Low Annexin A1 level in HTLV-1 infected patients is a potential biomarker for the clinical progression and diagnosis of HAM/TSP.

BACKGROUND: Human T-lymphotropic virus 1 (HTLV-1) is etiologically associated with the chronic inflammatory neurodegenerative disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) Annexin A1 (AnxA1) is an anti-inflammatory protein with proposed neuroprotective and anti-neuroinflammatory functions. We hypothesized that ANXA1 gene expression may be dysregulated in HTLV-1-infected HAM/TSP patients. METHODS: This study involved 37 individuals infected with HTLV-1, including 21 asymptomatic (AS) carriers and 16 with HAM/TSP, and a control group of 30 individuals negative for HTLV-1 and HTLV-2. For AS HTLV-1-positive and HAM/TSP patients, ANXA1 and formyl peptide receptor (FPR1, FPR2 and FPR3) expression and HTLV-1 proviral load (PVL) in peripheral blood cells were evaluated by real-time quantitative PCR (qPCR), and plasma AnxA1 levels were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: ANXA1 gene expression was increased in the AS group compared with the HAM/TSP and control groups, but the differences were not statistically significant. FPR1 gene expression was higher in patients with HTLV-1 than in controls (AS, p = 0.0032; HAM/TSP, p < 0.0001). Plasma AnxA1 levels were higher in the AS group than in the HAM/TSP group (p = 0.0045), and PVL was higher in patients with HAM/TSP than in AS individuals (p = 0.0162). The use of a combined ROC curve using Annexin 1 levels and proviral load significantly increased the sensitivity and specificity to predict progression to HAM/TSP (AUC = 0.851 and AUC = 0.937, respectively, to AUC = 1000). CONCLUSIONS: Our results suggest that AnxA1 may be dysregulated in HAM/TSP patients. Serological detection of AnxA1 in association with proviral load may provide a prognostic biomarker for HTLV-1-associated neurodegenerative disease.

Decreased serum Annexin A1 levels in Kawasaki disease with coronary artery aneurysm.

BACKGROUND: Kawasaki disease (KD) is an acute and systemic vasculitis whose etiology remains unclear. The most crucial complication is the formation of coronary artery aneurysm (CAA). Annexin A1 (ANXA1) is an endogenous anti-inflammatory agent and pro-resolving mediator involved in inflammation-related diseases. This study sought to investigate the serum ANXA1 levels in KD patients and further explore the relationship between ANXA1 and CAA, as well as additional clinical parameters. METHODS: Serum samples were collected from 95 KD patients and 39 healthy controls (HCs). KD patients were further divided into two groups: KD with CAAs (KD-CAAs) and KD non-CAAs (KD-NCAAs). Serum levels of ANXA1 and interleukin-6 (IL-6) were determined using enzyme-linked immunosorbent assays. RESULTS: Serum ANXA1 levels in the KD group were significantly lower than in the HC group. In particular, serum ANXA1 levels were substantially lower in the KD-CAA groups. Moreover, serum ANXA1 levels were positively correlated with N%, C-reactive protein (CRP), and IL-6 but negatively correlated with L% in the KD group. Positive correlations between serum ANXA1 levels and erythrocyte sedimentation rate (ESR), IL-6, and D-dimer (DD) were observed in the KD-CAA group. CONCLUSIONS: ANXA1 may be involved in the development of KD, and downregulation of ANXA1 may lead to the hypercoagulability seen in KD. IMPACT: For the first time, it was demonstrated that serum ANXA1 levels were significantly decreased in Kawasaki disease with coronary artery aneurysms. ANXA1 might be involved in the acute phase of Kawasaki disease. Low serum concentrations of ANXA1 might lead to the hypercoagulability stage in Kawasaki disease. ANXA1 might be a potential therapeutic target for patients with Kawasaki disease.

Leucine-rich alpha-2 glycoprotein 1, high mobility group box 1, matrix metalloproteinase 3 and annexin A1 as biomarkers of ulcerative colitis endoscopic and histological activity.

