Novel specific human and mouse stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10) antibodies for biochemical and immunohistochemical analyses.

Matrix metalloproteinases (MMP) are a family of more than 25 zinc-dependent enzymes that are centrally involved in cellular migration, tissue remodeling, cancer invasion and metastasis. Besides degrading extracellular matrix proteins, MMPs are crucial for growth factor and cytokine release and activation. At the same time, they can inactivate inflammatory mediators and enzymes themselves through protein degradation. Subclasses of MMPs include collagenases, gelatinases, stromelysins, membrane-bound MMPs, and others. With regard to the stromelysin subfamily, three members exist, e.g., stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), and stromelysin-3 (MMP-11). MMP-3, and MMP-10 share extensive similarities at the amino acid level that made it difficult to develop specific antibodies distinguishing between MMP-3 and MMP-10. Scrutinizing published data on and performing different analyses with detection of both stromelysins with commercially available or lab-made antibodies showed ambiguous results with regard to specificity of antibodies used to date. We developed new specific antibodies against the most divergent parts of the active forms of both proteins. We assessed the specificity of our novel specific anti-human and anti-mouse MMP-3 and MMP-10 antibodies in cell lysates and different human and murine skin tissues. Tests analyzing specificity of the novel antibodies included Western immunoblotting, immunofluorescence, and immunohistochemistry on paraffin sections. Analyses demonstrated specific detection of respective protein for human or mouse samples except for the anti-human MMP-3 antibody. The aim of this summary was to call attention the MMP research community to distinguish clearly between both enzymes. Our new specific anti-mouse MMP-3 and both MMP-10 antibodies allow us to address this detection problem and to enable comparative studies between both stromelysins with regard to their respective location and function in the tissue.

n-3 Fatty acids combined with flavan-3-ols prevent steatosis and liver injury in a murine model of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) affects 25% of adults and at present no licensed medication has been approved. Despite its complex patho-physiology, dietary strategies aiming at delaying or preventing NAFLD have taken a reductionist approach, examining the impact of single components. Accumulating evidence suggests that n-3 LC-PUFAs are efficacious in regulating lipogenesis and fatty acid oxidation. In addition, plant derived flavonoids are also emerging as a dietary strategy for NAFLD prevention, with efficacy attributed to their insulin sensitising and indirect antioxidant effects. Based on knowledge of their complementary molecular targets, we aimed to demonstrate that the combination of n-3 LC-PUFA (n-3) and flavan-3-ols (FLAV) prevents NAFLD. In a high-fat high-fructose (HF/HFr) fed C57Bl/6J mouse model, the independent and interactive impact of n-3 and FLAV on histologically defined NAFLD, insulin sensitivity, weight gain, intestinal and hepatic gene expression, intestinal bile acids were examined. Only the combination of FLAV and n-3 (FLAVn-3) prevented steatosis as evidenced by a strong reduction in hepatocyte ballooning. While FLAV reduced body (-28-30%), adipose tissue (-45-50%) weights and serum insulin (-22-25%) as observed following an intra-peritoneal glucose tolerance test, n-3 downregulated the expression of Srebf1 and the lipogenic genes (Acaca, Fasn). Significant impacts of interventions on intestinal bile acid metabolism, farnesoid X receptor (Fxr) signalling in the intestine and liver, and hepatic expression of fatty acid transporters (Fabp4, Vldlr, Cd36) were also evident. FLAVn-3 may be a novel intervention for NAFLD. Future research should aim to demonstrate its efficacy in the prevention and treatment of human NAFLD.

Imaging of compartmentalised intracellular nitric oxide, induced during bacterial phagocytosis, using a metalloprotein-gold nanoparticle conjugate.

