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Intracellular signalling in blood leucocytes shows multiple aberrations in acute pancreatitis (AP) complicated by organ dysfunction (OD). We studied whether the aberrations associate with severity of AP and occur in sepsis complicated by OD. The study comprises 14 sepsis patients (11 with shock), 18 AP patients (nine mild; six moderately severe; three severe) and 28 healthy volunteers. Within 48 h after admission to hospital, phosphorylation of nuclear factor-ĸB (NF-ĸB), signal transducers and activators of transcription (STATs) 1,3, and extracellular signal-regulated kinases 1/2 were measured from stimulated or non-stimulated leucocytes using phosphospecific whole blood flow cytometry. In sepsis, as compared with healthy subjects, phosphorylated NF-ĸB levels of monocytes promoted by bacterial lipopolysaccharides, tumour necrosis factor or Escherichia coli cells were lower (P < 0.001 for all), pSTAT1 levels of monocytes promoted by IL-6 were lower (P < 0.05 for all), and STAT3 was constitutively phosphorylated in monocytes, neutrophils and lymphocytes (P < 0.001 for all). In AP, severity was associated with proportions of pSTAT1-positive monocytes and lymphocytes promoted by IL-6 (P < 0.01 for both), constitutive STAT3 phosphorylation in neutrophils (P < 0.05), but not with any of the pNF-ĸB levels. Monocyte pSTAT3 fluorescence intensity, promoted by IL-6, was lower in sepsis and AP patients with OD than in AP patients without OD (P < 0.001). Collectively, signalling aberrations in sepsis with OD mimic those described previously in AP with OD. Possibility that aberrations in STAT1 and STAT3 pathways provide novel markers predicting evolution of OD warrants studies including patients presenting without OD but developing it during follow-up.

Ilmakunnas M, Hockerstedt K, Makisalo H, Siitonen S, Repo H, Pesonen EJ. Hepatic IL-8 release during graft procurement is associated with impaired graft function after human liver transplantation. Clin Transplant 2010: 24: 29–35. © 2009 John Wiley & Sons A/S. Abstract: 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) μmol/L vs. 30 (23–72) μmol/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.

Secondary immune response to tetanus toxoid (TT), monocyte function, and generation of suppressor cells were studied in patients with previous yersinia arthritis (YA) and healthy controls. A comparison of HLA-B27 positive and negative subjects revealed that leukocytes from the former showed significantly higher responses to TT but not to a variety of other antigens in a lymphocyte proliferation test, and higher rates of migration in a leukocyte migration inhibition assay in the absence of TT. The enhanced migration of leukocytes supports the concept that hyper-reactive neutrophils contribute to inflammatory symptoms in HLA-B27 positive subjects, irrespective of previous YA. No correlation was found between HLA-DR specificities and YA. However, HLA-DR2/HLA-B7 was associated with high suppressor cell activity and low serum levels of anti-TT-antibodies. This accords with the view that HLA-DR antigens play a role in the regulation of immune response.

Monosomy 7, a deletion of the long arm of chromosome 7, was shown in the neutrophils of peripheral blood in 5 of 6 patients with a myelodysplastic syndrome or leukemia who had monosomy 7 in their bone marrow cells. In a chemiluminescence assay the production of reactive oxygen species by neutrophils from patients was increased in whole blood and decreased in purified cells, which suggests that neutrophils with monosomy 7 tolerate poorly the cell purification procedures used. The in vitro migration of purified neutrophils obtained from patients with monosomy 7 was impaired. It is known that patients with monosomy 7 have an increased susceptibility to infections. It is possible that neutrophils with monosomy 7 are too easily triggered to a full-scale respiratory burst and thereby the cells exhaust their ability to eliminate invading microbes efficiently.

