Inhibition of neutrophil-dependent cytotoxicity for human endothelial cells by ACE inhibitors.

Angiotensin-converting enzyme inhibitors (ACEi) have immunomodulating properties and have been suggested to protect against endothelial injury, for example myocardial infarction and reperfusion injury. We tested whether two ACEi (captopril and enalapril), differing in a thiol group, protected human umbilical vein endothelial cells (HUVEC) from cytotoxicity induced by polymorphonuclear neutrophils (PMN) in vitro, when cells were activated by tumour necrosis factor-α (TNFα) or the arachidonate derivative lipoxin-A4 (LXA4 ), using separate cytotoxicity pathways. When (51) Cr labelled HUVEC were treated with captopril (0-500 µm) or enalapril (0-100 µm) for 2 h and then activated by TNFα (100 ng/ml) for 24 h, a significant, dose-dependent reduction of (51) Cr release was observed. Similarly, captopril reduced (51) Cr release when LXA4 (0.1 µm) was used to stimulate PMN for 4 h. Among previously defined mechanisms of significance for the cytotoxic reaction, expression of ICAM-1, but not intracellular Ca(2+) changes in PMN or PMN adherence to HUVEC, were reduced by ACEi treatment. Moreover, both ACEi inhibited HUVEC surface expression of TNFα receptor I (but not II). Thus, these ACEi, particularly captopril, interfere with PMN-induced cytotoxicity for endothelial cells by modulating pro-inflammatory surface receptors, which is a novel effect that might be explored for further therapeutic approaches.

Do peer educators make a difference? An evaluation of a youth-led HIV prevention model in Zambian Schools.

Restless Development's youth-led model places trained Volunteer Peer Educators (VPEs), aged 18-25 years, in schools to teach HIV prevention and reproductive health (RH). VPEs also run youth centers, extracurricular and community-based activities. This evaluation assesses (i) program effects on students' HIV/RH knowledge, attitudes and behaviors using a non-randomized quasi-experimental design among 2133 eighth and ninth grade students in 13 intervention versus 13 matched comparison schools and (ii) program costs. Intervention students had significantly higher levels of knowledge related to HIV [odds ratio (OR) 1.61, 95% confidence interval (CI) 1.18-2.19; P < 0.01] and RH (OR 1.71; 95% CI 1.21-2.49; P < 0.01), more positive attitudes toward people living with HIV and greater self-efficacy to refuse unwanted sex and access condoms. No evidence of differences in ever having had sex was found (28% in the intervention; 29% in the comparison schools). However, intervention students were more likely not to have had sex in the previous year (OR 1.26, 95% CI 1.03-1.56; P < 0.05) and to have had only one sex partner ever (OR 1.43, 95% CI 1.00-2.03; P < 0.05). The average annual cost of the program was US$21 per beneficiary. In conclusion, the youth-led model is associated with increased HIV and RH knowledge and self-efficacy and lowered levels of stigma and sexual risk-taking behaviors.

Rituximab in relapsing or refractory ANCA-associated vasculitis: a case series of 16 patients.

OBJECTIVES: Cyclophosphamide (CYC) and corticosteroids are generally considered standard induction therapy for anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). However, a subset of patients are refractory or intolerant to this treatment. Rituximab, a chimeric anti-CD20 antibody, has emerged as a second-line therapy, although controlled studies are scarce in patients with relapsing or refractory disease. METHODS: We report 16 patients with AAV who received rituximab for refractory or relapsing vasculitis having previously received CYC. The treatment protocols were 375 mg/m(2) × IV in five patients, 1000 mg × II in six patients, and 500 mg × II in five patients, all in combination with corticosteroids. A majority of patients used other concurrent immunosuppression, most commonly mycophenolate mofetil. Disease activity was assessed using the Birmingham Vasculitis Activity Score (BVAS 2003) at baseline and during follow-up together with C-reactive protein (CRP) and ANCA. Complete remission was defined as a BVAS score of 0 and partial remission as a reduction in BVAS of at least 50%. RESULTS: Twelve patients achieved complete remission, three patients partial remission, and one patient died during follow-up (median of 20 months, range 3-48 months). Six patients relapsed and received rituximab again. Four of these were positive for capture proteinase 3 (PR-3) ANCA but negative with conventional PR-3 ANCA upon retreatment. Seven patients had an adverse event, including two hepatitis B reactivations and one fatal sepsis. CONCLUSION: Treatment with rituximab in AAV was associated with prolonged remission in a subset of patients otherwise difficult to manage.