Nitric oxide (NO) plays an essential role within the immune system since it is involved in the break-down of infectious agents such as viruses and bacteria. The ability to measure the presence of NO in the intracellular environment would provide a greater understanding of the pathophysiological mechanism of this important molecule. Here we report the detection of NO from the intracellular phagolysosome using a fluorescently tagged metalloprotein-gold nanoparticle conjugate. The metalloprotein cytochrome c, fluorescently tagged with an Alexa Fluor dye, was self-assembled onto gold nanoparticles to produce a NO specific nanobiosensor. Upon binding of NO, the cytochrome c protein changes conformation which induces an increase of fluorescence intensity of the tagged protein proportional to the NO concentration. The nanobiosensor was sensitive to NO in a reversible and selective manner, and exhibited a linear response at NO concentrations between 1 and 300 µM. In RAW264.7γ NO- macrophage cells, the nanobiosensor was used to detect the presence of NO that had been endogenously generated upon stimulation of the cells with interferon-γ and lipopolysaccharide, or spontaneously released following treatment of the cells with a NO donor. Significantly, the nanobiosensor was shown to be taken up by the macrophages within phagolysosomes, i.e., the precise location where the NO, together with other species, destroys bacterial infection. The nanobiosensor measured, for the first time, increasing concentrations of NO produced during combined stimulation and phagocytosis of Escherichia coli bacteria from within localised intracellular phagolysosomes, a key part of the immune system.

Effect of tissue inhibitor of metalloproteinases 3 on DLK1 shedding in cultured human pre-adipocytes and implications for adipose tissue remodelling.

BACKGROUND: Metabolically unhealthy obesity is associated with adipose tissue inflammation and increased risk of type 2 diabetes. Dysfunctional adipose tissue remodelling has been implicated in development of metabolically unhealthy obesity, but the pathogenesis remains poorly characterised. We hypothesised that in health, tissue inhibitor of metalloproteinases 3 (TIMP3) modulates adipose tissue remodelling by regulating extracellular matrix turnover and shedding of the adipogenic regulator DLK1, but that in adipose tissue inflammation it might drive development of metabolically unhealthy obesity. METHODS: Primary pre-adipocyte and in-vitro-differentiated adipocyte cultures were established from abdominal subcutaneous adipose tissue donated by healthy women undergoing breast reconstruction. Cells were seeded onto collagen I, and subsequently treated with differentiation medium or tumour necrosis factor (TNF) alpha (50 ng/mL). Adenoviral transduction allowed TIMP3 overexpression. Media and lysates were collected for quantitative RT-PCR, and immunoblot and hydroxyproline release assays. Statistical analysis was performed with t testing or ANOVA. FINDINGS: Induction of differentiation in human pre-adipocytes reduced TIMP3 mRNA levels by 75% (n=3, p<0·0001). Hydroxyproline release by differentiating pre-adipocytes was 2·3 times greater than that by control-treated cells (mean 5·66 µg/mL [SD 0·77] vs 2·45 [0·36], p<0·0001) indicating greater collagen I degradation. TNF alpha reduced TIMP3 mRNA levels by 66% in in-vitro-differentiated adipocytes (n=3, p<0·0001); reduced TIMP3 expression was confirmed by western blot. Shedding of soluble DLK1 (sDLK1) by pre-adipocytes was increased by TNF alpha and by overexpression of adenovirally delivered TIMP3 compared with control conditions, as confirmed by immunoblot (n=3). Addition of recombinant human sDLK1 (500 pM) to pre-adipocyte cultures reduced adipogenesis, as assessed by oil red O staining (n=2). INTERPRETATION: We have shown that TIMP3 is downregulated in adipogenesis, and by inflammatory signals in adipocytes. Furthermore, TIMP3 modulates sDLK1 shedding and collagen I degradation. TIMP3 is known to inhibit ADAM17 (DLK1 sheddase) and MMP14 (implicated in extracellular matrix turnover). TIMP3 might therefore integrate inflammatory signals with adipose remodelling. Subversion of remodelling pathways by chronic adipose inflammation might lead to maladaptive adipose expansion and metabolically unhealthy obesity. FUNDING: British Heart Foundation, Diabetes Research and Wellness Foundation Open Funding 2011.

The role of metalloproteinases and their tissue inhibitors in adipose tissue remodelling and whole-body lipid distribution: a cross-sectional clinical study.

