A novel high-purity isolation method for human peripheral blood neutrophils permitting polymerase chain reaction-based mRNA studies.

In the past few years, the role of polymorphonuclear neutrophils (PMN) in specific immune responses has gained significance due to their ability to express a variety of immunoregulatory molecules. However, controversial results concerning the potential of neutrophils for cytokine production have been obtained by sensitive molecular biological techniques. This problem might be related to contaminating leukocytes in conventionally isolated neutrophil suspensions as outlined by our study. We have established a novel method yielding highly purified neutrophils by combining a discontinuous Percoll gradient with fluorescence activated cell sorting of CD16bright cells. The latter step exploits the exceptionally high expression of Fc gammaRIIIB on PMN. Neutrophils could be enriched to homogeneity (> 99.9%) with a viability exceeding 90%. Contamination with NK cells or other lymphocytes, monocytes and eosinophils could be excluded as evaluated by reverse transcription-polymerase chain reaction (RT-PCR) with primers for HLA-DR, c-fms and CD52. The transcriptional potential of such purified neutrophils was confirmed by their ability to express MHC class II molecules after stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF). Our method should permit studies of PMN at the mRNA level and future investigations concerning the specificity of immunoregulatory molecule synthesis by neutrophils.

An open-label trial of the selective cyclo-oxygenase-2 inhibitor, rofecoxib, in inflammatory bowel disease-associated peripheral arthritis and arthralgia.

BACKGROUND: Conventional non-steroidal anti-inflammatory drugs have been associated with an increased risk of exacerbation of inflammatory bowel disease. AIM: To evaluate, in a prospective, open-label study, the safety and efficacy of a 20-day regimen of the selective cyclo-oxygenase-2 inhibitor, rofecoxib, 12.5-25 mg/day, in inflammatory bowel disease patients with associated peripheral arthropathy and/or arthritis. METHODS: Patients with clinically inactive to mild inflammatory bowel disease and a joint pain score of at least two points on a scale ranging from zero (none) to four (very poor) were eligible. Response was defined by a decrease of at least two points in the arthralgia score. RESULTS: Of the 32 patients included, 26 (81%) were treated with rofecoxib, 25 mg/day, and six (19%) with rofecoxib, 12.5 mg/day. In three patients (9%), rofecoxib had to be withdrawn after a few days due to gastrointestinal complaints which ceased immediately after drug discontinuation. No flare of inflammatory bowel disease occurred. Thirteen of the 32 patients (41%) were responders and, overall, the arthralgia score decreased from two to one (P = 0.0001). CONCLUSIONS: This is the first prospective study on the use of a selective cyclo-oxygenase-2 inhibitor in inflammatory bowel disease patients with peripheral arthropathy and/or arthralgia. The promising safety and efficacy profile warrants further evaluation in controlled trials.

Recombinant tissue plasminogen activator during cardiopulmonary resuscitation in 108 patients with out-of-hospital cardiac arrest.

BACKGROUND: Thrombolytic therapy during cardiopulmonary resuscitation (CPR) is a controversial issue in emergency medicine practice. This study was conducted to determine whether administration of recombinant tissue plasminogen activator (rt-PA) in out-of-hospital cardiac arrest of non-traumatic aetiology improves CPR outcome. METHODS AND RESULTS: A retrospective chart review of 401 patients with out-of-hospital cardiac arrest who were resuscitated by the emergency medical services (EMS) during a 6 year period was performed. A total of 108 patients received rt-PA during CPR and were compared to 216 controls, closely matched according to baseline characteristics, arrival status and ECG findings. Administration of rt-PA was optional. Return of spontaneous circulation (ROSC) occurred in 76 patients under rt-PA treatment (70.4 vs. 51.0% in controls; P=0.001). Fifty-two patients from the lysis group survived the first 24 h (48.1 vs. 32.9% in controls; P=0.003), while 27 (25.9%) survived to discharge. Autopsy reports revealed major bleeding complications in six patients receiving rt-PA treatment. Fulminant intracranial haemorrhage was observed in one patient who received rt-PA and in two cases from the control group. CONCLUSIONS: Thrombolytic therapy may improve frequency of return of spontaneous circulation substantially and increase primary survival in patients with non-traumatic cardiac arrest. Serious bleeding complications are not frequently observed under rt-PA treatment.

In vivo effects of recombinant human interleukin-10 on lymphocyte phenotypes and leukocyte activation markers in inflammatory bowel disease.