Anti-apoA-1 IgG and oxidized LDL are raised in rheumatoid arthritis (RA): potential associations with cardiovascular disease and RA disease activity.

OBJECTIVE: To determine whether emerging cardiovascular risk factors such as anti-apolipoprotein A-1 (anti-apoA-1) immunoglobulin (Ig)G and oxidized low density lipoprotein (oxLDL) are associated with cardiovascular disease (CVD), carotid intima-media thickness (IMT), and disease activity in rheumatoid arthritis (RA). METHOD: We determined the aforementioned associations in 69 RA patients with disease duration of 5 years and 46 controls matched by age, sex, and smoking status. Anti-apoA-1 IgG and oxLDL were measured by enzyme-linked immunosorbent assay (ELISA). Carotid arteries were examined by ultrasound. Disease Activity Score calculated on 28 joints (DAS28) was used to assess disease activity. RESULTS: CVD prevalence was higher among RA patients than controls (17% vs. 2%, p = 0.01) but there was no difference in IMT (median: 0.67 vs. 0.66, p = 0.33). RA patients had a higher anti-apoA-1 IgG prevalence than controls (20% vs. 0%, p = 0.001). Anti-apoA-1 IgG and oxLDL levels were higher in cases than controls [median: 0.33 vs. 0.175 optical density (OD), p = 0.03; and 121 vs. 37.2 U/L, p = 0.0001, respectively]. Anti-apoA-1 IgG-positive patients had higher levels of oxLDL (median: 140.5 vs. 112 U/L, p = 0.01) than those tested negative. Receiver operating characteristic (ROC) curve analysis showed that only anti-apoA-1 IgG was a modest but significant predictor of CVD [area under the curve (AUC) = 0.65, p = 0.03] in RA patients. oxLDL was significantly associated with RA disease activity, whereas anti-apoA-1 IgG was not. CONCLUSIONS: Anti-apoA-1 IgG could be a marker of CVD in RA, whereas oxLDL levels seem to reflect RA disease activity. Other causes of CVD than a general increase in atherosclerosis (as determined by IMT measurements) including plaque stability may therefore be of importance to explain the increased incidence of CVD in RA.

Addition of infliximab compared with addition of sulfasalazine and hydroxychloroquine to methotrexate in patients with early rheumatoid arthritis (Swefot trial): 1-year results of a randomised trial.

BACKGROUND: New treatment strategies for early rheumatoid arthritis are evolving rapidly. We aimed to compare addition of conventional disease-modifying antirheumatic drugs (sulfasalazine and hydroxychloroquine) with addition of a tumour necrosis factor antagonist (infliximab) to methotrexate in patients with early rheumatoid arthritis. METHODS: We undertook a randomised trial in 15 rheumatology units in Sweden. We enrolled patients with early rheumatoid arthritis (symptom duration <1 year) and administered methotrexate (up to 20 mg per week). After 3-4 months, those who had not achieved low disease activity but who could tolerate methotrexate were randomly allocated by computer addition of either sulfasalazine and hydroxychloroquine or infliximab. Primary outcome was achievement of a good response according to European League Against Rheumatism (EULAR) criteria at 12 months. Patients were followed up to 24 months; here, we present findings at 12 months. Analysis was by intention to treat and we used non-responder imputation. The Swefot (Swedish Pharmacotherapy) study is registered in the WHO database at the Karolinska University Hospital, number CT20080004. FINDINGS: 487 patients were initially enrolled. Of 258 who had not achieved low disease activity with methotrexate, 130 were allocated sulfasalazine and hydroxychloroquine and 128 were assigned infliximab. 32 of 130 (25%) patients allocated sulfasalazine and hydroxychloroquine achieved the primary outcome compared with 50 of 128 (39%) assigned infliximab (risk ratio 1.59 [95% CI 1.10-2.30], p=0.0160). Adverse events were balanced fairly well between the two groups and accorded with known adverse events of the drugs used. No deaths occurred in either group. INTERPRETATION: In patients with early rheumatoid arthritis in whom methotrexate treatment failed, addition of a tumour necrosis factor antagonist to methotrexate monotherapy is clinically superior to addition of conventional disease-modifying antirheumatic drugs. FUNDING: Swedish Rheumatism Association, Schering-Plough.

