Biomedical subjects

H Repo

Publications and source records attributed to H Repo.

At least 19 recordsLinked to original sources

PubMed2006

Polymorphism at position +896 of the toll-like receptor 4 gene interferes with rapid response to treatment in rheumatoid arthritis.

The aim of this study was to determine whether the +896 A-->G substitution of the Toll-like receptor 4 (TLR4) gene, causing the Asp299-->Gly change in the extracellular domain of TLR4, influences treatment response in recent-onset rheumatoid arthritis. 169 patients with rheumatoid arthritis were genotyped from the Finnish Rheumatoid Arthritis Combination Therapy trial, in which they were treated either with only one disease-modifying antirheumatic drug (DMARD) with or without prednisolone (single group), or with three DMARDs and prednisolone (combination group). Patients homozygotic for the wild-type +896A allele were compared with those having the polymorphic G allele in terms of early clinical response (at 6 months) by the 28-joint Disease Activity Score (DAS28). 1 of 20 (5%; (95% (confidence interval (CI) 1 to 5)) patients of the single group with TLR4 +896AG or GG and 29 of 67 (43%; (95% CI 31 to 56)) patients with AA were in remission (p = 0.001). DAS28 of the single group with TLR4 +896AG or GG was higher than with AA (p = 0.019). In the combination group, remission rates and DAS28 values were comparable between the genotypes. The polymorphic TLR4 +896G allele may impair treatment response to single DMARD treatment in recent-onset rheumatoid arthritis.

Antirheumatic Agents↗

PubMed2005

Aberrant TNF secretion by whole blood in healthy subjects with a history of reactive arthritis: time course in adherent and non-adherent cultures.

BACKGROUND: The pathogenesis of reactive arthritis (ReA) apparently involves aberrations in innate immune functions such as monocyte tumour necrosis factor (TNF) generation. OBJECTIVE: To investigate TNF production in healthy subjects with previous yersinia triggered reactive arthritis. METHODS: The study comprised HLA-B27 positive subjects with previous reactive arthritis (B27+ReA+), and B27+ReA- and B27-ReA- subjects (n = 15 each). Whole blood TNF production was induced by lipopolysaccharide (LPS), which binds to CD14/TLR4 on the monocyte surface, or by a combination of phorbol 12-myristate 13-acetate (PMA) and Ca(2+) ionophore A23187, which activates monocytes independently of cell surface receptors. To further evaluate the possible role of adhesion mediated signalling on TNF production, blood samples were incubated in adherent or non-adherent conditions. TNF levels in culture supernatants were measured using an automated immunoassay analyser. The CD14(-159)C/T genotype was determined by a cycle minisequencing method. RESULTS: B27+ReA+ supernatants had higher TNF levels than B27+ReA- supernatants in PMA/A23187 wells in two hour (p = 0.004) and four hour cultures (p = 0.001). Rapid initial TNF release took place in adherent but not in non-adherent conditions. This adhesion associated difference was greater in the B27+ReA+ group than in the B27+ReA- or B27-ReA- group in response to PMA/A23187 (p values <0.001), and greater in the B27+ReA+ group than in the B27-ReA- group in response to LPS (p = 0.021). CD14(-159)T was associated allele dose dependently with an increase in the LPS induced TNF secretion allele (p = 0.030). CONCLUSIONS SUBJECTS: who have recovered from yersinia arthritis show enhanced TNF production, which may be regulated at the level of monocyte adhesion.

Adult↗

PubMed2005

Low circulating soluble interleukin 2 receptor level predicts rapid response in patients with refractory rheumatoid arthritis treated with infliximab.