BACKGROUND: Metabolically unhealthy obesity is associated with insulin resistance. Dysfunctional adipose tissue remodelling might explain features of this disorder, such as chronic white adipose tissue inflammation, adipocyte hypertrophy, and ectopic lipid deposition. Metalloproteinases and their tissue inhibitors (TIMPs) have been implicated in human adipose tissue remodelling. In a cross-sectional study, we investigated the association of adipose metalloproteinase and TIMP expression with whole-body lipid distribution and insulin resistance. METHODS: Healthy women undergoing elective surgery donated fasting blood samples (for calculation of homoeostasis model assessment of insulin resistance [HOMA2-IR], the primary outcome). At operation 2 cm(3) biopsy samples of subcutaneous and visceral adipose tissue were obtained. 1 cm(3) was fixed, paraffin-embedded, and stained for adipocyte size quantification, and RNA was extracted from the remaining tissue for quantitative RT-PCR analysis. The women also underwent whole-body MRI for analysis of fat distribution. FINDINGS: 26 women were recruited (mean age 50·3 years, SD 13·1) into five body-mass index categories (18·5-24·9 kg/m(2) [n=12, 46·1%], 25-29·9 [n=6, 23·1%], 30-34·9 [n=3, 11·5%], 35-39·9 [n=3, 11·5%], >40 [n=2, 7·8%]). Mean fasting glucose was 5·29 mmol/L (SD 0·66), mean fasting insulin 71·29 pmol/L (47·72), and mean HOMA2-IR 1·35 (0·91). HOMA2-IR correlated with body-mass index (r=0·73, p<0·0001), subcutaneous and visceral adipose tissue volumes (r=0·94 and r=0·87, respectively; both p<0·0001), and hepatic fat fraction (r=0·57, p=0·013). Visceral adipose tissue MMP14 expression correlated strongly with hepatic fat fraction (r=0·944, p<0·0001), HOMA2-IR (r=0·74, p=0·01), and visceral adipose tissue volume (r=0·74, p=0·036). Subcutaneous adipose tissue TIMP3 expression correlated with subcutaneous adipocyte area (r=0·72, p=0·029), but not with HOMA2-IR (r=-0·53, p=0·062). INTERPRETATION: The results suggest that metalloproteinases and TIMPs regulate adipose tissue remodelling and distribution. MMP14 has been implicated in collagen turnover in pre-adipocyte differentiation, whereas TIMP3 may modulate the shedding of DLK1, a regulator of adipogenesis. In our concurrent in-vitro study, we have shown that human adipocytes express metalloproteinases and TIMPs, and that their expression varies with inflammatory stimulation. These proteins might therefore integrate inflammatory signals with dysregulated adipose remodelling in metabolically unhealthy obesity. FUNDING: British Heart Foundation, Diabetes Research & Wellness Foundation Open Funding 2011.

Metalloproteinase-dependent and -independent processes contribute to inhibition of breast cancer cell migration, angiogenesis and liver metastasis by a disintegrin and metalloproteinase with thrombospondin motifs-15.

The ADAMTS proteinases are a family of secreted, matrix-associated enzymes that have diverse roles in the regulation of tissue organization and vascular homeostasis. Several of the 19 human family members have been identified as having either tumor promoting or suppressing roles. We previously demonstrated that decreased ADAMTS15 expression correlated with a worse clinical outcome in mammary carcinoma (e.g., Porter et al., Int J Cancer 2006;118:1241-7). We have explored the effects of A Disintegrin and Metalloproteinase with Thrombospondin motifs-15 (ADAMTS-15) on the behavior of MDA-MB-231 and MCF-7 breast cancer cells by stable expression of either a wild-type (wt) or metalloproteinase-inactive (E362A) protein. No effects on mammary cancer cell proliferation or apoptosis were observed for either form of ADAMTS-15. However, both forms reduced cell migration on fibronectin or laminin matrices, though motility on a Type I collagen matrix was unimpaired. Knockdown of syndecan-4 attenuated the inhibitory effects of ADAMTS-15 on cell migration. In contrast to its effects on cell migration, wt ADAMTS-15 but not the E362A inactive mutant inhibited endothelial tubulogenesis in 3D collagen gels and angiogenesis in the aortic ring assay. In experimental metastasis assays in nude mice, MDA-MB-231 cells expressing either form of ADAMTS-15 showed reduced spread to the liver, though lung colonization was enhanced for cells expressing wt ADAMTS-15. These studies indicate that extracellular ADAMTS-15 has multiple actions on tumor pathophysiology. Via modulation of cell-ECM interactions, which likely involve syndecan-4, it attenuates mammary cancer cell migration independent of its metalloproteinase activity; however, its antiangiogenic action requires catalytic functionality, and its effects on metastasis in vivo are tissue niche-dependent.

How psychiatrists should introduce themselves in the first consultation: an experimental study.

