Heikki Repo

Elevated levels of circulating cachectin/tumor necrosis factor in patients with acquired immunodeficiency syndrome☆

PURPOSE In order to evaluate the relationship between cachectin/tumor necrosis factor (TNF) and cachexia in the acquired immunodeficiency syndrome (AIDS), we studied the serum levels of endogenous cachectin/TNF in subjects with human immunodeficiency virus (HIV) infection. PATIENTS AND METHODS Fifty-three serum samples were obtained from 39 HIV-seropositive patients. The condition of each patient was clinically classified as either asymptomatic, lymphadenopathy syndrome (LAS), AIDS-related complex (ARC), or AIDS. Control sera were obtained from 29 healthy male blood donors. A double antibody radioimmunoassay was used to measure the serum levels of cachectin/TNF. RESULTS Cachectin/TNF levels were within the reference range of the control values in all (eight of eight) asymptomatic HIV-infected subjects and in 11 of 13 of the patients with LAS. In contrast, all patients with AIDS (nine of nine) and five of nine of the patients with ARC had raised levels of cachectin/TNF. Fluctuation of the levels of cachectin/TNF occurred during follow-up, but initially raised levels remained elevated. CONCLUSION Since cachectin/TNF suppresses lipoprotein lipase in adipocytes in vitro, and causes weight loss under experimental conditions, the findings of raised levels of cachectin/TNF in patients with AIDS may have relevance to the pathogenesis of cachexia.

Early prediction of organ failure by combined markers in patients with acute pancreatitis.

Several biological markers and clinical scoring systems have been used to predict the course of acute pancreatitis. Because organ failure is the most severe complication of the disease, prognostic markers and their combinations that would predict organ failure on hospital admission were sought.

Procalcitonin, soluble interleukin-2 receptor, and soluble E-selectin in predicting the severity of acute pancreatitis.

ObjectiveTo investigate whether marker(s) of systemic inflammation detect, at an early stage of acute pancreatitis, patients who may ultimately develop severe disease. DesignProspective study. SettingUniversity hospital emergency unit. PatientsThirty patients with mild acute pancreatitis (SEV0 group) and 27 with severe acute pancreatitis. Of the latter, 11 did not develop organ failure (SEV1 group), whereas the other 16 patients developed acute respiratory failure and 9 of them also developed renal failure (SEV2 group). InterventionsBlood samples were collected at admission to the hospital (T0), and at 12 hrs (T12) and 24 hrs (T24) after admission. Measurements and Main Results The plasma concentrations of procalcitonin (PCT), soluble E-selectin (sE-selectin), soluble interleukin-2 receptor (sIL-2R), and the serum concentration of C-reactive protein (CRP) were monitored. PCT levels at T0 were significantly higher in the SEV1 group (median 0.4 ng/mL, range 0.2–2.3) and the SEV2 group (0.8 ng/mL, 0.2–73.5) than in the SEV0 group (0.3 ng/mL, 0.1–3, p < .05 and p < .001, respectively). At T12, PCT level in the SEV2 group was significantly higher than that in the SEV1 group (2.2 ng/mL, 0.2–86.6 vs. 0.4 ng/mL, 0.3–2.8, p = .05), as it also was at T24 (2.2 ng/mL, 0.4–73.3 vs. 0.5 ng/mL, 0.3–4, p < .01). Among SEV2 patients, PCT concentration correlated negatively with the time elapsed between admission and the diagnosis of organ failure. At T12, sIL-2R levels of the SEV1 group (1011 U/mL, range 334–2211) and the SEV2 group (1495 U/ml, range 514–4526) both differed significantly from the SEV0 group (636 U/ml, range 356–1678, p < .05 and p < .001, respectively) as they also did at T24. Although CRP level in the SEV1 group at T12 did not differ from the SEV0 group, the difference between SEV2 (272 &mgr;g/mL, range 46–462) and SEV0 was significant (53 &mgr;g/mL, range 5–243, p < 0.01). sE-selectin levels did not differ between groups. ConclusionsAt admission to hospital, concentrations of PCT, but not those of CRP, sE-selectin, or sIL-2R, are higher in patients with severe acute pancreatitis than in patients with mild pancreatitis. PCT test had sensitivity of 94% and specificity of 73% for development of organ failure. PCT may be useful to identify the patients who benefit from novel therapies aimed at modifying the course of systemic inflammation.

Markers of inflammation in sepsis.

