Patients with acute pancreatitis complicated by organ dysfunction show abnormal peripheral blood polymorphonuclear leukocyte signaling.

BACKGROUND/OBJECTIVES: Circulating polymorphonuclear leukocytes (PMNLs) may contribute to development of organ dysfunction in acute pancreatitis (AP). We outlined aberrations in PMNL signaling profiles in patients with AP complicated by organ dysfunction and immune suppression. METHODS: Study comprised 13 patients treated at intensive care unit due to severe AP complicated by vital organ dysfunction. Mean proportion (SEM) of HLA-DR-positive monocytes was 55.0% (4.1%). 13 healthy volunteers served as reference subjects. Phosphorylation of PMNL NFκB, p38, ERK1/2 and STAT3, -5 and -6 was determined using whole blood flow cytometry. Transmigration of PMNLs was studied using endothelial EA-HY cell monolayer. RESULTS: Proportions of NFκB phosphorylation-positive PMNLs were lower in the patients' than in reference subjects' blood samples supplemented with tumor necrosis factor. p38 phosphorylation was normal while ERK1/2 phosphorylation was decreased. STAT3 was constitutively activated in five patients. Proportion of patients' pSTAT6-positive cells was normal while fluorescence intensity was decreased. STAT5 phosphorylation was normal. Transmigration of patients' PMNLs was increased. CONCLUSIONS: In patients with AP complicated by organ dysfunction proportion of pNFκB-positive PMNLs is decreased. This impairs patients' defense mechanisms against infection. Despite immune suppression, PMNL transmigration was increased and p38 phosphorylation capacity was not depressed, which may contribute to end organ inflammation and dysfunction.

Specific immunoassay reveals increased serum trypsinogen 3 in acute pancreatitis.

BACKGROUND: Trypsinogen 3 is a minor trypsinogen isoform in the pancreas. In contrast with trypsin 1 and 2, trypsin 3 degrades pancreatic secretory trypsin inhibitor, which may lead to an excess of active trypsin and acute pancreatitis (AP). We developed an immunoassay for trypsinogen 3 and studied whether an assay of serum trypsinogen 3 is of clinical utility in the diagnosis of AP. METHODS: Monoclonal antibodies were generated using recombinant human trypsinogen 3 as the antigen and used to establish a sandwich-type immunoassay. We analyzed serum trypsinogen 3 concentrations in 82 patients with AP and 63 patients with upper abdominal pain (controls). The reference interval was determined using serum samples from 172 apparently healthy individuals. RESULTS: The measuring range of the trypsinogen 3 assay was 1.0-250 µg/L. Intra- and interassay CVs were <11%, and cross-reactivity with other trypsinogen isoenzymes was <0.1%. The median trypsinogen 3 concentration in serum from healthy individuals was <1.0 µg/L, and the upper reference limit was 4.4 µg/L. We observed increased trypsinogen 3 concentrations in patients with mild (median 9.5 µg/L) and severe (15.0 µg/L) AP; in both groups, the concentrations were significantly higher than in controls (median <1.0 µg/L) (P < 0.0001). In ROC analysis, the area under the curve of trypsinogen 3 for separation between AP and controls was 0.90 (P < 0.0001). CONCLUSIONS: We established for the first time a specific immunoassay for trypsinogen 3 using monoclonal antibodies. Patients with AP were found to have increased serum concentrations of trypsinogen 3. The availability of this assay will be useful for studies of the clinical utility of trypsinogen 3.

Patients with acute pancreatitis complicated by organ failure show highly aberrant monocyte signaling profiles assessed by phospho-specific flow cytometry.