BACKGROUND: Treatment with infliximab induces a rapid therapeutic response in most patients with active rheumatoid arthritis. Factors predicting good response are not well known. OBJECTIVE: To study the predictive value of baseline level of soluble interleukin 2 receptor (sIL2R), a marker of lymphocyte activation, on the treatment response. METHODS: 24 patients with active rheumatoid arthritis received intravenous infusions of infliximab at study entry, at two weeks, at six weeks, and at eight week intervals thereafter. Outcome was evaluated at six weeks and 22 weeks. Clinical assessment and standard laboratory tests were made and the DAS28 disease activity score was calculated. Serum sIL2R level at entry was measured by automated immunoassay analyser (Immulite). The mean change in DAS28 score from entry to six weeks and 22 weeks was calculated and related to sIL2R level using baseline adjusted robust regression analysis. RESULTS: Baseline level of serum sIL2R (mean (SD), 621 (325) U/ml) did not correlate with baseline DAS28 score (r = 0.24 (95% confidence interval, -0.18 to 0.58)). At six weeks DAS28 scores improved, with a mean change of -2.53 (-3.08 to -1.98) (p<0.001). This change was predicted by low baseline sIL2R level (regression coefficient per 100 U/ml: 0.205 (0.003 to 0.407) (p = 0.047)). At 22 weeks the DAS28 scores improved, with a mean change of -2.26 (-2.75 to -1.77) (p<0.001). The change was not predicted by baseline sIL2R level. CONCLUSIONS: Low baseline sIL2R level may predict a rapid clinical response in patients with refractory rheumatoid arthritis treated with infliximab.

Adult↗

PubMed2005

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

BACKGROUND: 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. METHODS: Some 351 consecutive patients with acute pancreatitis were studied. Blood samples were taken within 12 h of admission. This case-control study included all 33 patients with organ failure and 99 matched controls without organ failure. Measurements included 19 prognostic markers and Acute Physiology And Chronic Health Evaluation (APACHE) II score. RESULTS: Plasma interleukin 10, serum glucose and serum calcium were identified as independent predictors of organ failure by logistic regression analysis. Calcium level correlated with clinical onset of organ failure. The combination of interleukin 10 (more than 50 pg/ml) or calcium (less than 1.65 mmol/l) was a significantly better predictor than any single marker or APACHE II score, with a sensitivity of 88 per cent, specificity 93 per cent and diagnostic odds ratio 94. CONCLUSION: Organ failure in acute pancreatitis can be predicted with high accuracy at hospital admission using a combination of plasma interleukin 10 and serum calcium measurements.

APACHE↗

PubMed2005

Granulocyte colony-stimulating factor therapy and systemic inflammation in critically ill patients.

OBJECTIVE AND DESIGN: The effect of the granulocyte colony-stimulating factor filgrastim on systemic inflammation was investigated in a prospective, randomized, placebo-controlled, double-blind study in critically ill patients. SUBJECTS: 59 critically ill patients were recruited within 48 h of intubation due to ventilatory insufficiency. TREATMENT: Subcutaneous dosage of placebo or 300 microg filgrastim once daily. METHODS: Serum samples were collected at study entry, and 1 and 3 days after the start (Day1 and Day3, respectively). Levels of soluble E-selectin (sE-selectin) and interleukin (IL)-10 were determined by ELISA, and those of IL-6, and soluble IL-2 receptor (sIL-2R) by Immulite chemiluminescence immunoassay. RESULTS: The median sE-selectin level decreased by day 3 significantly in the control group but not in the filgrastim group. The difference in the change between the study groups was significant (p = 0.049). IL-10 levels decreased significantly in the filgrastim group, tended to decrease in controls (p = 0.052), and the difference in the change tended to be significant (p = 0.058). IL-6 levels decreased in both groups comparably. sIL-2R levels were elevated and stable. CONCLUSIONS: Filgrastim prolongs endothelial activation and possibly inhibits development of immune suppression mediated by IL-10.

Adult↗

PubMed2005

Peripheral blood phagocyte CD14 and CD11b expression on admission to hospital in relation to mortality among patients with community-acquired infection.