Pathophysiology of sepsis is characterised by a whole body inflammatory reaction and concurrent activation of the hosts anti-inflammatory mechanisms. The balance between pro- and anti-inflammatory reactions is critical for the outcome of the patient. Strongly activated phagocytes and high levels of proinflammatory cytokines occur in patients who are at risk of developing circulatory shock and multiple organ dysfunction. Extensive anti-inflammatory reaction, which is characterised by the presence of high levels of circulating anti-inflammatory cytokines and impaired innate and adaptive immune functions, renders critically ill patients prone to secondary infections. Evaluation of the immune-inflammatory status on admission to the hospital may be helpful in the early identification of patients who are bound to develop organ dysfunction. Such patients could possibly benefit from a mode of therapy aimed at modifying the course of inflammatory response. The use of inflammatory markers may also improve diagnosis of severe infection. The present review summarises the studies on markers of inflammation and immune suppression used, first, as predictors of organ dysfunction in patients with systemic inflammation, and, second, as indicators of infection in adults and neonates.

Flow cytometric determination of CD11b upregulation in vivo

We describe a flow cytometric method to evaluate upregulation of peripheral blood neutrophil and monocyte integrin CD11b in vivo. To avoid spontaneous upregulation in vitro, buffy coat cells were separated on ice and all subsequent cell handling steps were carried out at 0-4 degrees C. Such leukocytes were 95-100% viable, as determined by PI staining. Buffy coat leukocytes were double-stained with CD11b PE-conjugated and CD14 FITC-conjugated monoclonal antibodies and, in addition, with the nucleic acid dye LDS-751. After staining, firstly, LDS-751 positive (+ve) leukocytes, and, secondly, CD14 +ve monocytes were collected in live mode. Aggregated and irrelevant cells were gated out on the basis of their LDS-751 staining pattern and cellular light scattering properties, and the CD11b expression on neutrophils and monocytes was determined. Upregulation of CD11b in vitro was significantly affected by factors such as cell handling temperature, pre-fixation of blood samples, and density gradient separation of the cells. Incubation of aliquots of buffy coat cell suspension supplemented with FMLP for 5 min or without FMLP supplement for 15 min at 37 degrees C significantly increased CD11b expression without affecting cell viability. We have demonstrated that CD11b is expressed at maximal levels on arthritic synovial fluid neutrophils and CD14 +ve cells, and at increased but submaximal levels on peripheral blood neutrophils and monocytes of patients recovering from sepsis. The results suggest that the method can be used to evaluate in vivo upregulation of CD11b.

Leukocyte migration agarose test for the assessment of human neutrophil chemotaxis. I. Effects of environmental factors on neutrophil migration under agarose.

To apply the leukocyte migration agrose 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 quantitative the neutrophil migration rate.

A prospective study of inflammation markers in patients at risk of indirect acute lung injury.

Systemic inflammation triggered by insults like sepsis and acute pancreatitis may play a role in development of indirect acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Because little is known about the course of systemic inflammation on the days preceding diagnosis of ARDS, we prospectively monitored immune inflammatory status in 52 patients at risk and we assessed the presence of ALI and ARDS on day 7 after admission to the intensive care unit. On admission, serum interleukin (IL) 8, IL-6, and soluble IL-2 receptor concentrations were significantly higher in patients with subsequent ALI (n = 18) than in patients without ALI (n = 30). During a 4-day follow-up, IL-8 and IL-6 levels of ALI patients remained high and those of non-ALI patients decreased. None of the markers discriminated ARDS patients (n = 9) from non-ARDS ALI patients (n = 9). Among 11 patients with acute pancreatitis, ALI patients had significantly higher IL-8, IL-6, and phagocyte CD11b expression levels than did non-ALI patients, whereas among 14 patients with massive transfusion, respective findings in ALI and non-ALI patients were comparable. Results give credence to the view that systemic inflammation plays a role in development of ALI triggered by pancreatitis, but not in that by massive transfusion. This finding, if confirmed in studies with sufficient statistical power, suggests that the patients with massive transfusion do not necessarily benefit from novel biotherapies aimed at altering the course of systemic inflammation.

Anticoagulant selection influences flow cytometric determination of CD11b upregulation in vivo and ex vivo

