Activated protein C retards recovery from coagulopathy in severe acute pancreatitis.

OBJECTIVES: Activated protein C (APC), an endogenous anticoagulant, has antithrombotic, fibrinolytic and anti-inflammatory properties. We recently conducted a controlled study (APCAP, activated protein C in severe acute pancreatitis) of APC treatment of patients with severe acute pancreatitis (SAP). Here we studied the effect of APC on the pivotal coagulation parameters of the surviving patients in the APCAP study. METHODS: The study consisted of 20 patients of whom 10 patients had received APC and 10 patients had received placebo. Coagulation parameters, physiological anticoagulants, thrombograms and circulating levels of IL-6 and CRP were determined on admission and at days 1, 3-4 and 6-7. RESULTS: During follow-up, the temporal levels of prothrombin time (PT) decreased and the temporal levels of thromboplastin time (TT) increased in placebo group (p< 0.001 for both), but not in APC group. The temporal levels of antithrombin (AT) increased less in APC group than in placebo group (p = 0.011). The shapes of the SAP patients' thrombograms were strongly deranged and were marginally affected by APC treatment. CONCLUSIONS: Coagulopathy in SAP, a complex phenomenon, is not alleviated by APC treatment. Rather, the patients receiving APC are heading toward normal homeostasis of coagulation slower than patients receiving placebo.

Early prediction of persistent organ failure by soluble CD73 in patients with acute pancreatitis*.

OBJECTIVE: New biomarkers are needed to better predict the severity of acute pancreatitis. CD73/ecto-5'-nucleotidase is an enzyme that generates adenosine, which dampens inflammation and improves vascular barrier function in several disease models. CD73 also circulates in a soluble form in the blood. We studied whether levels of soluble form of CD73 predict the development of organ failure in acute pancreatitis. DESIGN: A prospective cohort study of patients with acute pancreatitis from 2003 to 2007. SETTING: Admissions to the biggest tertiary care hospital in Finland. PATIENTS: One hundred sixty-one patients with acute pancreatitis, of which 107 were subclassified according to the revised Atlanta criteria into mild, 29 into moderately severe and 25 into severe. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Serum and blood cell samples were collected at admission. Protein levels of soluble form of CD73 in serum were determined using a novel enzyme-linked immunosorbent assay, activity of soluble form of CD73 using radioactive enzyme assays, and CD73 messenger RNA levels from leukocytes using quantitative polymerase chain reaction. Activity and protein concentration of soluble form of CD73, and messenger RNA level of CD73 all decreased along with the disease severity (p ≤ 0.01 for all). The activity of soluble form of CD73 at admission predicted the development of the severe pancreatitis in different groups of the patients. The area under the receiver-operating characteristic curve value for activity of soluble form of CD73 was 0.65 (95% CI, 0.51-0.80) among a subgroup of patients comprising moderately severe and severe disease, 0.79 (95% CI, 0.69-0.88) among all patients including mild pancreatitis, and 0.75 (95% CI, 0.60-0.89) among patients who had no signs of organ failure (modified Marshall score < 2) at admission. Especially, in the last-mentioned group, activity of soluble form of CD73 was better than C-reactive protein or creatinine in predicting the severe pancreat CONCLUSIONS: : Activity of soluble form of CD73 at admission to hospital has prognostic value in predicting the development of the severe form of acute pancreatitis.

Bloodstream infections among children with juvenile idiopathic arthritis: a prospective study from the onset of disease.

OBJECTIVES: To describe the incidence and nature of bloodstream infections (BSI) among children with juvenile idiopathic arthritis (JIA) followed-up prospectively from disease onset. METHODS: The Social Insurance Institution's (SII) national register on individuals with reimbursement for medication of chronic diseases was used to identify children with JIA from 2004 through 2011 and their medications. The National Infectious Disease Register (NIDR) collects data of all blood culture positive samples from all microbiology laboratories in Finland. We combined the NIDR and SII registers to identify JIA patients with BSI. Clinical and laboratory data of each JIA-BSI patient were collected from hospital records. RESULTS: There were 1604 JIA patients and 6630 person-years of follow-up. Five patients had BSI. During the first 5 years after diagnosis the cumulative emergence of BSI was 0.38% [95% confidence interval (CI) 0.16% to 0.92%]. The incidence rates were 7.5/10 000 follow-up years for JIA (95% CI 2.4-17.6) and 2.8/10 000 follow-up years for the age-matched general population (95% CI 2.7-2.9). The standardised incidence ratio was 3.0 (95% CI 1.2 to 7.2). The causative bacteria were Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli and Fusobacterium necrophorum. Three patients were on anti-rheumatic drugs, including two on TNF inhibitors. All patients responded rapidly to antimicrobial therapy and recovered uneventfully. CONCLUSIONS: Although BSI is rare among children with JIA, the incidence is 3-fold higher than among the general population.