OBJECTIVE: The LRG, HMGB1, MMP3 and ANXA1 proteins have been implicated in different inflammatory pathways in ulcerative colitis (UC), but their role as specific biomarkers of both endoscopic and histological activity has yet to be elucidated. In the present study, we aimed to evaluate the LRG1, HMGB1, MMP3 and ANXA1 as potential serum biomarkers for UC endoscopic and histological activity. METHODS: This cross-sectional study included UC patients under 5-ASA, and healthy controls (HC) undergoing colonoscopy. Blood and biopsy samples were obtained and endoscopic Mayo sub-score (Ms) was recorded for the UC patients. Intramucosal calprotectin as a marker of histologic activity was evaluated in all biopsy samples and serum LRG1, HMGB1, MMP3 and ANXA1 levels were measured in the blood samples. RESULTS: The HCs ANXA1 level was lower compared to that of the UC group [P = 0.00, area under the curve (AUC) = 0.881] and so was the HCs MMP3 level compared to that of patients (P = 0.00, AUC = 0.835). The HCs ANXA1 levels were also lower compared to these of the independent Ms groups, even to the Ms = 0 (P = 0.00, AUC = 0.913). UC endoscopic activity was associated with MMP3 levels (r = 0.54, P = 0.000) but not with ANXA1, LRG1 and HMGB1 levels CONCLUSION: Serum ANXA1 is a potential diagnostic biomarker of UC and serum MMP3 is a potential biomarker of UC endoscopic and histological activity.

Association of serum annexin A1 with treatment response and prognosis in patients with esophageal squamous cell carcinoma.

OBJECTIVE: Annexin A1 (ANXA1), a calcium-dependent phospholipid binding protein, is known to be regulated by microRNA-196a (miR-196a) in esophageal adenocarcinoma, and its high expression in tumor tissue is correlated with the poor prognosis of esophageal squamous cell carcinoma (ESCC). However, the role of ANXA1 in the serum of patients with ESCC remains unclear. MATERIALS AND METHODS: In this study, we used enzyme-linked immunosorbent assay to evaluate the levels of ANXA1 and real-time polymerase chain reaction to detect the expression of miR-196a in the serum of ESCC patients (healthy donors as controls) and evaluated the relationship between ANXA1 and clinical outcomes. RESULTS: The results showed that the level of serum ANXA1 in ESCC patients was significantly lower than that in controls (P = 0.001) but increased after chemoradiotherapy (P = 0.001). There was no correlation between the baseline level of serum ANXA1 and the short-term efficacy of treatment (P = 0.26) as well as the 1-year progression-free survival (PFS) (P = 0.094). However, there existed a significant correlation between the increases of serum ANXA1 expression and the 1-year PFS (P = 0.04). A higher increase (>2-fold of baseline) in the serum ANXA1 levels was correlated with a poorer PFS (hazard ratio = 3.096, 95% confidence interval 1.239-7.861). There was an inverse correlation between the expressions of miR-196a and ANXA1 in serum (Pearson's correlation of -0.54, P = 0.021). CONCLUSION: Our data revealed that the expression of serum ANXA1 in ESCC patients increases after chemoradiotherapy and the increased fold change in serum ANXA1 confers independent negative prognostic impact in ESCC. The higher the increase in serum ANXA1 levels, the poorer the outcome.

Defective formyl peptide receptor 2/3 and annexin A1 expressions associated with M2a polarization of blood immune cells in patients with chronic obstructive pulmonary disease.

BACKGROUND: Controversy exists in previous studies on macrophage M1/M2 polarization in chronic obstructive pulmonary disease (COPD). We hypothesized that formyl peptide receptor (FPR), a marker of efferocytosis and mediator of M1/M2 polarization, may be involved in the development of COPD. METHODS: We examined FPR 1/2/3 expressions of blood M1/M2a monocyte, neutrophil, natural killer (NK) cell, NK T cell, T helper (Th) cell, and T cytotoxic (Tc) cell by flowcytometry method in 40 patients with cigarette smoking-related COPD and 16 healthy non-smokers. Serum levels of five FPR ligands were measured by ELISA method. RESULTS: The COPD patients had lower M2a percentage and higher percentages of NK, NK T, Th, and Tc cells than the healthy non-smokers. FPR2 expressions on Th/Tc cells, FPR3 expressions of M1, M2a, NK, NK T, Th, and Tc cells, and serum annexin A1 (an endogenous FPR2 ligand) levels were all decreased in the COPD patients as compared with that in the healthy non-smokers. FPR1 expression on neutrophil was increased in the COPD patient with a high MMRC dyspnea scale, while FPR2 expression on neutrophil and annexin A1 were both decreased in the COPD patients with a history of frequent moderate exacerbation (≥ 2 events in the past 1 year). In 10 COPD patients whose blood samples were collected again after 1-year treatment, M2a percentage, FPR3 expressions of M1/NK/Th cells, FPR2 expression on Th cell, and FPR1 expression on neutrophil were all reversed to normal, in parallel with partial improvement in small airway dysfunction. CONCLUSIONS: Our findings provide evidence for defective FPR2/3 and annexin A1 expressions that, associated with decreased M2a polarization, might be involved in the development of cigarette smoking induced persistent airflow limitation in COPD.