OBJECTIVE AND DESIGN: Prognostic value of markers of systemic inflammation were evaluated in patients admitted to hospital. MATERIAL: The study comprises 327 patients with community- acquired infection verified on admission (n=290) or within 3-day follow-up (n=37). METHODS: On-admission levels of phagocyte CD11b/CD18 and CD14 expression were measured using whole blood flow cytometry. Clinical data were collected retrospectively from medical records. RESULTS: In univariate analysis, non-survivors as compared to survivors had higher age, lower arterial pressure, higher heart rate, and lower monocyte CD14 density. In multivariate analysis high age [relative mortality RR 1.05 (95% CI 1.01 to 1.08), p=0.016] and low CD 14 expression on monocytes [RR 7.49 (CI 1.63 to 34.33), p=0.01] remained predictive for the 28-day mortality. CONCLUSION: In patients with community-acquired infection, low on-admission level of monocyte CD14 is related to fatal outcome.

Adolescent↗

PubMed2005

The effect of albumin dialysis on cytokine levels in acute liver failure and need for liver transplantation.

In acute liver failure (ALF), detoxification capacity of liver cells is reduced and a variety of cytokines, immune modulators, and toxic substances are accumulating. Multiple organ failure in ALF has been associated with increased blood cytokine levels. We have used a blood purification system, molecular adsorbent recirculating system (MARS), which is based on removal of both protein bound and water-soluble substances and toxins in liver failure. In this study, we measured the effect of MARS therapy on plasma cytokine levels in 49 patients with ALF. Interleukin 6 (IL-6), IL-8, IL-10 and tumor necrosis factor (TNF)alfa were determined immediately before and after the first MARS therapy and after the last session using enzyme-linked immunosorbent assays. The overall survival of these ALF patients was 82% at 6 months; the native liver recovered in 26 cases, and 14 were successfully transplanted. All three interleukins were increased before the MARS treatment but only anti-inflammatory IL-10 was reduced significantly during therapy, which in this setting could be interpreted as a positive effect. We were not able to show constant decreases in proinflammatory cytokines, but only transient effects on IL-8 and IL-6. Surprisingly TNFalfa level was normal and did not change during therapy. In theory, MARS albumin dialysis may remove toxic substances from the blood circulation and thereby improve the possibilities of the liver to recover; however, of the measured cytokines only IL-10 decreased significantly.

Adolescent↗

PubMed2004

Laboratory markers of systemic inflammation as predictors of bloodstream infection in acutely ill patients admitted to hospital in medical emergency.

The aim of the present study was to determine whether the presence of an infectious focus or of fever alone can predict bloodstream infection and whether levels of C-reactive protein, procalcitonin, interleukin (IL)-6, IL-8, and soluble IL-2 receptor (sIL-2R) improve the diagnosis of community-acquired bloodstream infection. Markers of systemic inflammation were studied in 92 patients with community-acquired infection. On admission to hospital, 54 patients had an infectious focus, 25 had fever without an infectious focus, and 13 had neither. The presence of focus or fever predicted bloodstream infection (n=13 patients) with a sensitivity of 100% (95% confidence interval, 75-100), a specificity of 16% (95%CI, 9-26), a negative predictive value of 100% (95%CI, 75-100), and a positive predictive value of 16% (95%CI, 9-26). Positive predictive values of C-reactive protein, procalcitonin, IL-6, IL-8, and sIL-2R, all measured on admission, were also low (33-44%). Eight febrile patients in whom an infectious focus was found during a 3-day follow-up period had higher on-admission IL-6 (P=0.005) and sIL-2R (P=0.046) levels than did 17 febrile patients without an infectious focus. In conclusion, markers of systemic inflammation do not improve the diagnosis of community-acquired bloodstream infection; however, they may aid in identifying patients with fever due to occult infection.

Acute Disease↗

PubMed2004

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.

Acute Disease↗

PubMed2004

CARD15 frameshift mutation in patients with CROHN disease is associated with immune dysregulation.