BACKGROUND: How psychiatrists introduce themselves in the first consultation may influence the therapeutic relationship. There is no evidence about what type of introduction patients prefer. AIMS: To assess experimentally patients' preferences for how psychiatrists introduce themselves. METHOD: Twelve psychiatrists were filmed, each with three different introductions: stating name, profession and reason for consultation; the same, plus information on what will happen during the consultation; and the same, plus disclosure of a personal difficulty. Six randomly selected videos, of different psychiatrists, two of each type of introduction, were rated by each of 120 psychiatric in- and out-patients on Likert-type scales. RESULTS: Patients gave the most positive ratings to psychiatrists who introduced themselves with information about what will happen in the consultation rather than ones with briefer introductions or with additional personal disclosure (P = 0.002). Preferences were similar in different subgroups. CONCLUSIONS: Psychiatrists should introduce themselves with information about what they intend to do in the consultation, but without personal disclosure.

Dual regulation of metalloproteinase expression in chondrocytes by Wnt-1-inducible signaling pathway protein 3/CCN6.

OBJECTIVE: Wnt-1-inducible signaling pathway protein 3 (WISP-3)/CCN6 is mutated in progressive pseudorheumatoid dysplasia and may have effects on cartilage homeostasis. The aim of this study was to ascertain additional roles for WISP-3/CCN6 by determining its expression in osteoarthritic (OA) cartilage and by investigating its effects on cartilage-relevant metalloproteinase expression in immortalized (C-28/I2) and primary chondrocytes. METHODS: Cartilage steady-state levels of WISP-3/CCN6 messenger RNA and protein production were determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. WISP-3/CCN6 was overexpressed in C-28/I2 cells, and the resultant clones were analyzed by quantitative RT-PCR. The stable clones were analyzed by RT-PCR for metalloproteinase expression, and the signaling pathways involved were investigated using pharmacologic inhibition. The effects of WISP-3/CCN6 on metalloproteinase expression in primary chondrocytes were investigated using a small interfering RNA approach. RESULTS: WISP-3/CCN6 was highly expressed in OA cartilage compared with undamaged cartilage, at both the RNA and protein levels. WISP-3/CCN6 overexpression in C-28/I2 cells resulted in unexpected dual regulation of metalloproteinases; expression of the potent aggrecanase ADAMTS-5 was down-regulated 9-fold, while expression of MMP-10 was up-regulated 14-fold, and these responses were accentuated in the WISP-3/CCN6 clones grown in suspension. MMP-10 up-regulation was dependent on several MAPKs, but WISP-3/CCN6-mediated ADAMTS-5 repression was independent of these pathways and was partially relieved by activation of ß-catenin signaling. WISP-3/CCN6 also suppressed ADAMTS-5 expression in C-28/I2 cells treated with cytokines. In cytokine-treated primary chondrocytes, gene silencing of WISP-3/CCN6 resulted in enhanced ADAMTS-5 expression, while MMP-10 expression was suppressed. CONCLUSION: WISP-3/CCN6 was highly expressed in end-stage OA cartilage, suggesting a role for this growth factor in cartilage homeostasis. WISP-3/CCN6-induced repression of ADAMTS-5 expression and regulation of MMP-10 expression suggest complex and context-dependent roles for WISP-3/CCN6 in cartilage biology.

Psychological symptoms as long-term consequences of war experiences.

BACKGROUND/AIMS: War experiences can affect mental health, but large-scale studies on the long-term impact are rare. We aimed to assess long-term mental health consequences of war in both people who stayed in the conflict area and refugees. METHOD: On average 8 years after the war in former Yugoslavia, participants were recruited by probabilistic sampling in 5 Balkan countries and by registers and networking in 3 Western European countries. General psychological symptoms were assessed on the Brief Symptom Inventory and posttraumatic stress symptoms on the Impact of Event Scale-Revised. RESULTS: We assessed 3,313 interviewees in the Balkans and 854 refugees. Paranoid ideation and anxiety were the severest psychological symptoms in both samples. In multivariable regressions, older age, various specific war experiences and more traumatic experiences after the war were all associated with higher levels of both general psychological and posttraumatic stress symptoms in both samples. Additionally, a greater number of migration stressors and having only temporary legal status in the host country were associated with greater severity of symptoms in refugees. CONCLUSIONS: Psychological symptoms remain high in war-affected populations many years after the war, and this is particularly evident for refugees. Traumatic war experiences still predict higher symptom levels even when the findings have been adjusted for the influence of other factors.