Annexin A1 is elevated in patients with COPD and affects lung fibroblast function.

Purpose: Fibrosis in peripheral airways is responsible for airflow limitation in chronic obstructive pulmonary disease (COPD). Annexin A1 modulates several key biological events during inflammation. However, little is known about its role in airway fibrosis in COPD. We investigated whether levels of Annexin A1 were upregulated in patients with COPD, and whether it promoted airway fibrosis. Methods: We quantified serum Annexin A1 levels in never-smokers (n=12), smokers without COPD (n=11), and smokers with COPD (n=22). Correlations between Annexin A1 expression and clinical indicators (eg, lung function) were assessed. In vitro, human bronchial epithelial (HBE) cells were exposed to cigarette smoke extract (CSE) and Annexin A1 expression was assessed. Primary human lung fibroblasts were isolated from patients with COPD and effects of Annexin A1 on fibrotic deposition of lung fibroblasts were evaluated. Results: Serum Annexin A1 was significantly higher in patients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines stage III or IV than in those with GOLD stages I or II (12.8±0.8 ng/mL versus 9.8±0.7 ng/mL; p=0.016). Annexin A1 expression was negatively associated with airflow obstruction (forced expiratory volume in one second % predicted; r=-0.72, p<0.001). In vitro, Annexin A1 was significantly increased in CSE-exposed HBE cells in a time- and concentration-dependent manner. Annexin A1 promoted lung fibroblasts proliferation, migration, differentiation, and collagen deposition via the ERK1/2 and p38 mitogen-activated protein kinase pathways. Conclusion: Annexin A1 expression is upregulated in patients with COPD and affects lung fibroblast function. However, more studies are needed to clarify the role of Annexin A1 in airway fibrosis of COPD.

Annexin A1 attenuates microvascular complications through restoration of Akt signalling in a murine model of type 1 diabetes.

AIMS/HYPOTHESIS: Microvascular complications in the heart and kidney are strongly associated with an overall rise in inflammation. Annexin A1 (ANXA1) is an endogenous anti-inflammatory molecule that limits and resolves inflammation. In this study, we have used a bedside to bench approach to investigate: (1) ANXA1 levels in individuals with type 1 diabetes; (2) the role of endogenous ANXA1 in nephropathy and cardiomyopathy in experimental type 1 diabetes; and (3) whether treatment with human recombinant ANXA1 attenuates nephropathy and cardiomyopathy in a murine model of type 1 diabetes. METHODS: ANXA1 was measured in plasma from individuals with type 1 diabetes with or without nephropathy and healthy donors. Experimental type 1 diabetes was induced in mice by injection of streptozotocin (STZ; 45 mg/kg i.v. per day for 5 consecutive days) in C57BL/6 or Anxa1 -/- mice. Diabetic mice were treated with human recombinant (hr)ANXA1 (1 µg, 100 µl, 50 mmol/l HEPES; 140 mmol/l NaCl; pH 7.4, i.p.) or vehicle (100 µl, 50 mmol/l HEPES; 140 mmol/l NaCl; pH 7.4, i.p.). RESULTS: Plasma levels of ANXA1 were elevated in individuals with type 1 diabetes with/without nephropathy compared with healthy individuals (66.0 ± 4.2/64.0 ± 4 ng/ml vs 35.9 ± 2.3 ng/ml; p < 0.05). Compared with diabetic wild-type (WT) mice, diabetic Anxa1 -/- mice exhibited a worse diabetic phenotype and developed more severe cardiac (ejection fraction; 76.1 ± 1.6% vs 49.9 ± 0.9%) and renal dysfunction (proteinuria; 89.3 ± 5.0 µg/mg vs 113.3 ± 5.5 µg/mg). Mechanistically, compared with non-diabetic WT mice, the degree of the phosphorylation of mitogen-activated protein kinases (MAPKs) p38, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) was significantly higher in non-diabetic Anxa1 -/- mice in both the heart and kidney, and was further enhanced after STZ-induced type 1 diabetes. Prophylactic treatment with hrANXA1 (weeks 1-13) attenuated both cardiac (ejection fraction; 54.0 ± 1.6% vs 72.4 ± 1.0%) and renal (proteinuria; 89.3 ± 5.0 µg/mg vs 53.1 ± 3.4 µg/mg) dysfunction associated with STZ-induced diabetes, while therapeutic administration of hrANXA1 (weeks 8-13), after significant cardiac and renal dysfunction had already developed, halted the further functional decline in cardiac and renal function seen in diabetic mice administered vehicle. In addition, administration of hrANXA1 attenuated the increase in phosphorylation of p38, JNK and ERK, and restored phosphorylation of Akt in diabetic mice. CONCLUSIONS/INTERPRETATION: Overall, these results demonstrate that ANXA1 plasma levels are elevated in individuals with type 1 diabetes independent of a significant impairment in renal function. Furthermore, in mouse models with STZ-induced type 1 diabetes, ANXA1 protects against cardiac and renal dysfunction by returning MAPK signalling to baseline and activating pro-survival pathways (Akt). We propose ANXA1 to be a potential therapeutic option for the control of comorbidities in type 1 diabetes.