Acute pancreatitis (AP) is a common disease with wide variation of severity. The diagnosis of AP is usually based on high serum amylase or lipase values but the accuracy of these methods is considered unsatisfactory. One in five of the patients develops a severe disease and carries a considerable risk of development of organ failure and high mortality. Early detection of patients with severe AP and especially those with increased risk of organ failure is importance since such patients seem to benefit from treatment in an intensive care unit started as soon as possible after presentation. In addition to enzymological methods, increasing interest has been focused on laboratory markers reflecting the level of inflammatory response in AP. At present, in routine clinical work the most commonly used severity marker is serum C-reactive protein, the concentration of which rises too slowly to be used for early prediction of severity. New therapies aiming at modifying the course of systemic inflammation in AP are being developed and therefore monitoring the patient's immune inflammatory status is needed. In this review article we present the current knowledge of laboratory tests, which has been evaluated for diagnostic and prognostic purposes in AP.

Despite convincing results of studies in vitro, less is known about the effects of antioxidants on in vivo redox balance in humans. We developed a novel parameter of in vivo redox balance, and studied it and its relation to dental infections in 51 patients on medication for coronary heart disease (CHD) and 39 random controls matched for age group, sex, social class and locality. In vivo redox balance was the ratio of plasma antioxidant capacity, as measured with radical-trapping assay, to neutrophil respiratory burst capacity, as measured with whole blood chemiluminescence assay. Dental infections were quantitated with four rating scales. CHD patients had higher values than controls. Patients on acetosalicylic acid (ASA), diuretics or beta blockers, but not the ones on calcium channel blocker, had significantly higher redox balance than non-users. Combination of calcium channel blockers and ASA was associated with redox balance similar to taking beta blockers or diuretics. Diuretics and ASA were independent determinants of redox balance in multivariate analyses. Redox balance did not correlate with severity of dental infections (Spearman's r 0.06 to 0.11). The results contrast experimental data indicating that calcium channel blockers are as antioxidants superior to other cardiovascular drugs. Total antioxidant capacity in parallel with oxygen species production capacity should be considered in attempts to solve the antioxidant paradox.

Background Neutrophil subpopulations with enhanced oxidative reactivity have been described in a number of clinical and in vitro settings. In the dichlorofluorescin (DCFH) oxidation assay, it is essential to maintain cellular viability and plasma membrane integrity through all stages of sample preparation. The process of erythrocyte lysing is crucial because a number of commercial lysing reagents can increase leukocyte membrane permeability. Methods We assessed viability [propidium iodide (PI) method], DCFH oxidation, and CD11b expression of resting or in vitro–stimulated neutrophils exposed to six different red cell lysing procedures. Results Formaldehyde-containing reagents (Optilyse B, FACS Lyse, and Erythrolyse) but not hypotonic shock or ammonium chloride (NH4Cl) solutions rendered 91.4–99.8% of resting neutrophils PI positive, with concomitant reductions in dichlorofluorescein (DCF) fluorescence, suggesting efflux of the fluorochrome. However, when stimulated with N-formyl-methionyl-leucyl-phenylalanine or Yersinia enterocolitica and then treated with FACS Lyse or Erythrolyse, up to 69.9% of neutrophils remained PI negative and exhibited enhanced DCF fluorescence. CD11b expression of PI-positive and -negative neutrophils was comparable, suggesting that they were activated equally. Conclusions FACS Lyse and Erythrolyse can modify neutrophil plasma membrane integrity, whereas hypotonic shock and NH4Cl solutions retain cellular viability and are lysing methods of choice in evaluation of neutrophil respiratory burst by DCFH oxidation assay. Cytometry 43:290–296, 2001. © 2001 Wiley-Liss, Inc.

The authors optimized the flow cytometric dichlorofluorescin (DCFH)-oxidation assay for buffy coat neutrophil and monocyte respiratory burst activity. Sample handlings were minimized, monocytes identified with a CD14 antibody, and viability evaluated with propidium iodide. Sodium citrate was a better anticoagulant than heparin, with a more intense Yersinia enterocolitica (YER)-induced dichlorofluorescein (DCF)-fluorescence intensity and a higher proportion of DCF-positive cells. EDTA was unsuitable as an anticoagulant with reduced cell viability and poor DCF response. Exposure of cells to YER, phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (FMLP) elicited two neutrophil subpopulations, one with low and the other with high forward light scattering properties. FMLP induced only a marginal DCF response, but after YER or PMA, virtually all neutrophils responded with an increased DCF production. During optimal conditions, the resulting DCF- fluorescence histogram was two-peaked, and the subset of cells with increased forward light scattering properties corresponded to the cells with intense DCF-fluorescence. A similar heterogeneity was frequently but not always observed amongst monocytes. The results indicate that in the peripheral blood there are at least two neutrophil and monocyte populations. One is an effective responder to stimuli, the other exhibiting a moderate response only. Properly optimized, the DCFH-oxidation assay may be used for evaluating neutrophil and monocyte subsets in a clinical setting.