HGF/c-Met/ß1-integrin signalling axis induces tunneling nanotubes in A549 lung adenocarcinoma cells.

Tunneling nanotubes (TNTs) are thin cytoplasmic extensions involved in long-distance intercellular communication and can transport intracellular organelles and signalling molecules. In cancer cells, TNT formation contributes to cell survival, chemoresistance, and malignancy. However, the molecular mechanisms underlying TNT formation are not well defined, especially in different cancers. TNTs are present in non-small cell lung cancer (NSCLC) patients with adenocarcinoma. In NSCLC, hepatocyte growth factor (HGF) and its receptor, c-Met, are mutationally upregulated, causing increased cancer cell growth, survival, and invasion. This study identifies c-Met, ß1-integrin, and paxillin as novel components of TNTs in A549 lung adenocarcinoma cells, with paxillin localised at the protrusion site of TNTs. The HGF-induced TNTs in our study demonstrate the ability to transport lipid vesicles and mitochondria. HGF-induced TNT formation is mediated by c-Met and ß1-integrin in conjunction with paxillin, followed by downstream activation of MAPK and PI3K pathways and the Arp2/3 complex. These findings demonstrate a potential novel approach to inhibit TNT formation through targeting HGF/c-Met receptor and ß1-integrin signalling interactions, which has implications for multi-drug targeting in NSCLC.

Exploring Endotypes in Chronic Rhinosinusitis (ExpRess): Protocol for a cohort study.

BACKGROUND: Chronic Rhinosinusitis (CRS) affects approximately 1 in 10 UK adults and impacts quality of life quality of life significantly. Response to treatment may be driven by individual CRS endotypes and therefore work to delineate biomarker clusters that may separate responders from non-responders is needed. The ongoing MACRO three-arm parallel-group trial randomises adult CRS patients to endoscopic sinus surgery, macrolide therapy or placebo. AIM: This study aims to correlate CRS endotypes with clinical parameters from the ongoing MACRO trial, including olfactory function and outcomes in terms of response to treatment using core biomarkers sets. METHODS: Adult CRS patients enrolled into the MACRO trial will be recruited from participating UK otorhinolaryngology departments. Nasal tissue samples and swabs will be obtained in theatre or clinic from patients randomised to all three trial arms. Nasal tissue will be analysed with multiplex electrochemiluminescence for 32 cytokines including IL-5, IL-13, IgE and periostin. Bacterial swabs will be analysed using illumina miSeq 16S amplicon sequencing. Mean expression for each biomarker will be reported for treatment responder and non-responder groups. Correlation of biomarkers with MACRO trial outcome data such as endoscopic evaluation scores and quality-of-life improvement scores will be reported. DISCUSSION: Defining clear endotypes in CRS will contribute to refining patient pathways for the efficient use of clinical resources. This work may lay the groundwork for future studies to predict which patients might respond to medical or surgical therapy.

Systematic Review of Protein Biomarkers in Adult Patients With Chronic Rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is a heterogeneous condition characterized by differing inflammatory endotypes. The identification of suitable biomarkers could enable personalized approaches to treatment selection. OBJECTIVE: This study aimed to identify and summarize clinical studies of biomarkers in adults with CRS in order to inform future research into CRS endotypes. METHODS: We conducted systematic searches of MEDLINE and Web of Science from inception to January 30, 2022 and included all clinical studies of adult CRS patients and healthy controls measuring biomarkers using enzyme-linked immunosorbent assays or Luminex immunoassays. Outcomes included the name and tissue type of identified biomarkers and expression patterns within CRS phenotypes. Study quality was assessed using the National Institutes of Health quality assessment tool for observational cohort and cross-sectional studies. A narrative synthesis was performed. RESULTS: We identified 78 relevant studies involving up to 9394 patients, predominantly with CRS with nasal polyposis. Studies identified 80 biomarkers from nasal tissue, 25 from nasal secretions, 14 from nasal lavage fluid, 24 from serum, and one from urine. The majority of biomarkers found to distinguish CRS phenotypes were identified in nasal tissue, especially in nasal polyps. Serum biomarkers were more commonly found to differentiate CRS from controls. The most frequently measured biomarker was IL-5, followed by IL-13 and IL-4. Serum IgE, IL-17, pentraxin-3 and nasal phospho-janus kinase 2, IL-5, IL-6, IL-17A, granulocyte-colony stimulating factor, and interferon gamma were identified as correlated with disease severity. CONCLUSION: We have identified numerous potential biomarkers to differentiate a range of CRS phenotypes. Future studies should focus on the prognostic role of nasal tissue biomarkers or expand on the more limited studies of nasal secretions and nasal lavage fluid.We registered this study in PROSPERO (CRD42022302787).