BACKGROUND: Interleukin-10 (IL-10) exhibits potent anti-inflammatory and immunosuppressive activities in vitro. Recent data indicate that treatment with recombinant human IL-10 (rHuIL-10) Crohn's disease is safe and may induce clinical and endoscopic remission. The present study investigates the in vivo immunomodulatory properties of rHuIL-10 in inflammatory bowel disease (IBD). METHODS: As part of two randomized, double-blinded, placebo-controlled trials, repeated flow cytometric analyses of lymphocyte phenotypes (CD3, CD4, CD8, CD16 + 56, CD19) and activity markers (human leukocyte antigen [HLA]-DR; intercellular adhesion molecule-1 [ICAM-1]; IL-2-receptor alpha [IL-2R alpha]; high affinity receptor for immunoglobulin G [IgG; Fc gamma RI]) on T cells, monocytes, and neutrophils were performed in 17 patients with IBD who received rHuIL-10 (5, 10, or 20 micrograms/kg) or placebo, administered subcutaneously, once daily for 28 days. RESULTS: Minor changes were noted in CD3+, CD8+, and CD3+/CD16 + 56+ lymphocyte phenotypes, whereas absolute numbers of CD4+ lymphocytes and CD19+ cells increased. T-cell activation markers HLA-DR and IL-2R alpha were downregulated. rHuIL-10 did not influence HLA-DR expression on monocytes. ICAM-1 modulation on monocytes and neutrophils was mild and inconsistent. Fc gamma RI expression was upregulated on both neutrophils and monocytes. CONCLUSIONS: These data indicate that the immunosuppressive effects of rHuIL-10 treatment are partly different from its in vitro observed actions. The increase of the cytotoxicity-mediating Fc gamma RI points to potential immunostimulating properties of this cytokine.

The impact of intestinal resection on serum levels of anti-Saccharomyces cerevisiae antibodies (ASCA) in patients with Crohn's disease.

BACKGROUND: Antibodies to Saccharomyces cerevisiae (ASCA) are highly prevalent in sera of patients with Crohn's disease and have been proposed to identify subgroups of patients with a disabling disease course. AIM: To investigate the impact of intestinal resection on serum levels of ASCA in patients with Crohn's disease and the predictive value of ASCA levels on surgical recurrence. METHODS: Sera from 60 patients who underwent 'curative' intestinal resection due to stricturing and/or penetrating complications were collected preoperatively and during post-operative follow-up (week 2, months 4, 8 and 11 ± 1). Measurement of ASCA IgG and IgA isotypes were performed using ELISA. Re-operation rate was associated with ASCA status and serum levels. RESULTS: At baseline 44/60 (73%) of patients were rated as positive for ASCA IgG, 45/60 (75%) for ASCA IgA and 52/60 (87%) as positive for at least one of both. ASCA serum levels remained stable during first year from resection. After a median of 106 months 10 of 40 (25%) patients with long-term follow-up underwent one or more intestinal re-operations. Neither ASCA positivity nor absolute ASCA serum levels were predictive of surgical recurrence. CONCLUSIONS: Serum ASCA levels remain stable after curative intestinal resection in Crohn's disease. This indicates the persistence of both stimulus and immunological mechanism operative in the production of ASCA even after complete surgical resection of macroscopically inflamed intestinal tissue. After intestinal resection, neither ASCA positivity nor ASCA serum levels predict the risk of surgical recurrence during long-term follow-up.

Anti-Gal titers in healthy adults and inflammatory bowel disease patients.

INTRODUCTION: ALPHA-GAL is a glycoconjugate present on cell membranes of mammals and bacteria but not humans who display anti-Gal antibodies (AB) in high titers provoked by the commensal gut flora. In the present study, we sought to determine the longitudinal course of alpha-Gal specific AB titers of all isotypes over 8 weeks among healthy adult subjects. Furthermore, we hypothesized that inflammatory bowel disease (IBD) patients display increased anti-Gal titers. MATERIALS AND METHODS: We drew serum from healthy probands (n=20) weekly for 8 weeks and obtained plasma samples of from patients suffering from Crohn's disease (n=20) and ulcerative colitis (n=20). We measured anti-Gal ABs of all isotypes and total immunoglobulin (Ig) content using an enzyme-linked immunosorbent assay technique. For statistical evaluation of the longitudinal titers, we calculated confidence intervals for the slopes of a random intercept model, comparing variances between and within the probands. For group comparisons, we performed paired student t-tests and Pearson correlations. RESULTS: Alpha-Gal specific IgG, IgM, IgD, and IgA titers remained unvaried within a narrow range upon longitudinal observation. Most probands did not display alpha-Gal specific IgE ABs. Crohn's disease patients showed highly increased alpha-Gal-specific IgA titers compared with control subjects (P<.01). CONCLUSION: Apart from IgE, alpha-Gal-specific ABs of all isotypes remained constant over longer time periods in healthy subjects. Thus, significant titer changes actually represent increased antigen exposure and a specific anti-alpha-Gal response. Crohn's disease patients display increased anti-Gal IgA titers compared with healthy controls, which reflects a chronically impaired mucosal gut barrier in this patient cohort.

