DNA and dot blot for LL-37 immunoreactivity. RESULTS: Treatment with LL-37 (1 µmol/L) for 24 hours prevented LPS-induced stimulation of MCP-1 expression analyzed both on transcript and on protein levels. Stimulation with LL-37 (1 µmol/L) for 24 hours had no effect on toll-like receptor (TLR)2 and TLR4 transcript expression, suggesting that LL-37 acts downstream of the TLRs. Preincubation with LL-37 for 60 minutes followed by stimulation with LPS for 24 hours in the absence of LL-37 completely prevented LPS-evoked MCP-1 transcript expression, implying that LL-37 acts intracellularly and not via binding and neutralization of LPS. In PDL cells stimulated with LL-37 for 60 minutes, the peptide was internalized as demonstrated by immunocytochemistry, suggesting an intracellular mechanism of action. LL-37 immunoreactivity was observed both in the cytosol and in the nucleus. Downregulation of LPS-induced MCP-1 by LL-37 was not mediated by reduction in NF-κB activity as shown by unaltered expression of phosphorylated p65, phosphorylated p105, and IκBα NF-κB proteins in the presence of LL-37. Immunoreactivity for LL-37 was observed in PDL cell DNA treated with but not without 0.1 and 1 µmol/L LL-37 for 60 minutes in vitro. CONCLUSION: LL-37 abolishes LPS-induced MCP-1 production in human PDL cells through an intracellular, NF-κB-independent mechanism which probably involves direct interaction between LL-37 and DNA.

Globular C1q receptor (p33) binds and stabilizes pro-inflammatory MCP-1: a novel mechanism for regulation of MCP-1 production and function.

The protein gC1qR (globular C1q receptor), also named p33, was originally identified as a binding partner of the globular heads of C1q in the complement system. gC1qR/p33 is abundantly expressed in many cell types, but the functional importance of this protein is not completely understood. Here, we investigate the impact of gC1qR/p33 on the production and function of the pathophysiologically important chemokine monocyte chemoattractant protein-1 (MCP-1) and the underlying molecular mechanisms. Knockdown of gC1qR/p33 negatively regulated the production of MCP-1, but had no effect on the expression of transcript for MCP-1 in human periodontal ligament cells, suggesting a translational/post-translational mechanism of action. Laser scanning confocal microscopy showed considerable cytosolic co-localization of gC1qR/p33 and MCP-1, and co-immunoprecipitation disclosed direct physical interaction between gC1qR/p33 and MCP-1. Surface plasmon resonance analysis revealed a high-affinity binding (KD = 10.9 nM) between gC1qR/p33 and MCP-1. Using a transwell migration assay, we found that recombinant gC1qR/p33 enhances MCP-1-induced migration of human THP-1 monocytes, pointing to a functional importance of the interaction between gC1qR/p33 and MCP-1. An in vitro assay revealed a rapid turnover of the MCP-1 protein and that gC1qR/p33 stabilizes MCP-1, hence preventing its degradation. We propose that endogenous gC1qR/p33 physically interacts with MCP-1 causing stabilization of the MCP-1 protein and stimulation of its activity in human periodontal ligament cells, suggesting a novel gC1qR/p33-mediated pro-inflammatory mechanism of action.

Clinical relevance of integrin alpha 4 in gastrointestinal stromal tumours.

The molecular mechanisms for the dissemination and metastasis of gastrointestinal stromal tumours (GIST) are incompletely understood. The purpose of the study was to investigate the clinical relevance of integrin alpha 4 (ITGA4) expression in GIST. GIST transcriptomes were first compared with transcriptomes of other types of cancer and histologically normal gastrointestinal tract tissue in the MediSapiens in silico database. ITGA4 was identified as an unusually highly expressed gene in GIST. Therefore, the effects of ITGA4 knock-down and selective integrin alpha 4 beta 1 (VLA-4) inhibitors on tumour cell proliferation and invasion were investigated in three GIST cell lines. In addition, the prognostic role of ITGA4 expression in cancer cells was investigated in a series of 147 GIST patients with immunohistochemistry. Inhibition of ITGA4-related signalling decreased GIST cell invasion in all investigated GIST cell lines. ITGA4 protein was expressed in 62 (42.2%) of the 147 GISTs examined, and expression was significantly associated with distant metastases during the course of the disease and several adverse prognostic features. Patients whose GIST expressed strongly ITGA4 had unfavourable GIST-specific survival and overall survival compared to patients with low or no ITGA4 expression. Taken together, ITGA4 is an important integrin in the molecular pathogenesis of GIST and may influence their clinical behaviour.