The advantageous role of annexin A1 in cardiovascular disease.

The inflammatory response protects the human body against infection and injury. However, uncontrolled and unresolved inflammation can lead to tissue damage and chronic inflammatory diseases. Therefore, active resolution of inflammation is essential to restore tissue homeostasis. This review focuses on the pro-resolving molecule annexin A1 (ANXA1) and its derived peptides. Mechanisms instructed by ANXA1 are multidisciplinary and affect leukocytes as well as endothelial cells and tissue resident cells like macrophages and mast cells. ANXA1 has an outstanding role in limiting leukocyte recruitment and different aspects of ANXA1 as modulator of the leukocyte adhesion cascade are discussed here. Additionally, this review details the therapeutic relevance of ANXA1 and its derived peptides in cardiovascular diseases since atherosclerosis stands out as a chronic inflammatory disease with impaired resolution and continuous leukocyte recruitment.

Decreased plasma concentrations of brain-derived neurotrophic factor in preeclampsia.

BACKGROUND: Preeclampsia (PE) is a disease characterized by excessive maternal inflammatory response. Early studies suggested that brain-derived neurotrophic factor (BDNF) modulates inflammation. The main objective of this study was to investigate BDNF plasma concentrations in PE women and to compare with BDNF concentrations from normotensive pregnant women. We also investigated the association among the plasma concentrations of BDNF and inflammatory mediators, and maternal clinical features. METHODS: BDNF plasma concentrations were measured by ELISA in 38 PE women (17 early onset and 21 late onset) and in 20 normotensive pregnant women (Norm) matched for gestational age (Norm<34weeks: n=8; Norm≥34weeks: n=12). Correlation analyses between laboratory parameters and clinical characteristics were evaluated through Spearman's coefficients. RESULTS: BDNF concentration was lower in PE women than in normotensive pregnant women, but no difference was detected between the subgroups of PE women and normotensive pregnant women. BDNF correlated negatively with annexin A1, and positively with body mass index and diastolic blood pressure. No correlation was significant in normotensive pregnant women. CONCLUSIONS: Lower BDNF plasma concentrations and cross-talk between BDNF and AnxA1 signaling pathways might be involved in PE pathogenesis.

Human Blood Autoantibodies in the Detection of Colorectal Cancer.