Circulating cytokines in predicting development of severe acute pancreatitis.

INTRODUCTION: Severe acute pancreatitis (AP) is associated with high morbidity and mortality. Early prediction of severe AP is needed to improve patient outcomes. The aim of the present study was to find novel cytokines or combinations of cytokines that can be used for the early identification of patients with AP at risk for severe disease. METHODS: We performed a prospective study of 163 nonconsecutive patients with AP, of whom 25 had severe AP according to the revised Atlanta criteria. Admission serum levels of 48 cytokines and growth factors were determined using Bio-Plex Pro Human Cytokine Assay 21-plex and 27-plex magnetic bead suspension panels. Admission plasma levels of C-reactive protein (CRP), creatinine and calcium were measured for comparison. In subgroup analyses, we assessed the cytokine profiles of patients with severe AP (n = 14) who did not have organ dysfunction (OD) upon admission (modified Marshall score <2). RESULTS: Of 14 cytokines elevated in the severe AP group, interleukin 6 (IL-6) and hepatocyte growth factor (HGF) levels were independent prognostic markers of severe AP. IL-6, HGF and a combination of them predicted severe AP with sensitivities of 56.0%, 60.0% and 72.0%, respectively, and specificities of 90.6%, 92.8% and 89.9%, respectively. The corresponding positive likelihood ratio (LR+) values were 5.9, 8.3 and 7.1, respectively. The predictive values of CRP, creatinine and calcium were comparable to those of the cytokines. In subgroup analyses of patients with severe AP and without OD upon admission, we found that IL-8, HGF and granulocyte colony-stimulating factor (G-CSF) levels predicted the development of severe AP, with G-CSF being the most accurate cytokine at a sensitivity of 35.7%, a specificity of 96.1% and a LR+ of 9.1. CONCLUSIONS: IL-6 and HGF levels upon admission have prognostic value for severe AP which is similar to levels of CRP, creatinine and calcium. Although IL-6 and HGF, as either single or combined markers, were not perfect in identifying patients at risk for severe AP, the possibility that combining them with novel prognostic markers other than cytokines might improve prognostic accuracy needs to be studied. The accuracy of IL-8, HGF and G-CSF levels in predicting severe AP in patients without clinical signs of OD upon admission warrants larger studies.

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.

Detection of muramyl dipeptide-sensing pathway defects in monocytes of patients with Crohn's disease using phospho-specific whole blood flow cytometry.

Peripheral blood mononuclear cells of Crohn's disease (CD) patients with the common 1007fs mutation of the caspase recruitment domain-containing 15/nucleotide-binding oligomerization domain-containing 2 (CARD15/NOD2) gene show impaired nuclear factor kappa B (NF-κB) activation in response to muramyl dipeptide (MDP), as determined by Western blotting. We applied phospho-specific flow cytometry to examine NF-κB and p38 activation in whole blood monocytes of 16 CD patients with or without the 1007fs and previously described rare mutations of the CARD15 gene, and healthy reference subjects. Aliquots of whole blood were supplemented with MDP (0-1000 ng/mL), incubated for 10-40 min and processed for flow cytometry. Bacterial lipopolysaccharide (LPS) was used as a positive control agonist. We found that NF-κB and p38 phosphorylation induced by MDP was not detectable in monocytes of patients homozygous for the CARD15 1007fs mutation, while those induced by LPS were normal. We also determined MDP-induced NF-κB phosphorylation levels in nuclear extracts of mononuclear cells separated from blood using enzyme-linked immunosorbent assay (ELISA), and observed that the levels decreased in a 1007fs mutation-dose dependent manner. We conclude that phospho-specific whole blood flow cytometry provides a means to study phosphorylation of NF-κB and p38 in clinical samples and can be applied to screening of CD patients homozygous for the CARD15 1007fs mutation.

Double-blind, randomized, placebo-controlled study of three-month treatment with the combination of ofloxacin and roxithromycin in recent-onset reactive arthritis.