OBJECTIVES: To outline signaling profiles and transmigration capacity of monocytes of patients with severe acute pancreatitis. DESIGN: Prospective study. SETTING: University hospital intensive care unit. PATIENTS: Thirteen patients with severe acute pancreatitis. All patients had organ dysfunction (acute respiratory distress syndrome in 12, renal dysfunction in eight). Healthy volunteers served as reference subjects. INTERVENTIONS: Blood samples were collected after admission to the intensive care unit. MEASUREMENTS AND MAIN RESULTS: Phosphorylation of nuclear factor-kappaB and p38, signal transducers and activators of transcription (STATs) 1, 3, 5, and extracellular signal-regulated kinases 1/2 in appropriately stimulated and nonstimulated samples were studied using phospho-specific whole-blood flow cytometry. Monocyte chemotactic protein-1-induced transmigration of monocytes among mononuclear cells obtained by density gradient centrifugation was studied using Transwell cell culture inserts covered with confluent layer of endothelial EA-HY cells. Phosphorylation levels of nuclear factor-kappaB induced by tumor necrosis factor, bacterial lipopolysaccharide, muramyl dipeptide, Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis were significantly lower in patients' monocytes than monocytes of healthy reference subjects, whereas mitogen-activated protein kinase p38 phosphorylation levels were normal. Phosphorylation levels induced by interleukin-6 in STAT1 and STAT3 and by combination of phorbol 12-myristate 13-acetate and calcium ionophore A23187 in extracellular signal-regulated kinases 1/2, members of a mitogen-activated protein kinase family, were depressed in patients' monocytes, whereas phosphorylation levels induced by granulocyte-macrophage colony-stimulating factor in STAT5 was normal. In nonstimulated samples, phosphorylation levels were normal. The transmigration percentage of patients' monocytes was significantly lower than that of reference monocytes. CONCLUSIONS: In severe acute pancreatitis, monocytes show impaired nuclear factor kappaB and STAT1 activation, which may increase susceptibility to secondary infections. p38 activation is normal and STAT3 activation is depressed, which may contribute to maintenance of systemic inflammation. Extracellular signal-regulated kinases 1/2 activation is impaired, which may depress monocytes' transmigration and may consequently increase risk of infection. Monitoring of monocyte signaling profiles may aid in finding new therapeutic approaches and predictors of outcome of severe acute pancreatitis.

Acute pancreatitis with organ dysfunction associates with abnormal blood lymphocyte signaling: controlled laboratory study.

INTRODUCTION: Severe acute pancreatitis is associated with systemic inflammation, compensatory immune suppression, secondary infections, vital organ dysfunction, and death.Our study purpose was to delineate signaling profiles of circulating lymphocytes in acute pancreatitis complicated by organ dysfunction. METHODS: Sixteen patients with acute pancreatitis, dysfunction of vital organ(s), and immune suppression (proportion of HLA-DR Human Leukocyte Antigen - DR - positive monocytes < 80%) participated. Healthy volunteers served as reference subjects. Using phospho-specific whole blood flow cytometry we studied lymphocyte phosphorylation of nuclear factor-κB (NFκB), mitogen-activated protein kinases p38 and extracellular signal-regulated kinases (ERK)1/2, and signal transducers and activators of transcription (STATs) 1, 3, and 6. Statistical comparisons were performed with the Wilcoxon-Mann-Whitney test. RESULTS: In blood samples supplemented with tumor necrosis factor, E. coli or S. aureus, phosphorylation levels of NFκB were lower and levels of p38 were higher in patients with acute pancreatitis than healthy subjects. Low NFκB activation involved CD3+CD4+ and CD3+CD8+ lymphocytes. ERK1/2 phosphorylation induced by co-stimulation with phorbol 12-myristate 13-acetate and calcium ionophore A23187 was depressed in patients. STAT3 was constitutively activated in patients' CD3+CD4+ and CD3+CD8+ lymphocytes. Also, IL-6-induced STAT1 phosphorylation was impaired while IL-4-induced STAT6 phosphorylation was enhanced. CONCLUSIONS: Lymphocytes of patients with acute pancreatitis, organ dysfunction and immune suppression show impaired NFκB activation, which increases infection risk and enhanced p38 activation, which sustains inflammation. Secondly, they indicate constitutive STAT3 activation, which may favor Th17 lineage of CD4+ lymphocyte differentiation. Thirdly, they reveal impaired STAT1 activation and enhanced STAT6 activation, denoting a shift from Th1 towards Th2 differentiation.

Chemically modified tetracyclines (CMT-3 and CMT-8) enable control of the pathologic remodellation of human aortic valve stenosis via MMP-9 and VEGF inhibition.