Colorectal cancer (CRC) is the second most common malignancy in the western world. Early detection and diagnosis of all cancer types is vital to improved prognosis by enabling early treatment when tumours should be both resectable and curable. Sera from 3 different cohorts; 42 sera (21 CRC and 21 matched controls) from New York, USA, 200 sera from Pittsburgh, USA (100 CRC and 100 controls) and 20 sera from Dundee, UK (10 CRC and 10 controls) were tested against a panel of multiple tumour-associated antigens (TAAs) using an optimised multiplex microarray system. TAA specific IgG responses were interpolated against the internal IgG standard curve for each sample. Individual TAA specific responses were examined in each cohort to determine cutoffs for a robust initial scoring method to establish sensitivity and specificity. Sensitivity and specificity of combinations of TAAs provided good discrimination between cancer-positive and normal serum. The overall sensitivity and specificity of the sample sets tested against a panel of 32 TAAs were 61.1% and 80.9% respectively for 6 antigens; p53, AFP, K RAS, Annexin, RAF1 and NY-CO16. Furthermore, the observed sensitivity in Pittsburgh sample set in different clinical stages of CRC; stage I (n = 19), stage II (n = 40), stage III (n = 34) and stage IV (n = 6) was similar (73.6%, 75.0%, 73.5% and 83.3%, respectively), with similar levels of sensitivity for right and left sided CRC. We identified an antigen panel of sufficient sensitivity and specificity for early detection of CRC, based upon serum profiling of autoantibody response using a robust multiplex antigen microarray technology. This opens the possibility of a blood test for screening and detection of early colorectal cancer. However this panel will require further validation studies before they can be proposed for clinical practice.

Annexin A1 Is Increased in the Plasma of Preeclamptic Women.

BACKGROUND: Preeclampsia (PE) is a pregnancy disease associated with exacerbated inflammatory response. Annexin A1 (AnxA1) is a glucocorticoid-regulated protein endowed with anti-inflammatory and proresolving properties that has been much studied in various animal models of inflammation but poorly studied in the context of human inflammatory diseases. The main objective of this study was to measure AnxA1 levels in PE women and to compare those levels in normotensive pregnant and non-pregnant women. We evaluated the association among AnxA1, ultrasensitive C reactive protein (us-CRP) and soluble tumor necrosis factor alpha receptor type 1 (sTNF-R1) plasma levels of the study participants. METHODS: This study included 40 non-pregnant, 38 normotensive pregnant and 51 PE women. PE women were stratified in early (N = 23) and late (N = 28) subgroups, according to gestational age (GA) at onset of clinical symptoms. Protein AnxA1 and us-CRP plasma levels were determined by ELISA and immunoturbidimetric assays, respectively. Transcript levels of AnxA1 in peripheral blood mononuclear cells (PBMC) were measured by real time RT-PCR. RESULTS: Increased levels of AnxA1 coincided with higher us-CRP levels in the plasma of PE women. Pregnant women with early PE had higher levels of AnxA1 and us-CRP than normotensive pregnant women with GA <34 weeks. No significant difference was found for AnxA1 and us-CRP, comparing late PE and normotensive pregnant women with GA ≥ 34 weeks. AnxA1 mRNA levels in PBMC were similar among the studied groups. AnxA1 was positively correlated with sTNF-R1, but not with us-CRP. CONCLUSIONS: Our data show that increased AnxA1 levels were associated with a systemic inflammatory phenotype in PE, suggesting AnxA1 deregulation in PE pathogenesis. However, more studies are needed to clarify the role of AnxA1 and other proresolving molecules in the context of the systemic inflammatory response in this intriguing disease.

Investigation of circulating antibodies to ANXA1 in breast cancer.

Our recent work demonstrated that circulating levels of IgG antibody to linear peptide antigens derived from annexin A1 (ANXA1) were significantly increased in lung cancer. The present study was then undertaken to test whether circulating anti-ANXA1 antibodies were also altered in breast cancer. An enzyme-linked immunosorbent assay was developed in-house to determine circulating IgG against ANXA1-derived peptide antigens in 152 female patients with breast cancer and 160 female control subjects. Student's t test revealed that patients with breast cancer had significantly higher levels of anti-ANXA1 IgG than control subjects (t = 4.75, P < 0.0001). Receiver operating characteristic (ROC) analysis showed that the area under the ROC curve was 0.73 with 95% confidence interval (CI) 0.67-0.78, and the sensitivity of anti-ANXA1 IgG assay was 23.2% against the specificity of 90%. The levels of anti-ANXA1 IgG did not appear to be stage-dependent, and Pearson correlation analysis showed no correlation between the anti-ANXA1 IgG levels and the stages of breast cancer (r = -0.02, df = 149, P = 0.796). This work suggests that circulating IgG for ANXA1-derived peptide antigens may have both diagnostic and prognostic values for breast cancer although further screening is needed to identify more such peptide antigens derived from tumor-associated antigens.

Co-overexpression of Hsp90-ß and annexin A1 with a significantly positive correlation contributes to the diagnosis of lung cancer.