Lipopolysaccharide (LPS) prepared from a rough mutant of Salmonella typhimurium and deacylated enzymatically (dLPS) does not promote neutrophil adherence to human umbilical vein endothelial cells (HUVECs). This paper reports that similarly, a smooth form of LPS prepared from Yersinia enlerocolitica O:3, a serotype known to trigger reactive arthritis in humans, and treated with alkali (yersinia LPS-OH) failed to augment neutrophil adherence to HUVECs. Studies of the mechanism underlying the poor augmentation revealed that neither enzymatically deacylated LPS from Escherichia coli J5 (J5 dLPS) nor yersinia LPS-OH stimulated expression of endothelial cell adhesion molecules E-selectin, VCAM-1 and ICAM-1, whereas both Intact J5 LPS and yersinia LPS were stimulatory. Impaired up-regulation could not be explained by decreased binding of yersinia LPS-OH to HUVECs. Furthermore, 51Cr-labelled HUVECs treated with different concentrations of yersinia LPS-OH released 51Cr in the presence of anti-yersinia anti-0 antibody and complement. J5 dLPS and yersinia LPS-OH inhibited up-regulation of the adhesion molecules induced by J5 LPS and yersinia LPS but not that induced by tumour necrosis factor alpha. Taken together, the results suggest that although yersinia LPS-OH can depress development of acute inflammation by inhibiting up-regulation of etidothelial-cell adhesion molecules, sufiicient LPS-OH is bound to induce cell injury and thereby inflammation in the prescence of specific antibody and complement. The findings may have pathogenetic implications in yersinia-triggered reactive arthritis characterized by dissemination of yersinia LPS throughout the body.

To assess possible interactions between inflammation and activation of the anticoagulant protein C system during post-ischemic reperfusion protein C, APC, neutrophil L-selectin expression and myocardial myeloperoxidase activity (MPO) were measured in 19 patients undergoing cardiopulmonary bypass. After reperfusion for 10 min, APC to protein C ratio (APC/PC) increased from pre-reperfusion value 1.43 ± 0.12 (mean ± SEM) to 2.25 ± 0.29, p = 0.015. Negative correlations were observed between APC/PC and MPO activity (Spearman r −0.64, p = 0.007) and APC/PC and neutrophil L-selectin expression (r = −0.7, p = 0.007, demonstrating that post-ishemic protein C activation was associated with decreased neutrophil tissue sequestration. Thus, physiological protein C activation may be involved in regulation of the inflammatory injury during reperfusion of human ischemic coronary circulation. Am. J. Hematol. 67:210–212, 2001. © 2001 Wiley-Liss, Inc.

We conducted a double-blind, placebo-controlled, randomized study of 3-month treatment with lymecycline, a form of tetracycline, in reactive arthritis (ReA). Lymecycline therapy significantly decreased the duration of the illness in patients with Chlamydia trachomatis-triggered ReA, but not in other ReA patients. In 2 ReA patients, C trachomatis was found in the throat, an uncommon locale for this organism. Our results suggest that it is important to verify the triggering microbe and that it is beneficial to treat Chlamydia arthritis patients with a prolonged course of tetracycline.

We explored whether episodes stimulating leucocytes in vivo could be tracked from whole blood samples by monitoring activation of STAT1 by flow cytometry. The method was tested in hepatitis C patients (n = 9) that were on interferon (IFN)alpha regimen. CD14+ monocytes responded strongly to IFNalpha/gamma being sensitive indicators for recent immune activation. At 45 min after s.c. IFNalpha 91% of monocytes were phosphorylated STAT1+. The frequency of responding cells decreased to a base level within 6 h. Monocytes, however, had a long-term deficient phosphorylated STAT1 response to IFNalphain vitro that in patients on standard IFNalpha regimen lasted for 48 h. In patients on pegylated IFNalpha the phosphorylated STAT1 response was completely absent. We conclude that whole blood analysis of STAT1 activation by flow cytometry is applicable to monitor immune cells in patient material.