In a randomized, double-blind, placebo-controlled trial, 56 patients with recent-onset ReA [enteroarthritis, n = 47 (84%); uroarthritis, n = 9 (16%)] were randomly assigned to receive 200 mg ofloxacin and 150 mg roxithromycin twice daily (Combi, n = 26) or placebo (n = 30) for 3 months. Patients were assessed at entry, at 2 weeks, and at 1, 2, 3, 4, 5, and 6 months. The primary outcome measure was recovery from arthritis at 6 months, and secondary outcome measures were swollen and tender joint counts, Ritchie index, serum CRP level, erythrocyte sedimentation rate, and joint pain on a visual analogue scale at 6 months. After 6 months, 20 patients [77% (95% CI 56-91)] in Combi and 20 patients [67% (95% CI 47-83)] in placebo group had recovered from arthritis (p = 0.55), and all clinical and laboratory variables showed improvement with no statistically significant difference between groups. Adverse events were reported by 62% of the patients in the Combi versus 40% in the placebo group. In conclusion, outcome of ReA was good in both treatment groups. Three-month treatment with the combination of ofloxacin and roxithromycin had no advantage over placebo in patients with recent-onset ReA.

Thrombin generation in vitro and in vivo, and disturbed tissue factor regulation in patients with acute pancreatitis.

BACKGROUND: Being a central link between inflammation and coagulation, tissue factor (TF) and its inhibitor (TFPI) might be associated with the severity of acute pancreatitis (AP) and the development of organ failure (OF). METHODS: The study comprises 9 severe AP patients with OF and 24 reference patients (11 mild AP and 13 severe AP without OF). Plasma samples were collected on admission. TF-induced thrombin generation in plasma samples was studied using the thrombogram method. In vivo thrombin generation was estimated by prothrombin fragment F1+2. Free and total TFPI levels were measured. To evaluate coagulation status the activated partial thromboplastin time, prothrombin time, platelet count, D-dimer, fibrinogen, antithrombin (AT) 3 and protein C (PC) were determined. RESULTS: There was no significant difference in F1+2 levels between the patient groups. Patients with severe AP tended to show low platelet counts, PC and AT3 levels, and high D-dimer levels. In 11 patients the standard TF stimulation did not trigger thrombin generation in the thrombogram. All deaths occurred in these patients. Free TFPI levels and free/total TFPI ratios were significantly higher in these patients and in non-survivors. CONCLUSION: Failure of TF-initiated thrombin generation in the thrombogram assay explained by high levels of circulating free TFPI may be associated with OF and mortality in AP. and IAP.

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.

Low TNF-induced NF-kappaB and p38 phosphorylation levels in leucocytes in tumour necrosis factor receptor-associated periodic syndrome.

OBJECTIVE: TNF receptor-associated periodic syndrome (TRAPS) is a systemic autoinflammatory disorder caused by mutations in the type 1 TNF receptor (TNFRSF1A) gene. Because the pathomechanism of TRAPS may involve aberrant TNF-mediated intracellular signalling, we examined phosphorylation levels of nuclear factor kappaB (NF-kappaB) and p38 in response to TNF in 10 patients with three different TNFRSF1A mutations (C73R, C88Y and F112I). METHODS: Phosphorylation levels of NF-kappaB p65 and p38 were determined in fresh leucocytes stimulated with TNF (0-100 ng/ml) for 2.5-20 min and permeabilized for phospho-specific antibodies in a whole blood flow cytometry assay. As control agonists, we used bacterial lipopolysaccharide (LPS) and IFN-gamma, the latter mediating phosphorylation of the signal transducer and activator of transcription 1. Areas under curve values for dose-response and time course of NF-kappaB and p38 phosphorylation were calculated for the comparison of patients and reference subjects. RESULTS: NF-kappaB and p38 phosphorylation levels of monocytes, lymphocytes and neutrophils stimulated with TNF were significantly lower in TRAPS patients than in reference subjects. Phosphorylation levels induced by LPS, or by IFN-gamma, in patient and reference samples were comparable, indicating that the defect was confined to TNF-mediated signalling. CONCLUSIONS: In the three families studied, TRAPS was associated with low TNF-mediated signalling in leucocytes. This deficiency of the innate immune system may result in the activation of as yet unidentified compensatory regulatory mechanisms yielding the hyperinflammatory phenotype of TRAPS.

Hepatic IL-8 release during graft procurement is associated with impaired graft function after human liver transplantation.