Comparison of soluble ICAM-1, VCAM-1 and E-selectin levels in patients with episodic cluster headache and giant cell arteritis.

The pathophysiology of cluster headache (CH) is supposed to involve the lower posterior part of the hypothalamus, the trigeminal nerve, autonomic nerves and vessels in the orbital/retro-orbital region. The exact connection of this hypothalamic-trigemino-autonomic-vascular axis is not fully understood. The presence of inflammation in the perivascular tissue of the retro-orbital region has been presented as a possible mechanism behind the pain and the sympatheticoplegia sometimes observed during headache attacks. In a previous study we found neither increased levels of erythrocyte sedimentation rate, C-reactive protein or acute-phase reactants nor clinical signs of a generalized inflammatory disorder. However, these tests may not be sensitive enough to detect a focal inflammatory process in the retro-orbital region. In the present study, we analysed serum levels of three soluble adhesion molecules; soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble E-selectin (sE-selectin) in patients with episodic CH and in patients with biopsy-positive giant cell arteritis (GCA), a known vasculitic disorder of large and medium-sized arteries. A control group of healthy volunteers was also included. Within the CH group, sICAM-1, sVCAM-1 and sE-selectin showed an increasing trend in remission compared with the active CH period, but the difference was statistically significant for sE-selectin only. The mean sICAM-1 value was higher in patients with active GCA than in CH patients during the active cluster period. Compared with the healthy control group, the mean levels of soluble adhesion molecules in CH patients also tended to be higher, but statistically significantly so only for sVCAM-1. We hypothesize that CH is not a vasculitic disorder of the medium-sized arteries, but CH patients may have an immune response that reacts differently from that of healthy volunteers.

Interleukin-1beta influences the effect of infliximab on temporomandibular joint pain in rheumatoid arthritis.

OBJECTIVES: The aim of this study was to investigate the influence of plasma and synovial fluid tumour necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), IL-6, soluble TNF receptor II (TNF-sRII), IL-1 receptor antagonist (IL-1ra), soluble IL-1 receptor II (IL-1sRII) and IL-10 on the effect of the TNFalpha antibody infliximab on temporomandibular joint (TMJ) pain in patients with active rheumatoid arthritis (RA). METHODS: Fifteen patients with TMJ pain taking methotrexate were included in the study. The effect of intravenous infusions of infliximab was assessed after 14 or 22 weeks. TMJ resting and movement pain was assessed by a visual analogue scale (VAS) (0-100 mm) and samples of venous blood and TMJ synovial fluid were collected before and after treatment. RESULTS: The effect of infliximab on TMJ pain was influenced by pretreatment plasma levels of IL-1beta, IL-1ra, and IL-10 as well as pretreatment levels of TMJ synovial fluid IL-1sRII. High pretreatment levels of these cytokines and receptors as well as the presence of rheumatoid factor (RF) were associated with no or minor reduction in TMJ pain after treatment. CONCLUSIONS: Systemic treatment of RA with a combination of infliximab and methotrexate seems to be insufficient to alleviate TMJ pain in patients with RF or high pretreatment plasma levels of IL-1beta.

Adalimumab (Humira) restores clinical response in patients with secondary loss of efficacy from infliximab (Remicade) or etanercept (Enbrel): results from the STURE registry at Karolinska University Hospital.