Antibiotic susceptibility and serotype distribution of Streptococcus pneumoniae. Stable incidence of antibiotic-resistant isolates and common serotypes.

The serotype distribution and antibiotic susceptibility of 200 pneumococci isolated from clinical specimens in 1981 were determined. These results were compared with the susceptibility of organisms determined in 1974 and the serotype distribution of organisms determined in 1974 and 1977. Penicillin G, tetracycline, erythromycin, and chloramphenicol were tested in both 1974 and 1981. No isolates resistant to penicillin, erythromycin, or chloramphenicol were found, and the percent of isolates resistant to tetracycline and of moderate susceptibility to penicillin were similar in the 2 yr. An increasing incidence of pneumococci with decreased susceptibility to these antibiotics was not observed. Vancomycin was tested in 1981 only and no resistant isolates were identified. The distribution of serotypes during each time period was also similar. Our results establish a stable, low-level incidence of antibiotic-resistant pneumococci at our hospital, as well as a stable distribution of serotypes.

Antipneumococcal activities of RP 59500 (quinupristin-dalfopristin), penicillin G, erythromycin, and sparfloxacin determined by MIC and rapid time-kill methodologies.

Previous time-kill studies have shown that RP 59500 is rapidly bactericidal against pneumococci. To extend these findings, the activities of RP 59500, its two components RP 57669 RP 54476, penicillin G, erythromycin and sparfloxacin against 26 penicillin-susceptible, 25 penicillin-intermediate, and 25 penicillin-intermediate, and 25 penicillin-resistant pneumococci were determined by the agar dilution MIC and the time-kill testing methodologies within 10 min (ca. 0.2 h) and at 1 and 2 h. Respective agar dilution MICs at which 90% of isolates are inhibited for penicillin-susceptible, -intermediate, and -resistant strains were as follows: penicillin G, 0.03, 1, and 4 micrograms/ml;RP 59500, 1, 1, and 1 microgram/ml; RP 57669, 8, 32, and 16 micrograms/ml; RP 54476, > 128, > 128, and > 128 micrograms/ml; erythromycin, 0.06, 2, and > 128 micrograms/ml; and sparfloxacin, 1, 0.5, and 0.5 microgram/ml. RP 59500 was equally active (MIC at which 90% of isolates are inhibited, 1.0 microgram/ml) against erythromycin-susceptible and -resistant strains. Time-kill testing results showed that only RP 59500 at one to four times the MIC killed pneumococci at 0.2 h; RP 59500 was also the most active compound at 1 and 2 h. By comparison, penicillin and sparfloxacin at one, two, and four times the MICs reduced the original inoculum by > or = 1 log at 2 h for 46, 80, and 95% and for 50, 72, and 86% of strains, respectively. The killing activity of RP 59500 was the same against erythromycin-susceptible and -resistant strains. RP 57669, RP 54479, and erythromycin were either inactive or bacteriostatic at 2 h. Of all drugs tested, RP 59500 yielded the most rapid killing.

Donor dependent, interferon-gamma induced HLA-DR expression on human neutrophils in vivo.

Neutrophils are effector cells of innate immune responses. Stimulated by interferon-gamma (IFN-gamma) to express HLA-DR, neutrophils acquire accessory cell functions for superantigen-mediated T cell activation. In vitro HLA-DR induction on neutrophils varies in a functionally relevant way as levels of MHC class II expression and magnitude of neutrophil induced T cell responses are correlated functions. The aim of this study was to assess whether IFN-gamma induces HLA-DR on human neutrophils in a donor dependent fashion in vivo and to define regulatory events operative in MHC class II expression of neutrophils. In vivo administration of rhIFN-gamma in 55 patients with renal cell carcinoma resulted in a varying increase of HLA-DR on neutrophils. By setting a cut-off for response at>10% HLA-DR positive neutrophils, HLA-DR responders (51%) were as frequent as nonresponders (49%). In vivo kinetic studies revealed a peak expression of HLA-DR on neutrophils 48 h after rhIFN-gamma application, while nonresponders remained HLA-DR negative over a 72-h period. In vitro IFN-gamma stimulated neutrophils recapitulated the response profiles observed in vivo. No differences in IFN-gamma dependent CD64 and invariant chain expression, and IFN-gamma serum levels were observed among the response subgroups. HLA-DR mRNA was detected in neutrophils from rhIFN-gamma treated responders and nonresponders, HLA-DR protein solely in lysates of responder neutrophils. IFN-gamma stimulated HLA-DR expression on neutrophils is subject to donor dependent variations in vivo, which result from rather post-transcriptional than transcriptional regulation. Due to their abundance in inflammatory reactions heterogeneous HLA-DR expression by neutrophils could determine the outcome of superantigen-driven diseases.