Array profiling reveals contribution of Cthrc1 to growth of the denervated rat urinary bladder.

Bladder denervation and bladder outlet obstruction are urological conditions that cause bladder growth. Transcriptomic surveys in outlet obstruction have identified differentially expressed genes, but similar studies following denervation have not been done. This was addressed using a rat model in which the pelvic ganglia were cryo-ablated followed by bladder microarray analyses. At 10 days following denervation, bladder weight had increased 5.6-fold, and 2,890 mRNAs and 135 micro-RNAs (miRNAs) were differentially expressed. Comparison with array data from obstructed bladders demonstrated overlap between the conditions, and 10% of mRNAs changed significantly and in the same direction. Many mRNAs, including collagen triple helix repeat containing 1 ( Cthrc1), Prc1, Plod2, and Dkk3, and miRNAs, such as miR-212 and miR-29, resided in the shared signature. Discordantly regulated transcripts in the two models were rare, making up for <0.07% of all changes, and the gene products in this category localized to the urothelium of normal bladders. These transcripts may potentially be used to diagnose sensory denervation. Western blotting demonstrated directionally consistent changes at the protein level, with increases of, e.g., Cthrc1, Prc1, Plod2, and Dkk3. We chose Cthrc1 for further studies and found that Cthrc1 was induced in the smooth muscle cell (SMC) layer following denervation. TGF-ß1 stimulation and miR-30d-5p inhibition increased Cthrc1 in bladder SMCs, and knockdown and overexpression of Cthrc1 reduced and increased SMC proliferation. This work defines common and distinguishing features of bladder denervation and obstruction and suggests a role for Cthrc1 in bladder growth following denervation.

LL-37-induced human osteoblast cytotoxicity and permeability occurs independently of cellular LL-37 uptake through clathrin-mediated endocytosis.

The host defense peptide LL-37 is cytotoxic for bacteria but it has also been reported to reduce host cell viability through an intracellular mechanism. LL-37-evoked cytotoxicity may be involved in the loss of bone tissue in periodontitis which is an inflammatory disease characterized by high concentrations of LL-37 observed locally in the periodontal tissue at the inflammation process. Here, we showed that LL-37 reduced human osteoblast-like MG63 cell viability assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and increased plasma membrane permeability determined by measuring intracellular Ca2+ levels and lactate dehydrogenase (LDH) release. Treatment with chlorpromazine, a well-recognized inhibitor of clathrin-mediated endocytosis, reduced cellular uptake of synthesized LL-37 b y about 30% assessed by Western blotting and ELISA, while filipin, an inhibitor of caveolin-mediated endocytosis, had no effect. The chlorpromazine-induced attenuation of LL-37 uptake was not associated with modulation of LL-37-induced cytotoxicity and LL-37-evoked plasma membrane permeability. Clathrin heavy chain 2 is a major protein of the polyhedral coat of coated pits and vesicles encoded by clathrin heavy chain like 1 gene. Down-regulation of clathrin heavy chain like 1 gene activity by siRNA reduced uptake of LL-37 but did not affect LL-37-induced cytotoxicity and permeability. Thus, we show, using both a pharmacological approach and knockdown of clathrin heavy chain like 1 expression, that LL-37-induced MG63 cell cytotoxicity and permeability occurs independently of LL-37 uptake via clathrin-mediated endocytosis.

High clinical impact and diagnostic accuracy of EUS-guided biopsy sampling of subepithelial lesions: a prospective, comparative study.