BACKGROUND: Mutations in the caspase-activating recruitment domain 15 (CARD15) gene are associated with Crohn disease (CD). CARD15 is an intracellular receptor for bacterial lipopolysaccharides (LPS). LPS-induced activation of transfectants containing the frameshift mutation (1007fs) of CARD15 is impaired. The aim of this study was to investigate whether the presence of CARD15 1007fs affects activation of CD patients' own cells. Patients (4 homozygotes, 6 heterozygotes, and 6 wild-type) were matched according to clinical picture and medication. METHODS: Immune inflammatory status was evaluated by measuring monocyte HLA-DR and CD11b densities and the proportion of CD14dimCD16+ monocytes, and was found to be comparable in the three groups. Blood mononuclear cells were cultured overnight in serum-free medium alone, or the medium supplemented with LPS (0.1-10.0 ng/mL), a combination of IFN-gamma (100 IU/mL) and granulocyte-macrophage colony stimulating factor (GM-CSF) (5 ng/mL), or both. TNF and IL-10 levels in the culture supernatant were determined. RESULTS: LPS 0.1 or 1.0 ng/mL alone did not increase TNF levels. IFN-gamma/GM-CSF induced TNF release, and co-culture with LPS 1.0 or 10.0 ng/mL was strongly synergistic. CARD15 1007fs mutation was linked in a gene-dose-dependent manner to low TNF release induced by IFN-gamma/GM-CSF (P value for linear trend = 0.001). The degree of synergism in co-culture was normal or high, suggesting that 1007fs did not depress responses to LPS. IL-10 levels were not related to CARD15 1007fs. CONCLUSIONS: In CD patients, CARD15 1007fs is associated in a gene-dose-dependent manner to low mononuclear cell TNF release by IFN-gamma/GM-CSF but does not impair TNF release by LPS. This type of immune dysregulation may influence susceptibility to and/or phenotype of CD.

Adult↗

PubMed2004

Cellular and humoral markers of systemic inflammation in acute reactive arthritis and early rheumatoid arthritis.

OBJECTIVE: To compare systemic inflammation in reactive arthritis (ReA), rheumatoid arthritis (RA), and sepsis using novel markers of systemic inflammation, and to study whether they are helpful in distinguishing between ReA and RA. METHODS: In 28 patients with acute ReA, 16 patients with early untreated RA, and 25 patients with blood culture-positive sepsis, phagocyte CD 11b expression was measured by flow cytometry, serum procalcitonin (PCT) levels by immunoluminometric assay, and soluble E-selectin (sE-selectin) levels by enzyme-linked immunosorbent assay (ELISA). RESULTS: Neutrophil and monocyte CD11b expression and serum levels of PCT and sE-selectin were higher in patients with sepsis than patients with ReA or RA, or in healthy subjects (all p < 0.01). They were comparable in healthy subjects, ReA, and RA. CONCLUSION: Patients with acute ReA and early RA have normal CD11b expression levels on phagocytes and normal PCT and sE-selectin levels in serum. Elevated levels suggest possible sepsis.

Acute Disease↗

PubMed2002

Raltitrexed treatment promotes systemic inflammatory reaction in patients with colorectal carcinoma.

We studied longitudinally inflammatory reactions and serum C-reactive protein (S-CRP) levels in 52 colorectal cancer patients treated with a median of six 3-weekly cycles of raltitrexed 1.5-3.0 mg m(-2) combined with oral carmofur (1-hexylcarbomoyl-5-fluorouracil) 300-400 mg m(-2) on cycle days 2-14. Thirty-nine (75%) of these patients had fever on days 2 to 9 after receiving raltitrexed, 49 (94%) had fatigue Gr. > or = 1, and 49 (94%) elevated S-CRP without a documented infection. The systemic inflammatory composite score (consists of body temperature, fatigue, S-CRP, interleukin-6 (S-IL-6), S-IL-8, and tumour necrosis factor-alpha (S-TNF alpha) levels) was calculated in a cross-sectional one-cycle study involving 60 colorectal cancer patients treated with single-agent raltitrexed, raltitrexed and carmofur, or 5-fluorouracil-based chemotherapy (n=20 in each group). The median S-CRP, S-IL-6, and S-TNF alpha levels were higher 7 days after giving raltitrexed (57 vs 23 mg l(-1), 64 vs 10 ng l(-1), and 11 vs 10 ng l(-1), respectively) or raltitrexed+carmofur (142 vs 10 mg l(-1), 64 vs 10 ng l(-1), and 16 vs 9 ng l(-1), respectively) than at baseline (P<0.01 for each comparison), but not when 5-fluorouracil-based regimens were administered. These findings suggest that colorectal cancer patients treated with raltitrexed may develop drug-related systemic inflammation, which may be difficult to discriminate from infection.