OBJECTIVES: To determine whether the tumour necrosis factor-alpha (TNF-alpha) antagonist adalimumab (Humira) can be efficacious after secondary loss of efficacy (i.e. loss of clinical response in patients who had initially demonstrated clinical response) to infliximab (Remicade) or etanercept (Enbrel). PATIENTS AND METHODS: We studied 36 patients from the Stockholm TNF-alpha follow-up registry (STURE) who received adalimumab after secondary loss of efficacy to infliximab (group A, n = 27) or etanercept (group B, n = 9), and 26 patients who were started on adalimumab as the first TNF-alpha antagonist (group C). RESULTS: In group A, the baseline disease activity score 28 (DAS28) at infliximab institution was 5.5+/-0.2. During infliximab treatment, the mean best DAS28 was 3.7+/-0.2 (p<0.001), but increased to 5.2+/-0.3 when infliximab was stopped. After 3 months on adalimumab, the mean DAS28 decreased to 4.5+/-0.3 (p<0.003), and then to 4.2+/-0.2 at 6 months (p<0.001). In group B, the baseline DAS28 at etanercept institution was 6.6+/-0.5. During etanercept treatment, the mean best DAS28 was 4.6+/-0.5 (p<0.01), but increased to 5.7+/-0.4 by the time etanercept was stopped. After 3 months on adalimumab, the mean DAS28 decreased to 4.8+/-0.3 (p<0.005), and to 4.1+/-0.2 at 6 months (p<0.001). In group C, the mean baseline DAS28 was 5.6+/-0.3. After 6 months of adalimumab therapy, the DAS28 decreased to 3.5+/-0.4 (p<0.001). ACR20 responses with adalimumab in groups A, B, and C were similar (70-78%). CONCLUSIONS: For patients with secondary loss of efficacy from infliximab or etanercept, switching to adalimumab can restore a good clinical response.

Genetic markers for the efficacy of tumour necrosis factor blocking therapy in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a genetically complex disease where the response to different treatments varies greatly between different patients. This is the case with the tumour necrosis factor (TNF) blocking agents, where 20-40% of patients have been described as non-responders. No predictive markers exist as yet for the prognosis of response. OBJECTIVE: To analyse whether polymorphisms of several cytokine genes are associated with the responsiveness to TNF blockade with etanercept. METHODS: 123 patients with active RA were treated with etanercept and response rates were determined after three months using American College of Rheumatology (ACR)20 and disease activity score (DAS)28 response criteria. Genotyping was done for TNF (-308 TNFA), interleukin (IL)10 (-1087 IL10), transforming growth factor (TGF)beta1 (codon 25 TGFB1), and IL1 receptor antagonist (intron 2 IL1RN). RESULTS: 24 patients (20%) were defined as non-responders owing to their failure to fulfil any of the ACR20 or DAS28 response criteria. None of the recorded alleles was alone significantly associated with responsiveness to treatment. However, a certain combination of alleles (-308 TNF1/TNF1 and -1087 G/G) was associated with good responsiveness to etanercept (p<0.05). In addition, a combination of alleles influencing interleukin 1 receptor antagonist (IL1Ra) and TGFbeta1 production (A2 allele for IL1RN and rare C allele in codon 25 of TGFB1 gene) was associated with non-responsiveness (p<0.05). CONCLUSION: Genetic polymorphisms, which may influence the balance of pro- and anti-inflammatory cytokines of relevance for the course of RA, are associated with clinical responsiveness to etanercept treatment.

Anti-tumour necrosis factor (TNF)-alpha therapy (etanercept) down-regulates serum matrix metalloproteinase (MMP)-3 and MMP-1 in rheumatoid arthritis.

OBJECTIVES: Matrix metalloproteinases (MMPs) are cytokine-modulated enzymes that play an important role in the pathogenesis of rheumatoid arthritis (RA) by inducing bone resorption and cartilage destruction. This study evaluated the modulation of serum and synovial MMPs and their inhibitor, tissue inhibitor of matrix metalloproteinases (TIMP)-1, by therapy with soluble tumour necrosis factor (TNF) alpha receptor (etanercept). METHODS: Serum samples were collected from 60 RA patients at baseline and after 8 or 12 weeks of treatment. Paired synovial biopsies were obtained from 11 patients at two time points, before and after 8 weeks of treatment. We measured serum levels of MMP-1, MMP-3 and TIMP-1 by ELISA. Immunohistological analysis of synovial tissue was performed using monoclonal antibodies specific for MMP-1, MMP-3 and TIMP-1. RESULTS: Etanercept therapy significantly down-regulated serum levels of MMP-3 and MMP-1 in parallel with the reduction in inflammatory parameters (C-reactive protein concentration and erythrocyte sedimentation rate) in RA patients. Baseline pretreatment serum levels of MMP-3 correlated with changes in clinical disease activity during therapy. No consistent changes in serum level of TIMP-1 were observed, while ratios of MMP-1 and MMP-3 to TIMP-1 were down-regulated following etanercept treatment. Immunohistochemical analyses revealed great interindividual variability, with generally a high level of expression of MMP and low expression of TIMP. No significant change in the pattern or number of positive cells occurred during therapy. CONCLUSIONS: In RA patients, etanercept therapy down-regulates serum levels of MMP-3 and MMP-1 and the ratio between MMPs and TIMP-1. This may be an important mechanism for the prevention of future development of joint damage.