AIM: Hsp90-ß and annexin A1 have been demonstrated to be associated with tumorigenesis. However, the effect of Hsp90-ß and annexin A1 in lung cancer remains poorly understood. In this research, the correlation of Hsp90-ß and annexin A1 in lung cancer patients were analyzed. METHODS: The expression levels of Hsp90-ß and annexin A1 were examined by immunohistochemistry and ELISA. RESULTS: Lung cancer tissues and serum exhibited higher co-expression of Hsp90-ß and annexin A1 than control groups (p < 0.05). Hsp90-ß and annexin A1 could discriminate lung cancer from the control groups (sensitivity of Hsp90-ß was 80.2% in tissues and 96% in serum; specificity of Hsp90-ß was 80% in tissues and 83.33% in serum; sensitivity of annexin A1 was 68.76% in tissues and 95.23% in serum; specificity of annexin A1 was 75% in tissues and 85.7% in serum) and multi-index combined detection had a better diagnostic value. CONCLUSION: The expression levels of Hsp90-ß and annexin A1 positively correlated and such co-overexpression of Hsp90-ß and annexin A1 contributed to lung cancer diagnosis.

Alterations in the profile of blood neutrophil membrane receptors caused by in vivo adrenocorticotrophic hormone actions.

Elevated levels of adrenocorticotrophic hormone (ACTH) mobilize granulocytes from bone marrow into the blood, although these neutrophils are refractory to a full migratory response into inflamed tissues. Here, we investigated the dependence of glucocorticoid receptor activation and glucocorticoid-regulated protein annexin A1 (ANXA1) on ACTH-induced neutrophilia and the phenotype of blood neutrophil after ACTH injection, focusing on adhesion molecule expressions and locomotion properties. ACTH injection (5 µg ip, 4 h) induced neutrophilia in wild-type (WT) mice and did not alter the elevated numbers of neutrophils in RU-38486 (RU)-pretreated or ANXA1(-/-) mice injected with ACTH. Neutrophils from WT ACTH-treated mice presented higher expression of Ly6G⁺ANXA1(high), CD18(high), CD62L(high), CD49(high), CXCR4(high), and formyl-peptide receptor 1 (FPR1(low)) than those observed in RU-pretreated or ANXA1(-/-) mice. The membrane phenotype of neutrophils collected from WT ACTH-treated mice was paralleled by elevated fractions of rolling and adherent leukocytes to the cremaster postcapillary venules together with impaired neutrophil migration into inflamed air pouches in vivo and in vitro reduced formyl-methionyl-leucyl-phenylalanine (fMLP) or stromal-derived factor-1 (SDF-1α)-induced chemotaxis. In an 18-h senescence protocol, neutrophils from WT ACTH-treated mice had a higher proportion of ANXAV(low)/CXCR4(low), and they were less phagocytosed by peritoneal macrophages. We conclude that alterations on HPA axis affect the pattern of membrane receptors in circulating neutrophils, which may lead to different neutrophil phenotypes in the blood. Moreover, ACTH actions render circulating neutrophils to a phenotype with early reactivity, such as in vivo leukocyte-endothelial interactions, but with impaired locomotion and clearance.

Dysregulation of anti-inflammatory annexin A1 expression in progressive Crohns Disease.

BACKGROUND: Development of inflammatory bowel disease (IBD) involves the interplay of environmental and genetic factors with the host immune system. Mechanisms contributing to immune dysregulation in IBD are not fully defined. Development of novel therapeutic strategies is focused on controlling aberrant immune response in IBD. Current IBD therapy utilizes a combination of immunomodulators and biologics to suppress pro-inflammatory effectors of IBD. However, the role of immunomodulatory factors such as annexin A1 (ANXA1) is not well understood. The goal of this study was to examine the association between ANXA1 and IBD, and the effects of anti-TNF-α, Infliximab (IFX), therapy on ANXA1 expression. METHODS: ANXA1 and TNF-α transcript levels in PBMC were measured by RT PCR. Clinical follow up included the administration of serial ibdQs. ANXA1 expression in the gut mucosa was measured by IHC. Plasma ANXA1 levels were measured by ELISA. RESULTS: We found that the reduction in ANXA1 protein levels in plasma coincided with a decrease in the ANXA1 mRNA expression in peripheral blood of IBD patients. ANXA1 expression is upregulated during IFX therapy in patients with a successful intervention but not in clinical non-responders. The IFX therapy also modified the cellular immune activation in the peripheral blood of IBD patients. Decreased expression of ANXA1 was detected in the colonic mucosa of IBD patients with incomplete resolution of inflammation during continuous therapy, which correlated with increased levels of TNF-α transcripts. Gut mucosal epithelial barrier disruption was evident by increased plasma bacterial 16S levels. CONCLUSION: Loss of ANXA1 expression may support inflammation during IBD and can serve as a biomarker of disease progression. Changes in ANXA1 levels may be predictive of therapeutic efficacy.