In experimental models, brain death induces inflammatory cascades, leading to reduced graft survival. Thus far, factors prior to graft preservation have gained less attention in clinical setting. We studied pre-preservation inflammatory response and its effects on graft function in 30 brain dead liver donors and the respective recipients. Before donor graft perfusion, portal and hepatic venous blood samples were drawn for phagocyte adhesion molecule expression and plasma cytokine determinations. Donor intensive care unit stay correlated with donor C-reactive protein (R = 0.472, p = 0.013) and IL-6 (R = 0.419, p = 0.026) levels, and donor (R = 0.478, p = 0.016) and recipient gamma-glutamyl transferase (R = 0.432, p = 0.019) levels. During graft procurement, hepatic IL-8 release was observed in 17/30 donors. Grafts with hepatic IL-8 release exhibited subsequently higher alkaline phosphatase [319 (213-405) IU/L vs. 175 (149-208) IU/L, p = 0.006] and bilirubin [101 (44-139) micromol/L vs. 30 (23-72) micromol/L, p = 0.029] levels after transplantation. Our findings support the concept that inflammatory response in the brain dead organ donor contributes to the development of graft injury in human liver transplantation.

APCAP--activated protein C in acute pancreatitis: a double-blind randomized human pilot trial.

INTRODUCTION: Previous human studies have shown low activity of protein C (APC) in severe acute pancreatitis (SAP). This, together with the findings in animal models, suggests that activated protein C (APC) may protect against pancreatic injury and ameliorate the disease. We, therefore, evaluated its effect on multiple organ dysfunction (MOD) measured by the SOFA (Sequential Organ Failure Assessment) and on organ-failure-free days, and the safety of APC in SAP. METHODS: A prospective double blind randomized pilot study was use. The study occurred in one university hospital tertiary intensive care unit (ICU) with eight beds. The patients were chosen according to the following inclusion criteria: 1) Those admitted to the hospital < 96 h from the onset of pain, 2) Those who had a three-fold increase in serum amylase over normal upper range or/and in whom computed tomography (CT) verification of SAP was noted, 3) Those who had one or more organ dysfunction (OD), and 4) Those in whom less than 48 hours had passed since their first OD. Of a total of 215 adult patients with SAP screened between June 2003 and August 2007, 158 fulfilled the study inclusion criteria. After exclusions 32 patients were randomized to the study. The intervention consisted of APC (N = 16) administered intravenously for 96 hours with a dose of 24 µg/kg/hour or placebo (N = 16) with a similar infusion rate. The sample size for the study was calculated according to the primary end-point: the change in SOFA during study drug infusion (Days 0 and 5). Comparisons between the study groups were performed using patient-related changes and calculation of difference in means (DIM, 95% CIs) and regarding categorical variables with Fisher's exact test. For all comparisons P < 0.05 was considered significant. RESULTS: No serious bleeding was detected clinically or by CT scans in either group. No significant difference in SOFA score change between the APC and placebo groups was found (difference in means (DIM) +2.3, 95% CI -0.7 to +5.3). Treatment with APC was associated with an increase in serum levels of both total and conjugated bilirubin. No differences in ventilator-free days, in renal replacement therapy-free days, in vasopressor-free days, or in days alive outside the hospital were detected. CONCLUSIONS: No serious bleeding or differences in the evolution of MOD were detected between APC and the placebo. Instead we found an increase in serum bilirubin in the APC group compared to the placebo group in patients with SAP. TRIAL REGISTRATION: ClinicalTrials.gov NCT01017107.

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.

Novel CARD15/NOD2 mutations in Finnish patients with Crohn's disease and their relation to phenotypic variation in vitro and in vivo.

BACKGROUND: Three mutations (R702W, G908R, and 1007fs) of the CARD15/NOD2 gene associate with Crohn's disease (CD). Despite a strong linkage of CD to the inflammatory bowel disease (IBD) 1 region, only 16% of the Finnish CD patients carry 1 of these 3 mutations, pointing to the possibility of yet undetected founder mutations in the genetically isolated Finns. The aim of this study was to screen for CARD15 mutations in Finnish CD patients and to assess their functional consequences and relation to clinical phenotype. METHODS: We performed CARD15 mutation screening in 240 CD probands. For functional studies, blood mononuclear cells were cultured alone or with muramyl dipeptide (MDP) and IL-8 levels were determined. RESULTS: We identified 30 different variants, including 12 new ones. Allele frequencies for the R702W, G908R, and 1007fs mutations were 3.3%, 0.4%, and 4.8%, respectively. The 1007fs variant was the only 1 associated significantly with CD. Five novel variants (R38M, W355X, P727L, W907R, R1019X) were found in 5 patients. The biochemical nature of these new mutations, data obtained by cross-species comparisons, as well as low IL-8 production favors their pathogenic role. All 5 patients with novel mutations presented a complicated form of ileal or ileocolonic disease. CONCLUSIONS: In conclusion, we identified 5 novel CARD15 mutations with an apparent pathophysiological role, but could not identify a putative Finnish founder mutation. It is still possible that regulatory mutations present in the flanking or intronic areas of the CARD15 gene contribute to the genetic susceptibility of CD. Homozygosity or compound heterozygosity for CARD15 gene mutations must be considered especially in complicated CD patients.