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.

Migration of polymorphonuclear leukocytes (PMN) was studied in six healthy subjects with neutropenia (peripheral blood neutrophil count less than or equal to 1.5 X 10(9)/1). Determined as migration differentials, chemotactic and chemokinetic responsiveness tended to be higher in the neutropenic group. Despite this slight tendency to increased responsiveness in vitro, migration in vivo proceeded more slowly in the neutropenic group than in the control group: at 12 hours the leukocyte counts in the skin chamber media were significantly lower, whereas at 24 hours they were nearly normal. Our results show that PMN migration in healthy neutropenic subjects is different from that in healthy non-neutropenic subjects. These differences should be taken into account in the evaluation of the role of aberrant PMN migration in the development of infectious episodes in a neutropenic patient.

We studied the effects of different variables on luminol-enhanced chemiluminescence (CL) of whole blood, induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP), opsonized zymosan particles (OZP), or phosphate buffered saline (PBS). Use of correction factor based on blood neutrophil count and hemoglobin concentration decreased interindividual variation and improved normality of the frequency distributions of CL responses. In FMLP- and PBS-induced CL, day-to-day variation was not significant, but it was significant in OZP-induced CL. Although storage of blood samples for 8 hours decreased CL, the decrease was not evident in a study of initial activation, i.e. CL measured at one minute after application of FMLP. Our results suggest that FMLP-induced initial activation is well suited for epidemiological studies because it is easy to determine and is affected neither by day-to-day variation nor by storage of the blood samples for a few hours.

Several studies have failed to show an association between Shigella sonnei dysentery and reactive arthritis. We describe 3 patients who had reactive arthritis and a recent or concurrent S sonnei infection. To our knowledge, this is only the second study to suggest this association. We propose that S sonnei should be considered as a triggering agent for reactive arthritis.

Chemotaxis, chemokinesis, and spontaneous locomotion of polymorphonuclear leukocytes (PMNs) of yersinia arthritis (YA) patients and healthy subjects with or without HLA-B27 were studied by agarose assay and membrane filter technique. HLA-B27 positive healthy control subjects and yersinia patients showed significantly higher response to chemotactic stimulus than HLA-B27 negative controls. Consequently, the high response was associated with HLA-B27 irrespective of YA. The high responder PMNs may contribute to the more severe inflammatory symptoms in YA patients with HLA-B27.

Chemotactic and chemokinetic migration of polymorphonuclear leukocytes in sera from patients with previous yersinia arthritis and from healthy subjects with or without HLA--B27 were studied by the leading front method. Irrespective of yersinia arthritis, zymosan-activated sera from subjects who were HLA--B27 positive were significantly more chemokinetic than were HLA--B27 negative zymosan-activated sera from healthy control subjects. The chemotactic activities of zymosan-activated sera that were HLA--B27 positive or negative, as determined by chemotactic increments, were much the same. The results suggest that zymosan-activated serum that is HLA--B27 positive stimulates random migration of polymorphonuclear leukocytes, but not their directional migration, more than does HLA--B27 negative serum that has been zymosan activated. This may contribute to accumulation of polymorphonuclear leukocytes at the site of inflammation in vivo and thereby to inflammatory symptoms in yersinia arthritis patients with HLA--B27.

To apply the leukocyte migration agarose test (LMAT) to the in vitro assessment of human neutrophil chemotaxis, effects of different culture conditions on neutrophil migration under agarose were studied. Presence of either serum or human serum albumin (HSA) in the culture medium was necessary for detectable neutrophil migration. HSA was preferred since heat-stabile chemotactic agents were found to be generated from fresh serum in the presence of agarose. Additional CO2 in the assay milieu could be replaced by decreasing the NaHCO3 concentration of the culture medium. Both the directed and the spontaneous migration rates of neutrophil leukocytes increased when the concentration of agarose was decreased. Area and distance of migration and cumulative cell count of migrated neutrophil leukocytes were suitable for quantitating the neutrophil migration rate.