Plasma levels in sepsis patients of annexin A1, lipoxin A4, macrophage inflammatory protein-3a, and neutrophil gelatinase-associated lipocalin.

BACKGROUND: The relationship between the various cytokine responses that occur during sepsis remains controversial. Emerging evidence indicates that the proinflammatory and anti-inflammatory responses are regulated simultaneously from the beginning of sepsis. However, the roles of the novel anti-inflammatory mediators annexin (Anx)A1 and lipoxin (LX)A4 and the proinflammatory cytokines neutrophil gelatinase-associated lipocalin (NGAL) and macrophage inflammatory protein (MIP)-3a have been studied. METHODS: In this study, the plasma levels of AnxA1, LXA4, NGAL, MIP-3a, interleukin (IL)-8 and IL-6 in patients with sepsis were determined on admission to the intensive care unit. The patients were classified into survivors and non-survivors based on their outcome on day 28. RESULTS: AnxA1 and LXA4 levels were decreased in sepsis patients compared with control patients, whereas the levels of the proinflammatory cytokines MIP-3a, NGAL, IL-8, and IL-6 were elevated. Furthermore, a significantly higher level of MIP-3a was detected in nonsurviving patients compared with surviving patients (p < 0.05), whereas there were no significant differences between these two groups for the levels of the other mediators. Correlation analysis demonstrated that only NGAL level was closely correlated with the level of IL-6. Univariate analysis indicated that the levels of MIP-3a and IL-8 were independent factors associated with patient survival, but this was not confirmed by the multivariate analysis. CONCLUSION: AnxA1 and LXA4 plasma levels were found to be decreased in sepsis patients, whereas the levels of MIP-3a and NGAL were found to be elevated. This warrants further study in order to determine the clinical implications of these changes.

Expression of annexin-1 in equine leucocytes and the effects of the N-terminal annexin-1 peptide, Ac2-26, on equine neutrophil superoxide production.

N-terminal peptides derived from the anti-inflammatory peptide, annexin-1, inhibit neutrophil function but can also induce pro-inflammatory effects. Although equine annexin-1 has been sequenced, its cellular expression and properties have not been reported. This study has examined whether annexin-1 is present in equine leucocytes and how the N-terminal peptide, Ac2-26, affects equine neutrophil superoxide production. Annexin-1 expression in equine neutrophils and mononuclear cells and the ability of Ac2-26 to activate neutrophil p42/44 MAPK were determined by immunoblotting. Equine neutrophil superoxide production was measured by the reduction of cytochrome (cyt) C following stimulation with Ac2-26 and the formyl peptide receptor (FPR) agonists, FMLP, WKYMVm and WKYMVM. Responses were examined in the presence of the pan-FPR antagonist, BOC-2, and the role of p42/44 MAPK in agonist-induced effects was determined using PD98059. The effect of Ac2-26 on superoxide production in response to serum-treated zymosan (STZ) was also investigated, and the roles of FPR and p42/44 MAPK ascertained. Annexin-1 was detected in both equine neutrophils and mononuclear cells using a polyclonal rabbit anti-human annexin-1 antibody. Ac2-26 (5x10(-5)M) induced superoxide production in cytochalasin B-primed (48+/-8 versus 21+/-9 (unstimulated cells) nmol cyt C/10(6) neutrophils) and un-primed cells (37+/-10 versus 11+/-5 nmol cyt C/10(6) neutrophils). FMLP and WKYMVm, but not WKYMVM, also caused superoxide production in primed neutrophils, suggesting the response was mediated by FPR receptor binding. This was supported by the marked inhibitory effect of BOC-2 on the responses to Ac2-26 and FMLP although, interestingly, the effects of WKYMVm were not significantly reduced (50+/-5 (WKYMVm) versus 45+/-5 (WKYMVm+BOC-2) nmol reduced cyt C/10(6) neutrophils). Inhibition of p42/44 MAPK activation with PD98059 significantly attenuated superoxide production in response to Ac2-26, FMLP and WKYMVm and Western blotting showed that Ac2-26 induced p42/44 MAPK activation. At a concentration which did not cause superoxide production, Ac2-26 (10(-5)M) significantly reduced the response to STZ (84+/-17% inhibition). This inhibitory effect was attenuated by both BOC-2 and PD98059. These results suggest that if activation of equine leucocytes in vivo leads to the release and subsequent cleavage of annexin-1, the N-terminal peptides formed could bind to neutrophil FPR and decrease free radical production in response to particulate stimuli. This could help to reduce local tissue damage but, as Ac2-26 can also stimulate superoxide production at higher concentrations in an FPR-dependent manner, the amount of free radical production may depend on the concentration of peptide present.