We recently devised three-colour flow cytometric assay for evaluating expression of CD11b on neutrophils and monocytes in circulation. Artefactual upregulation of CD11b ex vivo was minimized by cooling blood samples on ice. In this communication we further characterize the method in terms of different anticoagulants. EDTA was less optimal than ACD or heparin because (i) saturating concentrations of CD11b antibody (clone D12) were not achieved with resting cells; (ii) CD11b fluorescence intensity of synovial fluid cells, i.e., in vivo activated cells expressing CD11b at high levels, was significantly lower in EDTA plasma, and (iii) EDTA mediated more cell damage at 37 degrees C, as determined by PI staining. The fluorescence data suggested that D12 antibody binding was dependent on divalent cations. Saturating concentrations in the presence of EDTA in medium were easily obtained with synovial fluid cells and peripheral blood phagocytes activated with chemotactic peptide FMLP, suggesting that cell activation decreased cation concentrations required for D12 antibody binding. Using another CD11b antibody (2MPL19c), whose binding proved to be cation independent, it was shown that CD11b upregulation was not affected by EDTA. ACD was superior to heparin and phenylalanylprolylarginyl chloromethyl ketone (PPACK), a thrombin inhibitor, because cell counts were significantly lower in heparinized samples in cold, and in PPACK-anticoagulated samples treated with LPS at 37 degrees C. Kinetics of L-selectin shedding was similar in heparin and ACD, suggesting that cell loss did not derive from differences in cell activation. In comparison of buffy coat cell assay and whole blood assay, neutrophil CD11b expression was similar but background fluorescence was significantly higher in whole blood preparations. This implies that nonspecific antibody binding may occur more in whole blood assay, whereas in the buffy coat cell assay, sample manipulation procedures may slightly increase CD11b antibody binding, but not control antibody binding. Finally, it was confirmed that warming from 0 degrees C, but not from room temperature, to 37 degrees C increased CD11b expression significantly on neutrophils, and it was further shown that monocytes undergo similar changes. Cooling did not upregulate CD11b, and completely prevented LPS-induced upregulation. In conclusion, the results support use of ACD in evaluating CD11b expression; if EDTA is used, it is important to make sure that binding of CD11b antibody selected does not require presence of divalent cations in medium.

Decreased HLA (human leucocyte antigen)-DR expression on peripheral blood monocytes predicts the development of organ failure in patients with acute pancreatitis

Immune suppression plays an important role in the pathogenesis of acute pancreatitis. Monocyte expression of HLA (human leucocyte antigen)-DR, a cellular marker of immune suppression, was determined in relation to the development of organ dysfunction in patients with acute pancreatitis. A total of 310 consecutive patients with acute pancreatitis, admitted to a university hospital within 72 h of pain onset, were studied; 194 (63%) had mild disease (group I), 87 (28%) had severe disease without organ dysfunction (group II), and 29 (9%) had severe disease with organ dysfunction (group III). HLA-DR expression, defined both as the proportion of monocytes that were HLA-DR-positive and as monocyte HLA-DR fluorescence intensity, was determined at admission, using whole-blood flow cytometry. Of the patients in group III, 13 (45%) developed organ dysfunction within 24 h of admission. The proportion of HLA-DR-positive monocytes and monocyte HLA-DR density were both related to the severity of pancreatitis (P<0.001 for linear trend). In predicting organ dysfunction, the sensitivity, specificity and positive-likelihood ratio for the proportion of HLA-DR-positive monocytes were 83% [95% CI (confidence interval) 64-94%], 72% (67-77%) and 3.0 respectively, and for monocyte HLA-DR density the respective values were 69% (49-85%), 84% (79-88%) and 4.3. In conclusion, monocyte HLA-DR expression predicts the development of organ dysfunction that occurs early in patients with acute pancreatitis.

Plasma anti‐inflammatory cytokines and monocyte human leucocyte antigen‐DR expression in patients with acute pancreatitis

Background: Immune suppression plays a role in the pathogenesis of acute pancreatitis. The purpose was to describe plasma anti‐inflammatory cytokines and blood monocyte human leucocyte antigen (HLA)‐DR expression, a cellular marker of immune suppression, in relation to clinical outcome in acute pancreatitis. Methods: We studied 74 patients with acute pancreatitis admitted within 72 h after symptom onset; 27 had mild disease and 47 severe disease, of whom 20 developed organ failure. Plasma cytokine concentrations and monocyte HLA‐DR density were determined at admission and 1, 2, 3, 7, 14 and 21 days later. Results: The levels of interleukin‐1 receptor antagonist, interleukin‐6 and interleukin‐10 correlated inversely to monocyte HLA‐DR expression; each marker correlated with disease severity. Interleukin‐4, ‐11 and ‐13 levels were low. Organ failure occurred at median 36 h (range 8 to 158) after admission and was predicted at admission by the combination of interleukin‐6 and interleukin‐10 with sensitivity of 95%, specificity of 88% and positive likelihood ratio of 7.6 (95% confidence interval 3.3 to 17). Patients with secondary infections had a lower proportion of HLA‐DR positive monocytes than did controls at day 14 (median: 32% versus 65%; n = 7) and at day 21 (median: 49% versus 83%; n = 6), P < 0.05 each. In the organ failure group, HLA‐DR expression did not differ between survivors and non‐survivors. Conclusions: Determining the severity of anti‐inflammatory reaction at admission and monitoring the course of immune suppression provide a means for predicting clinical outcome in acute pancreatitis.