BACKGROUND: In a tertiary center setting we aimed to study the diagnostic accuracy and clinical impact of EUS-guided biopsy sampling (EUS-FNB) with a reverse bevel needle compared with that of fine needle aspiration (EUS-FNA) in the work-up of subepithelial lesions (SEL). METHODS: All patients presenting with SELs referred for EUS-guided sampling were prospectively included in 2012-2015. After randomization of the first pass modality, dual sampling with both EUS-FNB and EUS-FNA was performed in each lesion. Outcome measures in an intention-to-diagnose analysis were the diagnostic accuracy, technical failures, and adverse events. The clinical impact was measured as the performance of additional diagnostic procedures post-EUS and the rate of unwarranted resections compared with a reference cohort of SELs sampled in the same institution 2006-2011. RESULTS: In 70 dual sampling procedures of unique lesions (size: 6-220 mm) the diagnostic sensitivity for malignancy and the overall accuracy of EUS-FNB was superior to EUS-FNA compared head-to-head (90 vs 52%, and 83 vs 49%, both p < 0.001). The adverse event rate of EUS-FNB was low (1.2%). EUS-FNB in 2012-2015 had a positive clinical impact in comparison with the reference cohort demonstrated by less cases referred for an additional diagnostic procedure, 12/83 (14%) vs 39/73 (53%), p < 0.001, and fewer unwarranted resections in cases subjected to surgery, 3/48 (6%) vs 12/35 (34%), p = 0.001. CONCLUSIONS: EUS-FNB with a reverse bevel needle is safe and superior to EUS-FNA in providing a conclusive diagnosis of subepithelial lesions. This biopsy sampling approach facilitates a rational clinical management and accurate treatment.

The host defense peptide LL-37 is detected in human parotid and submandibular/sublingual saliva and expressed in glandular neutrophils.

The human host defense peptide, LL-37, is an important player in the first line of defense against invading microorganisms. LL-37 and its precursor, hCAP18, have been detected in unstimulated whole saliva but no reports showing hCAP18/LL-37 in isolated, parotid, and/or submandibular/sublingual saliva have been presented. Here, we measured the levels of hCAP18/LL-37 in human parotid and submandibular/sublingual saliva and investigated the expression of hCAP18/LL-37 in parotid and submandibular gland tissue. Parotid and submandibular/sublingual saliva was collected from healthy volunteers, and the levels of hCAP18/LL-37 in saliva were analyzed by dot blot, ELISA, and western blotting. Cellular expression of hCAP18/LL-37 in human parotid and submandibular glands was investigated by immunohistochemistry. Immunoreactivity for hCAP18/LL-37 was detected in both parotid and submandibular/sublingual saliva of all individuals. The concentration of hCAP18/LL-37 was similar in parotid and submandibular/sublingual saliva, and was determined by densitometric scanning of each dot and normalization to the total protein concentration of each sample, and by ELISA. Double immunohistochemistry revealed that intravascular neutrophils of both parotid and submandibular glands express hCAP18/LL-37. For the first time, we demonstrate hCAP18/LL-37 in isolated human parotid and submandibular/sublingual saliva and expression of hCAP18/LL-37 in glandular intravascular neutrophils, indicating that neutrophils of the major salivary glands contribute to the LL-37 content of whole saliva.

Polyamine concentration is increased in thoracic ascending aorta of patients with bicuspid aortic valve.