[Blockade of TNFalpha--new therapeutic principle in severe rheumatoid arthritis]. / Blockad av TNF alpha--ny behandlingsprincip vid svår reumatoid artrit.

TNF-alpha is a proinflammatory cytokine. It has a key function in the inflammatory cascade both systemically and locally in the inflamed joints of patients affected by rheumatoid arthritis (RA). Treatment with two different "biological" drugs that block the proinflammatory capacity of TNF-alpha has recently been approved by the European drug authorities. This paper discusses experience gained in clinical trials and during the first year of treatment in Sweden using infliximab (anti-TNF-alpha monoclonal antibodies) and etanercept (recombinant TNF-alpha receptor fusion protein).

Effects of antirheumatic gold salts on cytokine-induced neutrophil-dependent cytotoxicity for human endothelial cells.

BACKGROUND AND METHODS: To assess whether gold-containing disease-modifying antirheumatic drugs influenced the ability of human polymorphonuclear granulocytes (PMN) or nitric oxide (NO) to cause injury to human umbilical vein endothelial cells (HUVEC) in vitro (measured as release of 51Cr) after HUVEC had been activated with interleukin-1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF-alpha). RESULTS: IL-1 beta and TNF-alpha caused a prominent PMN-mediated 51Cr release that was dose-dependently reduced when auranofin (AF) or gold sodium aurothiomalate (GSTM) were added to PMN and HUVEC in the assay system. This protective effect remained, with stimulus- and drug-specific variations, when HUVEC alone were treated with the drugs. Likewise, when HUVEC cytotoxicity was induced by exogenous NO (donated by S-nitroso-acetyl-penicillamine [SNAP]), AF and GSTM inhibited the injury significantly. Some observations indicated that PMN agonists, generated by TNF-alpha-activated HUVEC (e.g., IL-8), were modulated by the antirheumatic drugs. First, addition of AF, but not GSTM, reduced the generation of IL-8 by 65%. Secondly, TNF-alpha-activated HUVEC triggered a rapid, transient rise of cytosolic Ca2+ concentrations in previously quiescent PMN; this rise was obliterated by prior treatment of HUVEC with AF (but not with GSTM). When TNF-alpha-induced endothelial cytotoxicity was provoked by PMN lysates, AF and GSTM inhibited this injury significantly. CONCLUSIONS: Thus, in this in vitro model of vasculitis, AF and GSTM reduced IL-1 beta- and TNF-alpha-induced and PMN-dependent HUVEC injury by interfering with intercellular signaling systems and cytotoxicity conferred by NO and PMN granula constituents.

Effects of anti-rheumatic gold salts on NF-kappa B mobilization and tumour necrosis factor-alpha (TNF-alpha)-induced neutrophil-dependent cytotoxicity for human endothelial cells.

We have previously shown that the gold-containing disease-modifying anti-rheumatic drugs, auranofin (AF) and gold sodium aurothiomalate (GSTM) reduce human umbilical vein endothelial cell (HUVEC) adhesion molecule expression and neutrophil (PMN) adherence. AF diminishes E-selectin and intercellular adhesion molecule-1 (ICAM-1) on cytokine-activated HUVEC, while GSTM decreases only E-selectin. Since tight adhesion is critical for PMN to damage EC, we tested whether these drugs modulated human PMN-mediated injury to TNF-alpha-activated HUVEC in vitro (as measured by 51Cr release). Here we show that TNF-alpha caused a prominent PMN-mediated cytotoxicity that was dose-dependently reduced when AF and GSTM were added to the assay system. We also found that a potent inhibitor of NF-kappaB, pyrrolidine dithiocarbamate (PDTC) in a dose-dependent manner impaired TNF-alpha-induced cytotoxicity, indicating a role of NF-kappaB activation in cytokine-induced endothelial injury. To examine the effects of AF and GSTM on TNF-alpha-induced NF-kappaB activation this was measured in HUVEC nuclear extracts by an electrophoretic mobility shift assay. AF, but not GSTM, decreased TNF-alpha-induced NF-kappaB activation in HUVEC. Thus, in this in vitro model of vasculitis, AF and GSTM dose dependently reduced TNF-alpha-mediated neutrophil-dependent cytotoxicity for HUVEC, and AF, but not GSTM, inhibited NF-kappaB mobilization, thereby providing possible mechanisms for effects of AF and GSTM.