Adenocarcinoma↗

PubMed2002

Early dexamethasone decreases expression of activation markers on neutrophils and monocytes in preterm infants.

AIM: To investigate the effects of early dexamethasone administration on activation of circulating neutrophils and monocytes in preterm infants with respiratory distress syndrome requiring treatment with surfactant. METHODS: Neonates (n = 30) with respiratory distress were randomized to receive dexamethasone (DEX group, 29.1 +/- 1.2 wk, 1223 +/- 156 g, n = 15) from the first postnatal day, or to serve as controls (control group, 29.2 +/- 1.4 wk, 1250 +/- 148 g, n = 15). Dexamethasone was given as a 4 d course (0.5 mg kg(-1) on postnatal days 1 and 2, and 0.25 mg kg(-1) on days 3 and 4). Polymorphonuclear leucocyte (PMN) and monocyte surface expression of CD11b, L-selectin and CD14 was quantified with flow cytometry, and plasma macrophage-inflammatory protein-1alpha (MIP-1alpha) with an enzyme-linked immunosorbent assay. Blood samples were collected on days 1, 2-3 and 5-7. RESULTS: In the DEX group 1/15, and in the control group 7/15 developed bronchopulmonary dysplasia (p < 0.04). PMN CD11b (median 100, range 70-190 vs 154, 96-213, p=0.01), monocyte CD14 (235, 102-433 vs 355, 219-533, p=0.01) and plasma MIP-1alpha (20 ng l(-1), 20-32 vs 37 ng l(-1), 20-70, p = 0.005) were lower in the DEX group at days 2-3. All adhesion molecule expression and plasma MIP-1alpha levels were comparable at days 5-7, with the exception of monocyte L-selectin expression levels, which remained lower in the DEX group. CONCLUSION: In preterm infants with respiratory distress syndrome, early dexamethasone causes downregulation of PMN and monocyte activation. This may attenuate pulmonary inflammation and improve pulmonary outcome.

Anti-Inflammatory Agents↗

PubMed2002

Infectious background of patients with a history of acute anterior uveitis.

OBJECTIVE: To study the infectious background of patients with a history of acute anterior uveitis (AAU) and healthy control subjects. METHODS: Sixty four patients with previous AAU and 64 sex and age matched controls were studied. Serum antibodies to Salmonellae, Yersiniae, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Campylobacter jejuni, and Borrelia burgdorferi were measured using enzyme linked immunosorbent assay (ELISA), and antibodies to Chlamydia trachomatis and Chlamydia pneumoniae by microimmunofluorescence test. Peripheral blood mononuclear cells (PBMCs), separated by density gradient centrifugation, were studied for Salmonella and Yersinia antigens by means of an immunofluorescence test, and for C pneumoniae DNA with a polymerase chain reaction (PCR). RESULTS: Neither prevalence nor levels of single microbial antibodies studied differed between the patients and control subjects, or between subgroups of patients created on the basis of clinical characteristics. In logistic regression analysis, the high number of recurrences (>10) of AAU was independently related to the presence of single or multiple bacterial antibodies (p=0.04). None of the PBMC samples of the patients were positive for Yersinia or Salmonella antigens. C pneumoniae PCR was positive in a patient who was negative for C pneumoniae antibodies. CONCLUSION: Although neither the prevalence nor the levels of single microbial antibodies studied differed between the patients and the controls, current data suggest that the presence of single or multiple antibodies in patients with many recurrences of AAU compared with patients with none or few recurrences may be a sign of repeated infections, antigen persistence, or raised innate immune responsiveness.

Acute Disease↗

PubMed2002

Circulating soluble E-selectin in early rheumatoid arthritis: a prospective five year study.