Distinct functions of natural ADAM-15 cytoplasmic domain variants in human mammary carcinoma.

Adamalysins [a disintegrin and metalloproteinase (ADAM)] are a family of cell surface transmembrane proteins that have broad biological functions encompassing proteolysis, adhesion, and cell signal regulation. We previously showed that the cytoplasmic domain of ADAM-15 interacts with Src family protein tyrosine kinases and the adaptor protein growth factor receptor binding protein 2 (Grb2). In the present study, we have cloned and characterized four alternatively spliced forms of ADAM-15, which differ only in their cytoplasmic domains. We show that the four ADAM-15 variants were differentially expressed in human mammary carcinoma tissues compared with normal breast. The expression of the individual isoforms did not correlate with age, menopausal status, tumor size or grade, nodal status, Nottingham Prognostic Index, or steroid hormone receptor status. However, higher levels of two isoforms (ADAM-15A and ADAM-5B) were associated with poorer relapse-free survival in node-negative patients, whereas elevated ADAM-15C correlated with better relapse-free survival in node-positive, but not in node-negative, patients. The expression of ADAM-15A and ADAM-15B variants in MDA-MB-435 cells had differential effects on cell morphology, with adhesion, migration, and invasion enhanced by expression of ADAM-15A, whereas ADAM-15B led to reduced adhesion. Using glutathione S-transferase pull-down assays, we showed that the cytoplasmic domains of ADAM-15A, ADAM-15B, and ADAM-15C show equivalent abilities to interact with extracellular signal-regulated kinase and the adaptor molecules Grb2 and Tks5/Fish, but associate in an isoform-specific fashion with Nck and the Src and Brk tyrosine kinases. These data indicate that selective expression of ADAM-15 variants in breast cancers could play an important role in determining tumor aggressiveness by interplay with intracellular signaling pathways.

Consequences of untreated posttraumatic stress disorder following war in former Yugoslavia: morbidity, subjective quality of life, and care costs.

AIM: To assess long-term mental health outcomes in people who suffer from war-related posttraumatic stress disorder (PTSD) but do not receive appropriate treatment. METHODS: We interviewed 264 subjects from former Yugoslavia, who lived in Croatia, Serbia, Germany, and the United Kingdom. All of them had suffered from PTSD at some point following the war, but never received psychiatric or psychological treatment. The interviews took place on average 10.7+/-3.0 years after the war-related trauma. Outcomes were current PTSD on the Clinician Administered PTSD Scale for Diagnostic and Statistical Manual of Mental Disorders-IV, subjective quality of life (SQOL) on the Manchester Short Assessment of Quality of Life, and care costs. Socio-demographic characteristics, the level of traumatic war-events, and aspects of the post-war situation were tested for association with outcomes. RESULTS: Current PTSD was diagnosed in 83.7% of participants, the mean SQOL score was 4.0+/-0.9, and mean care costs in the last 3 months exceeded euro1100 in each center. Older age, more traumatic war-events, lower education, and living in post-conflict countries were associated with higher rates of current PTSD. Older age, combat experience, more traumatic war-events, being unemployed, living alone, being housed in collective accommodation, and current PTSD were independently associated with lower SQOL. Older age and living in Germany were linked to higher costs of formal care. CONCLUSION: People with untreated war-related PTSD have a high risk of still having PTSD a decade after the traumatic event. Their SQOL is relatively low, and they generate considerable care costs. Factors that have been reported as influencing the occurrence of PTSD also appear relevant for recovery from PTSD. Current PTSD may impair SQOL independently of social factors.

Interplay between metalloproteinases and cell signalling in blood brain barrier integrity.