Cluster headache is not associated with signs of a systemic inflammation.

OBJECTIVE: To investigate whether there is clinical or biochemical evidence for a transient systemic inflammation during active periods of cluster headache. METHODS: Twenty-seven male and female consecutively selected patients with episodic cluster headache filled in questionnaires aiming at detecting any concurrent systemic vasculitic or rheumatoid disease. They were physically examined by both a neurologist and a rheumatologist independent of each other. Blood and urine samples were taken one to three times during an active cluster period and once in remission. The following analyses were performed: hemoglobin, erythrocyte sedimentation rate, C-reactive protein, complete blood counts including differential counts, creatinine, albumin, creatine kinase, electrophoreses of serum (with haptoglobin, orosomucoid, IgG, IgM), von Willebrand's factor, antinuclear antibodies, rheumatoid factor, cytoplasmic antineutrophil cytoplasmic autoantibodies, perinuclear antineutrophil cytoplasmic autoantibodies, and routine urinary tests. An age- and sex-matched control group of 99 consecutive patients attending the Outpatient Department of Neurology for symptoms/diseases other than severe headache completed the same questionnaire as the patient group. RESULTS: Only one patient with cluster headache showed clinical signs (livedo reticularis) that could have been due to an ongoing systemic vasculitis. Most symptoms were equally or even more prevalent in the control group than among the patients with cluster headache. However, cold feet were about twice as prevalent among female patients with cluster headache than in the control group. This was considered due to their smoking habits. Laboratory tests showed no statistically significant differences between the active cluster periods and remission. There were some slightly abnormal values in single laboratory tests, some of which were probably due to concurrent upper respiratory infections. The findings of laboratory tests for one patient could have been due to nephritis. All patients were negative for cytoplasmic antineutrophil cytoplasmic autoantibodies and perinuclear antineutrophil cytoplasmic autoantibodies. CONCLUSIONS: These results were taken as evidence that no systemic inflammation is present during the active cluster headache period. However, whether a local retro-orbital inflammation underlies the pathophysiology of cluster headache remains obscure.

No effect of carvedilol on nitric oxide generation in phagocytes but modulation of production of superoxide ions.

Since carvedilol has been claimed to possess antioxidative effects, this drug might affect functional responses, including nitric oxide (NO) generation, of polymorphonuclear neutrophils (PMN) and macrophages. When we assessed the effects of carvedilol on PMN responses in vitro, we observed that carvedilol dose dependently modulated generation of superoxide ions by NADPH oxidase when induced by the formylpeptide formyl-methionyl-leucyl-phenylalanine (fMLP) or the phorbol ester phorbol myristate acetate. This effect was not coupled to diminished phospholipase C activity. In contrast to the effect on NADPH oxidase, neither the fMLP-elicited NO generation by PMN nor the response of the murine macrophage cell line J774 to lipopolysaccharide was affected. There was no evidence from cell-free assay systems that carvedilol is a scavenger for superoxide ions or NO. Moreover, carvedilol did not affect other reactions dependent on NO, e.g. spontaneous or fMLP-stimulated PMN migration or lipoxin A(4)-, fMLP-, or A23187-induced neutrophil cytotoxicity for human umbilical vein endothelial cells. Thus, these effects point to the possibility that carvedilol modulates the NADPH oxidase of PMN but leaves the nitric oxide synthase of phagocytes intact.

Prednisolone inhibits cytokine-induced adhesive and cytotoxic interactions between endothelial cells and neutrophils in vitro.