Cytokine profiles in acute liver failure treated with albumin dialysis.

Cytokines are released within the liver in response to hepatic injury, and acute liver failure (ALF) triggers systemic inflammation. Pro-inflammatory (tumor necrosis factor-alpha [TNF-alpha] and interleukin-8 [IL-8]) and anti-inflammatory (interleukin-10 [IL-10] and interleukin-6 [IL-6]) cytokines and the lymphocyte activation marker (interleukin-2-soluble receptor alpha chain [IL-2sRalpha]) were monitored in 49 ALF patients considered for liver transplantation and treated with albumin dialysis (molecular adsorbent recirculating system [MARS]). Twenty-six patients were categorized by clinical outcome as "good" (native liver recovered) and 23 as "poor" (patient bridged to liver transplantation or deceased). MARS did not clearly affect cytokine profiles during treatment; only IL-10 levels decreased in the whole patient population and mostly in patients with the worst prognosis. In the good outcome group, IL-8 and IL-6 levels decreased during treatment; on the contrary, in poor outcome patients IL-6 levels even increased. Initial IL-2sRalpha levels were higher in poor outcome patients relative to the good outcome subset. Cytokine profiles seem to differ in ALF according to patient outcome. A deeper understanding of cytokine patterns during pathogenesis could reveal prognostic markers and aid the development of immunomodulating ALF therapies.

Baseline JAK phosphorylation profile of peripheral blood leukocytes, studied by whole blood phosphospecific flow cytometry, is associated with 1-year treatment response in early rheumatoid arthritis.

BACKGROUND: We found recently that baseline signal transducer and activator of transcription 3 phosphorylation in peripheral blood CD4+ T cells of patients with early rheumatoid arthritis (RA) is associated with treatment response to synthetic disease-modifying antirheumatic drugs (DMARDs). This prompted us to study the baseline phosphorylation profiles of Janus kinases (JAKs) in blood leukocytes with respect to treatment response in early RA. METHODS: Thirty-five DMARD-naïve patients with early RA provided blood samples for whole blood flow cytometric determination of phosphorylation of JAKs in CD4+ and CD8+ T cells, CD19+ B cells, and CD14+ monocytes. Treatment response was determined after 1 year of treatment with synthetic DMARDs, with remission defined as absence of tender and swollen joints and normal erythrocyte sedimentation rate. Exact logistic regression was used to investigate the association of baseline variables with treatment response. Ninety-five percent CIs of means were estimated by bias-corrected bootstrapping. RESULTS: High JAK3 phosphorylation in CD4+ and CD8+ T cells, CD19+ B cells, and CD14+ monocytes and low JAK2 phosphorylation in CD14+ monocytes were significantly associated with remission following treatment with synthetic DMARDs. CONCLUSIONS: Baseline JAK phosphorylation profile in peripheral blood leukocytes may provide a means to predict treatment response achieved by synthetic DMARDs among patients with early RA.

Upregulated but insufficient generation of activated protein C is associated with development of multiorgan failure in severe acute pancreatitis.

INTRODUCTION: Disturbed protein C (PC) pathway homeostasis might contribute to the development of multiple organ failure (MOF) in acute pancreatitis (AP). We therefore evaluated circulating levels of PC and activated protein C (APC), evaluated monocyte deactivation in AP patients, and determined the relationship of these parameters to MOF. PATIENTS AND METHODS: Thirty-one patients in the intensive care unit were categorized as cases (n = 13, severe AP with MOF) or controls (n = 18, severe AP without MOF). Blood samples were drawn every second day to determine the platelet count, the levels of APC, PC, and D-dimer, and the monocyte HLA-DR expression using flow cytometry. The APC/PC ratio was used to evaluate turnover of PC to APC. RESULTS: During the initial two weeks of hospitalization, low PC levels (<70% of the adult mean) occurred in 92% of cases and 44% of controls (P = 0.008). The minimum APC level was lower in cases than in controls (median 85% versus 97%, P = 0.009). Using 87% as the cut-off value, 8/13 (62%) cases and 3/18 (17%) controls showed reduced APC levels (P = 0.021). A total of 92% of cases and 50% of controls had APC/PC ratios exceeding the upper normal limit (P = 0.013). Plasma samples drawn before MOF showed low PC levels and high APC/PC ratios. HLA-DR-positive monocytes correlated with PC levels (r = 0.38, P < 0.001) and APC levels (r = 0.27, P < 0.001), indicating that the PC pathway was associated with systemic inflammation-triggered immune suppression. CONCLUSION: PC deficiency and decreased APC generation in severe AP probably contributed to a compromised anticoagulant and anti-inflammatory defence. The PC pathway defects were associated with the development of MOF. The data support feasibility of testing the use of APC or PC to improve the clinical outcome in AP.