Polyamines are cationic molecules synthesized via a highly regulated pathway, obtained from the diet or produced by the gut microbiota. They are involved in general molecular and cellular phenomena that play a role also in vascular disease. Bicuspid aortic valve (BAV) is a congenital malformation associated to a greater risk of thoracic ascending aorta (TAA) aneurysm, whose pathogenesis is not yet well understood. We focused on differential analysis of key members of polyamine pathway and on polyamine concentration in non-dilated TAA samples from patients with either stenotic tricuspid aortic valve (TAV) or BAV (diameter ≤ 45 mm), vs. normal aortas from organ donors, with the aim of revealing a potential involvement of polyamines in early aortopathy. Changes of gene expression in TAA samples were evaluated by RT-PCR. Changes of ornithine decarboxylase 1 (ODC1), a key enzyme in polyamine formation, and cationic amino acid transporter 1 (SLC7A1/CAT-1) expression were analyzed also by Western blot. ODC1 subcellular localization was assessed by immunohistochemistry. Polyamine concentration in TAA samples was evaluated by HPLC. BAV TAA samples showed an increased concentration of putrescine and spermidine vs. TAV and donor samples, together with a decreased mRNA level of polyamine anabolic enzymes and of the putative polyamine transporter SLC7A1/CAT-1. The catabolic enzyme spermidine/spermine N1-acetyltransferase 1 showed a significant mRNA increase in TAV samples only, together with a decreased concentration of spermine. The decreased expression of SLC7A1/CAT-1 and ODC1 mRNAs in BAV corresponded to increased or unchanged expression of the respective proteins. ODC was located mainly in smooth muscle cell (SMC) nucleus in TAV and donor samples, while it was present also in SMC cytoplasm in BAV samples, suggesting its activation. In conclusion, BAV, but not TAV non-dilated samples show increased polyamine concentration, accompanied by the activation of a regulatory negative feedback mechanism.

Pyk2 inhibition promotes contractile differentiation in arterial smooth muscle.

Modulation from contractile to synthetic phenotype of vascular smooth muscle cells is a central process in disorders involving compromised integrity of the vascular wall. Phenotype modulation has been shown to include transition from voltage-dependent toward voltage-independent regulation of the intracellular calcium level, and inhibition of non-voltage dependent calcium influx contributes to maintenance of the contractile phenotype. One possible mediator of calcium-dependent signaling is the FAK-family non-receptor protein kinase Pyk2, which is activated by a number of stimuli in a calcium-dependent manner. We used the Pyk2 inhibitor PF-4594755 and Pyk2 siRNA to investigate the role of Pyk2 in phenotype modulation in rat carotid artery smooth muscle cells and in cultured intact arteries. Pyk2 inhibition promoted the expression of smooth muscle markers at the mRNA and protein levels under stimulation by FBS or PDGF-BB and counteracted phenotype shift in cultured intact carotid arteries and balloon injury ex vivo. During long-term (24-96 hr) treatment with PF-4594755, smooth muscle markers increased before cell proliferation was inhibited, correlating with decreased KLF4 expression and differing from effects of MEK inhibition. The Pyk2 inhibitor reduced Orai1 and preserved SERCA2a expression in carotid artery segments in organ culture, and eliminated the inhibitory effect of PDGF stimulation on L-type calcium channel and large-conductance calcium-activated potassium channel expression in carotid cells. Basal intracellular calcium level, calcium wave activity, and store-operated calcium influx were reduced after Pyk2 inhibition of growth-stimulated cells. Pyk2 inhibition may provide an interesting approach for preserving vascular smooth muscle differentiation under pathophysiological conditions.

SLUG transcription factor: a pro-survival and prognostic factor in gastrointestinal stromal tumour.

BACKGROUND: The SLUG transcription factor has been linked with the KIT signalling pathway that is important for gastrointestinal stromal tumour (GIST) tumourigenesis. Its clinical significance in GIST is unknown. METHODS: Influence of SLUG expression on cell proliferation and viability were investigated in GIST48 and GIST882 cell lines. The association between tumour SLUG expression in immunohistochemistry and recurrence-free survival (RFS) was studied in two clinical GIST series, one with 187 patients treated with surgery alone, and another one with 313 patients treated with surgery and adjuvant imatinib. RESULTS: SLUG downregulation inhibited cell proliferation, induced cell death in both cell lines, and sensitised GIST882 cells to lower imatinib concentrations. SLUG was expressed in 125 (25.0%) of the 500 clinical GISTs evaluated, and expression was associated with several factors linked with unfavourable prognosis. SLUG expression was associated with unfavourable RFS both when patients were treated with surgery alone (HR=3.40, 95% CI=1.67-6.89, P=0.001) and when treated with surgery plus adjuvant imatinib (HR=1.83, 95% CI=1.29-2.60, P=0.001). CONCLUSIONS: GIST patients with high tumour SLUG expression have unfavourable RFS. SLUG may mediate pro-survival signalling in GISTs.