Expression of annexin A1 mRNA in peripheral blood from oral squamous cell carcinoma patients.

Several studies have been suggesting annexin A1 protein as an active player in tumorigenesis of many organs. Nevertheless, its tumor biomarker role has been mainly studied in tissues by immunohistochemistry or cell culture. Hence, in this investigation, the peripheral blood from 27 oral squamous cell carcinoma (OSCC) patients and 25 negative control individuals were examined by quantitative real-time PCR. Down-regulated ANXA1 expression at mRNA level was observed in OSCC samples (p=0.026). Significantly diminished mRNA levels correlated to age, sex and the anatomical site of the tumor lesion were observed. Moreover, the ROC curve analysis revealed the performance of ANXA1 expression as a suitable biomarker for patients with oral cavity cancer, especially those with 60years of age or older and/or women. For the first time, ANXA1 mRNA is revealed as blood-based biomarker, and its adoption for complementary non-invasive diagnosis of OSCC is suggested. These results suggest that, beyond the anti-inflammatory function, annexin A1 may also play a tumor suppressor role in peripheral blood cells, such as leukocytes.

Differential modulation of annexin I binding sites on monocytes and neutrophils.

Specific binding sites for the anti-inflammatory protein annexin I have been detected on the surface of human monocytes and polymorphonuclear leukocytes (PMN). These binding sites are proteinaceous in nature and are sensitive to cleavage by the proteolytic enzymes trypsin, collagenase, elastase and cathepsin G. When monocytes and PMN were isolated independently from peripheral blood, only the monocytes exhibited constitutive annexin I binding. However PMN acquired the capacity to bind annexin I following co-culture with monocytes. PMN incubation with sodium azide, but not protease inhibitors, partially blocked this process. A similar increase in annexin I binding capacity was also detected in PMN following adhesion to endothelial monolayers. We propose that a juxtacrine activation rather than a cleavage-mediated transfer is involved in this process. Removal of annexin I binding sites from monocytes with elastase rendered monocytes functionally insensitive to full length annexin I or to the annexin I-derived pharmacophore, peptide Ac2-26, assessed as suppression of the respiratory burst. These data indicate that the annexin I binding site on phagocytic cells may have an important function in the feedback control of the inflammatory response and their loss through cleavage could potentiate such responses.

Autoantigen components recognizable by scleroderma sera are exported via ectocytosis of fibroblasts.

Previously, we have demonstrated that ectocytosis, a unique cell trafficking process to export a specific subset of cellular proteins in the form of membrane vesicles, can be triggered from human skin fibroblasts cultured in a three-dimensional collagen lattice upon stress relaxation. The same culturing system was employed in the present study using fibroblasts isolated from patients with systemic sclerosis (SSc). To see whether any putative intracellular autoantigens causing SSc might escape out of cells by way of ectocytosis, the same stress-relaxation method was used to induce a synchronized ectocytosis among cultured cells. Membrane vesicles released by scleroderma fibroblasts were subsequently isolated, resolved on SDS-PAGE and immunoblotted with sera from 89 patients with various autoimmune diseases and 11 normal volunteers. Three major polypeptides with apparent mol. wts of 12-14, 32-34 and 70-80 kDa are prominent bands on both SDS-PAGE and immunoblots. The 32-34 kDa polypeptide has been further identified as a member of the annexin protein family, while the 70-80 kDa protein has been shown to be topoisomerase I, as judged by its reactivity to patients' sera and a rabbit polyclonal antibody, and as also judged by a functional assay. In conclusion, our results suggest that ectocytosis might be one of the potential pathways for cells to export intracellular antigens and subsequently cause autoimmune reactions.