The Blood-Brain Barrier (BBB) is a highly specialised interface separating the Central Nervous System (CNS) from circulating blood. Dysregulation of the BBB is a key early event in pathological conditions such as inflammation, in which the entry of activated leukocytes into the CNS is facilitated by BBB breakdown. The metzincin family of metalloproteinases (MPs) is one of the major contributors to BBB permeability as they cleave endothelial cell-cell contacts and underlying basal lamina components. However, the mechanisms by which MPs regulate BBB integrity has not yet been fully elucidated. The aim of this review is to provide an overview of pathways by which MPs could regulate the BBB in the context of neuroinflammation.

Low density lipoprotein from patients with Type 2 diabetes increases expression of monocyte matrix metalloproteinase and ADAM metalloproteinase genes.

AIMS: Type 2 diabetes is characterised by increased plasma concentrations of pro-inflammatory cytokines [such as tumour necrosis factor - alpha; TNF-alpha] and soluble forms of adhesion molecules involved in leukocyte - endothelial interactions. These molecules are synthesised as transmembrane proteins and the plasma soluble forms are generated by ectodomain cleavage from the cell surface by members of the ADAM [adisintegrin and metalloproteinase] proteinase family. We hypothesised that plasma low density lipoprotein [LDL] from subjects with Type 2 diabetes would influence in vitro monocytic ADAM and matrix metalloproteinase [MMP] gene expression differently compared to control LDL. METHODS: We examined relative mRNA expression by real time PCR in a monocytic cell line [THP-1] cultured for 4, 8 and 24 hrs with human plasma LDL derived from subjects with [n = 5] or without [n = 4] Type 2 diabetes. Gene expression for MMP-1 and 9, and ADAM - 8, 15, 17 and 28 was studied. RESULTS: Type 2 diabetes LDL significantly increased gene expression of MMP - 1 [p < 0.01] MMP - 9 [p < 0.001], and ADAM 17 [p < 0.05], - 28 [p < 0.01] and - 15 [p < 0.01] compared to control LDL. Type 2 diabetes LDL had disparate effects on inhibitors of MMP. CONCLUSION: These data suggest that Type 2 diabetes LDL could lead to increased adhesion molecule and TNF alpha cell surface shedding, and vascular plaque instability, by promoting increased expression of ADAM and MMP genes.

Monocyte matrix and ADAM metalloproteinase expression in type 2 diabetes after aspirin therapy.

The matrix metalloproteinase system (MMP and the TIMP inhibitors), and the ADAM metalloproteinases, have roles in maintaining vascular plaque stability and the shedding of cell surface molecules, such as TNF-alpha and adhesion molecules; aspirin suppresses MMP expression and ADAM activity from some cell lines in vitro. In a randomised prospective controlled study, we examined peripheral venous monocyte MMP-9, TIMP-1 and ADAM mRNA levels, and protein expression, in subjects with type 2 diabetes (n=10) and controls (n=14) before and after oral aspirin therapy (150mg daily for 14 days) or no active intervention. Baseline monocyte TIMP-1 mRNA levels were significantly lower in the diabetes group (p=0.0014), although monocyte MMP-9 mRNA, and MMP-9 and TIMP-1 protein expression after culture did not differ significantly between groups. Plasma MMP-9 (p=0.027) and TIMP-1 (p=0.016) concentrations were significantly greater, and the ratio of plasma TIMP-1:MMP-9 concentrations significantly lower, in the diabetes group (p=0.023). ADAM mRNA levels did not differ significantly between groups and oral aspirin therapy had no significant effect on any variable. Type 2 diabetes is characterised by reduced monocyte TIMP-1 mRNA levels, and a lower plasma MMP-9 to TIMP-1 protein ratio compared to controls, a pattern that would promote coronary plaque instability if reproduced within vascular plaque. Monocyte ADAM mRNA levels do not differ between group and oral aspirin has no significant effect on these variables.

Cytokine stimulated vascular cell adhesion molecule-1 (VCAM-1) ectodomain release is regulated by TIMP-3.