OBJECTIVE: Tetracycline derivatives affect many cellular functions relevant to chronic cardiovascular pathologies, including cell proliferation, migration and matrix remodelling. Accordingly, we sought to determine whether they may modulate the pathologic characteristics known to be significantly involved in human aortic valve stenosis, such as gelatinase production, apoptosis, expression of vascular endothelial growth factor (VEGF) and tumour necrosis factor-alpha (TNF-alpha). METHODS: The effects of tetracycline derivatives (tetracycline and CMTs-3, -5, -8) on MMP-2 and -9 and their endogenous tissue inhibitor (TIMP-1 and -2) production profiles in explanted human aortic valve pieces were examined by means of gelatine zymography and reverse zymography. Chemiluminescent ELISA was performed to assess VEGF and TNF-alpha concentrations in the medium, and in order to evaluate programmed cell death, in situ labelling of the 3'-ends of the DNA fragments generated by apoptosis-associated endonucleases was performed. RESULTS: CMT-3 and -8 lowered the MMP-9 and VEGF levels significantly in a drug-, dose-, and time-dependent manner. MMP-2 and TIMPs remained unchanged, emphasizing the specificity of CMTs to MMP-9 production on the one hand and restoring the beneficial equilibrium of MMP-9 and TIMPs on the other. Tetracycline was the only drug with a significant impact on net gelatinolytic activity, suggesting that the effect of tetracycline is more extensive concerning total MMP activity. CONCLUSIONS: Tetracycline derivatives may have therapeutic effects on the pathologic remodellation of advanced human aortic stenosis through the inhibition of MMP-9 and VEGF production.

Evidence for an altered balance between matrix metalloproteinase-9 and its inhibitors in calcific aortic stenosis.

BACKGROUND: Recently, aortic valve stenosis has been demonstrated to exhibit increased expression of certain matrix metalloproteinases (MMPs), and this has relevantly raised the question about possible interdependency between these and their tissue inhibitors. We sought to assess the expression of elastolytic MMPs and their inhibitors (TIMPs) in nonrheumatic aortic stenosis. METHODS: The study comprised 30 stenotic and six noncalcified human aortic valves. To measure the expression levels and the amount and molecular forms of gelatinases (MMP-2, MMP-9) and TIMPs (1, 2), in situ hybridization, gelatin zymography, and reverse zymography were carried out. Antielastin staining by a monoclonal BA-4 antibody was performed to investigate the changes of one of the main substrates of these MMPs, and to substantiate the nature of the putative MMP- synthesizing cell. The cases were also immunostained with an antibody to alpha-smooth muscle actin. Inflammatory cell characterization was managed by monoclonal mouse antibodies (UCHL-1, L26, and PGM-1). RESULTS: Compared with the controls, the calcific valves showed increased mRNA expression and activation of MMP-9, and this was associated with typical characteristics of valve disease. MMP-2 mRNA production was rare, but proMMP-2 protein was detected in all valves. In agreement with the interdependency between MMP-9 and its inhibitors, a suggestive imbalance came out in diseased valves. CONCLUSIONS: The disproportion between MMP-9 and its tissue inhibitors may favor a persistent MMP activation state within the calcific valve and likely contribute to the valvular remodeling process in the setting of developing aortic stenosis.

Calcification and cellularity in human aortic heart valve tissue determine the differentiation of bone-marrow-derived cells.

Human bone-marrow-derived mesenchymal stem cells (MSC) are responsible the remodeling of human tissue. However, damaged aortic valves are lack the ability to regenerate which is an active cell-mediated process. Diseased aortic valve remodeling has similarities even to bone formation. In this study, the prerequisites for cultured MSCs to undergo osteoblastic differentiation on aortic valves were explored. An ex vivo model using a human aortic valve microenvironment was developed. The expression of type I procollagen, alkaline phosphatase activity, osteocalcin secretion and osteocalcin immunostaining were studied to evaluate the induction of osteogenesis of the MSCs on noncalcified and calcified human aortic valves. Aortic valves were exposed to freeze-thaw injury to devitalize valves in order to separately study the role of valve matrix vs. endothelial cells in the explants. Thus, valves were assigned to 1 of 4 treatment groups: noncalcified uninjured valves, calcified uninjured valves, noncalcified injured and calcified injured. Finally, valves were decalcified to separately explore the effect of a calcified matrix on the osteogenesis. In this co-culture system, the noncalcified uninjured valves inhibited osteogenesis of MSCs, whereas the calcified valves promoted differentiation towards osteoblastic lineage. Devitalization of the valve matrix inflicted a significant increase in the osteogenesis of co-cultured MSCs. Calcified matrix in the valves seemed to have a role in the spontaneous osteogenesis of the MSCs. This spontaneous matrix induced differentiation of MSCs into osteoblast lineage could not be inhibited by pravastatin, indomethacin or tetracycline. In conclusion, these results suggest that interactions between MSCs and aortic valve matrix components and cells modulate MSC phenotype in this environment. Further studies are required to characterize this interesting phenomenon in greater detail.