STAT6 and STAT1 Pathway Activation in Circulating Lymphocytes and Monocytes as Predictor of Treatment Response in Rheumatoid Arthritis.

OBJECTIVE: To find novel predictors of treatment response to disease-modifying antirheumatic drugs (DMARDs), we studied activation of STAT (signal transducers and activators of transcription) 6 and 1 in circulating leukocytes of patients with rheumatoid arthritis (RA). METHODS: 19 patients with untreated recent-onset RA, 16 patients with chronic RA irresponsive to synthetic DMARDs and 37 healthy volunteers provided blood samples for whole blood flow cytometric determination of intracellular STAT6 and STAT1 phosphorylation, expressed as relative fluorescence units, in response to IL-4 and IFN-γ, respectively. Phosphorylation was restudied and treatment response (according to European League Against Rheumatism) determined after 1-year treatment with synthetic DMARDs in recent-onset RA and with biological DMARD in synthetic DMARD-irresponsive RA. Estimation-based exact logistic regression was used to investigate relation of baseline variables to treatment response. 95% confidence intervals of means were estimated by bias-corrected bootstrapping and the significance between baseline and follow-up values was calculated by permutation test. RESULTS: At baseline, levels of phosphorylated STAT6 (pSTAT6) induced by IL-4 in monocytes were higher in those who achieved good treatment response to synthetic DMARDs than in those who did not among patients with untreated RA (OR 2.74, 95% CI 1.05 to 9.47), and IFN-γ -stimulated lymphocyte pSTAT1 levels were higher in those who achieved good treatment response to a biological drug than in those who did not among patients with chronic RA (OR 3.91, 95% CI 1.12 to 20.68). During follow-up, in recent-onset RA patients with good treatment response to synthetic DMARDS, the lymphocyte pSTAT6 levels decreased (p = 0.011), and, consequently, the ratio of pSTAT1/pSTAT6 in lymphocytes increased (p = 0.042). CONCLUSION: Cytokine-stimulated STAT6 and STAT1 phosphorylation in circulating leukocytes was associated with treatment response to DMARDs in this pilot study. The result, if confirmed in larger studies, may aid in developing personalized medicine in RA.

Circulating nucleosomes as predictive markers of severe acute pancreatitis.

BACKGROUND: The components of nucleosomes, which contain DNA and histones, are released into the circulation from damaged cells and can promote inflammation. We studied whether the on-admission levels of circulating nucleosomes predict the development of severe acute pancreatitis (AP), in particular among the patients who present without clinical signs of organ dysfunction. METHODS: This is a prospective study of 74 AP patients admitted to Helsinki University Hospital from 2003 to 2007. Twenty-three patients had mild, 27 moderately severe, and 24 severe AP as defined by the revised Atlanta criteria. 14/24 severe AP patients had no sign of organ dysfunction on admission (modified marshall score <2). Blood samples were obtained on admission and the plasma levels of nucleosomes were measured using enzyme-linked immunosorbent assay. RESULTS: The on-admission levels of nucleosomes were significantly higher in severe AP than in mild or moderately severe AP (p < 0.001 for all), higher in non-survivors (n = 8) than in survivors (p = 0.019), and correlated with the on-admission levels of C-reactive protein (p < 0.001) and creatinine (p < 0.001). Among the AP patients who presented without organ dysfunction, the on-admission nucleosome level was an independent predictor of severe AP (p = 0.038, gender-adjusted forward-stepping logistic regression). CONCLUSIONS: Circulating nucleosome levels may be helpful in identifying, on admission to hospital, the AP patients who present without clinical signs of organ dysfunction, and, yet, are bound to develop organ dysfunction during hospitalization.