BACKGROUND: Soluble E-selectin (sE-selectin) is a marker of activation of vascular endothelium. OBJECTIVE: To examine serum levels of sE-selectin in a cohort of 85 patients with early rheumatoid arthritis (RA) followed up for five years. METHODS: sE-selectin levels were assessed annually using an enzyme linked immunosorbent assay (ELISA) and related to simultaneously obtained clinical and laboratory measures. Joint inflammation was evaluated by active joint count, functional status by Health Assessment Questionnaire (HAQ), and radiographic findings in hands and feet by the Larsen method. Laboratory tests included serum C reactive protein (CRP) level, erythrocyte sedimentation rate, blood haemoglobin level, white blood cell count (WBC), and platelet count. Area under the curve (AUC) was calculated for each variable, and Jonckheere's test for ordered alternatives was applied to assess significance of association between sE-selectin AUC tertiles and other variables. Baseline sE-selectin tertiles were related to change in Larsen score and HAQ score at five years. Odds ratios (OR) with 95% confidence interval (CI) were calculated using univariate and multivariate logistic regression. RESULTS: sE-selectin levels were associated with CRP level (p=0.012), WBC (p=0.037), active joint count (p=0.019), progression of joint destruction (p=0.038), and HAQ score at five years (p=0.021), but not with extra-articular symptoms or comorbidity. Baseline sE-selectin levels in the third tertile predicted the HAQ score at five years (OR 4.18, 95% CI 1.15 to 15.22). sE-selectin levels of patients did not differ significantly from those of healthy control subjects. CONCLUSION: The degree of activation of vascular endothelium is associated with activity and outcome of early RA.

Adolescent↗

PubMed2002

Systemic inflammation and innate immune response in patients with previous anterior uveitis.

AIM: To determine the presence of systemic inflammation and innate immune responsiveness of patients with a history of acute anterior uveitis but no signs of ocular inflammation at the time of recruitment. METHODS: Tumour necrosis factor alpha (TNF-alpha) production in response to bacterial lipopolysaccharide (LPS) was studied using whole blood culture assay; levels of TNF-alpha in culture supernatants, and soluble interleukin 2 receptor (sIL-2R) in serum were determined by chemiluminescent immunoassay (Immulite); monocyte surface expression of CD11b, CD14, and CD16 and the proportion of monocyte subsets CD14(bright)CD16(-) and CD14(dim)CD16(+) were studied with three colour whole blood flow cytometry; and serum C reactive protein (CRP) levels were determined using immunonephelometric high sensitivity CRP assay. RESULTS: The CRP level (median, interquartile range) was significantly higher in 56 patients with previous uveitis than in 37 controls (1.59 (0.63 to 3.47) microg/ml v 0.81 (0.32 to 2.09) microg/ml; p=0.008). The TNF-alpha concentration of the culture media per 10(5) monocytes was significantly higher in the patient group than in the control group in the presence of LPS 10 ng/ml (1473 (1193 to 2024) pg/ml v 1320 (935 to 1555) pg/ml; p=0.012) and LPS 1000 ng/ml (3280 (2709 to 4418) pg/ml v 2910 (2313 to 3358) pg/ml; p=0.011). The background TNF-alpha release into the culture media was low in both groups. CD14 expression of CD14(bright)CD16(-) monocytes, defined as antibody binding capacity (ABC), was similar for the patients and controls (22,839 (21,038 to 26,020) ABC v 21,657 (19,854 to 25,646) ABC). CONCLUSIONS: Patients with previous acute anterior uveitis show high innate immune responsiveness that may play a part in the development of ocular inflammation.

Adult↗

PubMed2001

Evaluation of red blood cell lysing solutions in the study of neutrophil oxidative burst by the DCFH assay.

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 (NH(4)Cl) 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 NH(4)Cl solutions retain cellular viability and are lysing methods of choice in evaluation of neutrophil respiratory burst by DCFH oxidation assay.

Cell Membrane↗

PubMed2001

Activated protein C and inflammation in human myocardium after heart surgery.

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.

Adult↗