Clinical relevance of serum interleukin-6 in Crohn's disease: single point measurements, therapy monitoring, and prediction of clinical relapse.

OBJECTIVE: To investigate the clinical relevance of interleukin-6 (IL-6) serum levels in patients with Crohn's disease (CD), single point IL-6 measurements in sera from consecutive CD patients and healthy donors (HD), as well as longitudinal measurements during the course of steroid therapy for active CD were performed. Patients with steroid-induced remission were followed until clinical relapse. METHODS: One hundred thirty-six CD patients without steroid or other immunosuppressive treatment within 2 months and surgical procedures within 3 months before study entry were investigated; 63 patients with active CD were enrolled into the follow-up program. Clinical activity was evaluated by the Crohn's disease activity index (CDAI) and serum IL-6 levels measured by enzyme-linked immunosorbent assay. RESULTS: IL-6 serum levels were significantly elevated in CD patients compared to HD (p < 0.001). In individual patients serum IL-6 levels correlated with corresponding CDAI scores in a subgroup referred to as primarily inflammatory patients presenting without bowel stenosis, previous intestinal resection, or concomitant inflammatory disorders (r = 0.72, p < 0.001). Primarily inflammatory patients displayed higher serum IL-6 levels (median: 6.0 pg/ml; range: 1.3-25) than CD patients with bowel stenosis (median: 2.0; range: 1.3-4.9; p < 0.01) or extensive intestinal resection (median: 1.5; range: 1.3-13.7; p < 0.001). Longitudinally measured serum IL-6 levels reflected the clinical response during steroid therapy and predicted clinical relapse after steroid-induced remission at week 9 of the treatment protocol. CONCLUSIONS: Serum IL-6 is a clinically relevant parameter for CD that correlates with inflammatory activity and implies a prognostic value after steroid-induced remission.

Tissue-dependent factors affect gene delivery to tumors in vivo.

Systemic application of surface-shielded transferrin-polyethylenimine/DNA complexes leads to predominant DNA uptake and gene expression in Neuro2a tumors in syngeneic A/J mice. Similarly, high expression levels were found in Huh-7 and HepG2 human tumor xenografts in SCID mice after systemic application of surface-shielded EGF-PEG-PEI/DNA complexes. Significant DNA uptake but low gene expression were found in the M-3 melanoma while no DNA uptake and no gene expression were found in KB, 518A2, A549, and SW480 xenograft tumor models. To elucidate the reasons for these differences, the tumors were analyzed for vascularization and infiltration of macrophages. Neuro2a, Huh-7, and HepG2 tumors are well vascularized, with a high density of partially immature blood vessels and low numbers of infiltrating macrophages. The M-3 melanoma is well vascularized correlating with significant DNA uptake, however, necrosis and intensive infiltration by macrophages lead to rapid degradation of DNA. In contrast, the KB, 518A2, A549, and SW480 tumors are poorly vascularized, correlating with undetectable DNA uptake and gene expression. Using two different vector systems the data indicate that gene delivery to tumors in vivo is affected by tissue-dependent factors. Uptake of DNA into the tumor depends on vascularization of the tumor, while necrosis and macrophage infiltration may facilitate degradation of the DNA.

Influence of topically and systemically active steroids on circulating leukocytes in Crohn's disease.

OBJECTIVE: Budesonide, although only topically active, is effective in the treatment of Crohn's disease. This study was performed to compare the clinical efficacies of budesonide and prednisolone in relation to the activation status of circulating leukocytes. METHODS: Twenty-four patients with active Crohn's disease were randomized to treatment with either budesonide or 6-methylprednisolone. Clinical response was monitored by the Crohn's disease activity index, C-reactive protein, and orosomucoid. Expression of CD25 and CD71 on T cells and CD64 on neutrophils was determined by flow cytometry. The release of TNF-alpha and IL-1beta by peripheral blood mononuclear cells was measured by ELISA. RESULTS: After 2 wk of treatment a clinical response was observed in both groups, but it was more accentuated in patients treated with prednisolone. At baseline an upregulation of CD71 and CD64, but not CD25, was found in active patients. Prednisolone significantly decreased the expression of CD64 and the release of TNF-alpha and IL-1beta, but did not alter the expression of CD25 and CD71. Budesonide treatment failed to exert any effect on circulating leukocytes. CONCLUSIONS: The inability of budesonide to downregulate activated circulating leukocytes may contribute to the somewhat lower clinical efficacy of this topical steroid in the treatment of active Crohn's disease.