OBJECTIVES: Vascular cell adhesion molecule-1 (VCAM-1) is a cell surface adhesion molecule involved in the recruitment of leukocytes to endothelial cells on arterial walls during the pathogenesis of atherosclerosis. The soluble ectodomain of VCAM-1 (sVCAM-1) is proteolytically released from the cell surface into the circulation, a process which is up-regulated in patients with cardiovascular or inflammatory disease. Here we investigate mechanisms involved in sVCAM-1 generation in response to cytokine stimulation. METHODS: VCAM-1 ectodomain release into the conditioned media of MCEC-1 murine endothelial cells and cells grown from primary aortic explants from timp3-/- mice and wild-type littermates was measured by sandwich ELISA and Western blot after stimulation with tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), or the phorbol ester PMA. Protease expression was inhibited (knocked down) with siRNA and validated using real-time PCR. RESULTS: Proinflammatory cytokines IL-1beta and TNFalpha up-regulated VCAM-1 ectodomain release from the MCEC-1 cells, and this was dependant on p38 and mitogen-activated protein kinases (MAP kinases) and inhibited by the matrix metalloproteinase (MMP) inhibitor BB94 and tissue inhibitor of metalloproteinase (TIMP)-3, but not TIMP-1 or TIMP-2. Timp-3-/- cells exhibited greater VCAM-1 ectodomain release following cytokine stimulation than TIMP-3-expressing cells. Additionally, cytokine stimulation of MCEC-1 cells was shown to cause down-regulation of TIMP-3 expression. Knockdown of the metalloproteinase ADAM17, but not ADAM10 or ADAM12, gene expression reduced cytokine-stimulated VCAM-1 shedding. CONCLUSIONS: TIMP-3 regulates the release of sVCAM-1 from cytokine-stimulated endothelial cells, which is mediated by ADAM17.

Monocyte matrix metalloproteinase production in Type 2 diabetes and controls--a cross sectional study.

BACKGROUND: Coronary plaque rupture may result from localised over expression of matrix metalloproteinases (MMPs) within the plaque by infiltrating monocyte-macrophages. As MMP expression can be promoted by the modified lipoproteins, oxidative stress and hyperglycaemia that characterises Type 2 diabetes, we hypothesised that peripheral monocytes in these patients, exposed to these factors in vivo, would demonstrate increased MMP production compared to controls. METHODS: We examined peripheral venous monocyte expression of MMP and tissue inhibitor of metalloproteinase-1 (TIMP-1) in 18 controls and 22 subjects with Type 2 diabetes and no previous cardiovascular complications. RESULTS: No significant difference in MMP-1, 3 or 9 or TIMP-1 production was observed between control and diabetes groups. CONCLUSIONS: Monocyte MMP-1, 3, and 9, and TIMP-1, production are not abnormal in Type 2 diabetes. This data cannot be extrapolated to monocyte-macrophage behaviour in the vessel wall, but it does suggest MMP and TIMP-1 expression prior to monocyte infiltration and transformation are not abnormal in Type 2 diabetes.

Plasma matrix metalloproteinases, low density lipoprotein oxidisability and soluble adhesion molecules after a glucose load in Type 2 diabetes.

BACKGROUND: Acute hyperglycaemia is an independent cardiovascular risk factor in Type 2 diabetes which may be mediated through increased oxidative damage to plasma low density lipoprotein, and in vitro, high glucose concentrations promote proatherogenic adhesion molecule expression and matrix metalloproteinase expression. METHODS: We examined these atherogenic risk markers in 21 subjects with Type 2 diabetes and 20 controls during an oral 75 g glucose tolerance test. Plasma soluble adhesion molecule concentrations [E-selectin, VCAM-1 and ICAM-1], plasma matrix metalloproteinases [MMP-3 and 9] and plasma LDL oxidisability were measured at 30 minute intervals. RESULTS: In the diabetes group, the concentrations of all plasma soluble adhesion molecules fell promptly [all p < 0.0001] related principally to glycaemic excursions, but such changes also occurred in the control group. Plasma MMP-3 and -9 concentrations were lower [p < 0.05], and LDL oxidisability greater [p < 0.01] in the diabetes group but did not change in either group. There was a direct relationship between plasma MMP-9 and s ICAM-1 in the controls [r = 0.62; p = 0.006] perhaps suggesting a functional relationship between s ICAM-1 shedding and MMP-9. CONCLUSIONS: A glucose load leads to a rapid fall in plasma soluble adhesion molecule concentrations in Type 2 diabetes and controls, perhaps reflecting reduced generation of soluble from membrane forms during enhanced leukocyte-endothelial adhesion or increased hepatic clearance, without changes in plasma matrix metalloproteinase concentrations or low density lipoprotein oxidisability. These in vivo findings are in contrast with in vitro data.