We assessed whether prednisolone influenced the ability of human polymorphonuclear neutrophils (PMN) to adhere to and cause lysis of human umbilical vein endothelial cells (HUVEC) in vitro (as measured by the release of 51Cr). Pretreatment of the endothelium with IL-1beta or tumour necrosis factor-alpha (TNF-alpha) caused prominent endothelial E-selectin expression and endothelial hyperadhesiveness for neutrophils, as well as PMN-mediated cytotoxicity. All these processes were dose-dependently reduced when prednisolone was added to the assay system. This protective effect remained when HUVEC alone were pretreated with the drug prior to washing and cytokine activation. Likewise, when HUVEC cytotoxicity was induced by the nitric oxide (NO) donor S-nitroso-acetyl-penicillamine (SNAP), prednisolone reduced cell injury significantly. In contrast, prednisolone did not interfere with signalling systems between TNF-alpha-stimulated HUVEC and quiescent PMN such as IL-8 generation and release of cytosolic Ca2 + in the PMN. Thus, in this in vitro model of vasculitis, prednisolone dose-dependently reduced cytokine-induced E-selectin expression and HUVEC hyperadhesiveness for neutrophils, as well as reducing neutrophil-dependent cytotoxicity against HUVEC via NO-dependent steps.

Prednisolone interferes with neutrophil adhesion and neutrophil mediated endothelial injury.

Adhesion of leukocytes to vascular endothelium is a crucial step in inflammation. This interaction may result in damage of the endothelial cells (EC). We evaluated the effects of prednisolone on adhesive interactions between human polymorphonuclear neutrophil granulocytes (PMN) and human umbilical vein endothelial cells (HUVEC) as well as PMN mediated cytotoxicity to HUVEC (as release of 51chromium), mediated by N-formyl-methionyl-leucyl-phenylalanine (fMLP), lipoxin A4 (LXA4), and the calcium ionophore A23187 in vitro. Prednisolone dose-dependently interfered with adhesion and cytotoxicity induced by fMLP. Prednisolone (at 10 microM) led to a 39% reduction of adhesion and an almost complete inhibition of cytotoxicity, mainly by effects on the PMN. Prednisolone also interfered with cytotoxicity induced by LXA4 by effects on PMN as well as on HUVEC. Adhesion and cytotoxicity induced by the calcium ionophore A23187 was not affected in any way by prednisolone. Thus, in these in vitro models of vasculitis, prednisolone interferes with adhesive and cytotoxic interactions induced by receptor-dependent agonists. These protective effects of prednisolone might explain some of the beneficial effects of glucocorticoids in the treatment of vasculitis.

Biochemical characterization and crystal structure determination of human heart short chain L-3-hydroxyacyl-CoA dehydrogenase provide insights into catalytic mechanism.

Human heart short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) catalyzes the oxidation of the hydroxyl group of L-3-hydroxyacyl-CoA to a keto group, concomitant with the reduction of NAD+ to NADH, as part of the beta-oxidation pathway. The homodimeric enzyme has been overexpressed in Escherichia coli, purified to homogeneity, and studied using biochemical and crystallographic techniques. The dissociation constants of NAD+ and NADH have been determined over a broad pH range and indicate that SCHAD binds reduced cofactor preferentially. Examination of apparent catalytic constants reveals that SCHAD displays optimal enzymatic activity near neutral pH, with catalytic efficiency diminishing rapidly toward pH extremes. The crystal structure of SCHAD complexed with NAD+ has been solved using multiwavelength anomalous diffraction techniques and a selenomethionine-substituted analogue of the enzyme. The subunit structure is comprised of two domains. The first domain is similar to other alpha/beta dinucleotide folds but includes an unusual helix-turn-helix motif which extends from the central beta-sheet. The second, or C-terminal, domain is primarily alpha-helical and mediates subunit dimerization and, presumably, L-3-hydroxyacyl-CoA binding. Molecular modeling studies in which L-3-hydroxybutyryl-CoA was docked into the enzyme-NAD+ complex suggest that His 158 serves as a general base, abstracting a proton from the 3-OH group of the substrate. Furthermore, the ability of His 158 to perform such a function may be enhanced by an electrostatic interaction with Glu 170, consistent with previous biochemical observations. These studies provide further understanding of the molecular basis of several inherited metabolic disease states correlated with L-3-hydroxyacyl-CoA dehydrogenase deficiencies.