Early discontinuation of empirical antibiotic treatment in neutropenic patients with acute myeloid leukaemia and high-risk myelodysplastic syndrome.

INTRODUCTION: Current guidelines advocate empirical antibiotic treatment (EAT) in haematological patients with febrile neutropenia. However, the optimal duration of EAT is unknown. In 2011, we have introduced a protocol, promoting discontinuation of carbapenems as EAT after 3 days in most patients and discouraging the standard use of vancomycin. This study assesses the effect of introducing this protocol on carbapenem and vancomycin use in high-risk haematological patients and its safety. METHODS: A retrospective before-after study was performed comparing a cohort from 2007 to 2011 (period I, before restrictive EAT use) with a cohort from 2011 to 2014 (period II, restrictive EAT use). Neutropenic episodes related to chemotherapy or stem cell transplantation (SCT) in patients with acute myeloid leukaemia (AML) or high-risk myelodysplastic syndrome (MDS) were analysed. The primary outcome was the use of carbapenems and vancomycin as EAT during neutropenia, expressed as days of therapy (DOT)/100 neutropenic days and analysed with interrupted time series (ITS). Also the use of other antibiotics was analysed. Safety measurements included 30-day mortality, ICU admittance within 30 days after start of EAT and positive blood cultures with carbapenem-susceptible microorganisms. RESULTS: Three hundred sixty-two neutropenic episodes with a median duration of 18 days were analysed, involving 201 patients. ITS analysis showed decreased carbapenem use with a step change of - 16.1 DOT/100 neutropenic days (95% CI - 26.77 to - 1.39) and an overall reduction of 21.6% (8.7 DOT/100 neutropenic days). Vancomycin use decreased with a step change of - 13.7 DOT/100 neutropenic days (95% CI - 23.75 to - 3.0) and an overall reduction of 54.7% (14.6 DOT/100 neutropenic days). The use of all antibiotics combined decreased from 155.6 to 138 DOT/100 neutropenic days, a reduction of 11.3%. No deaths directly related to early discontinuation of EAT were identified, also no notable difference in ICU-admission (9/116 in period I, 9/152 in period II) and positive blood cultures (4/116 in period I, 2/152 in period II) was detected. CONCLUSION: The introduction of a protocol promoting restrictive use of EAT resulted in reduction of carbapenem and vancomycin use and appears to be safe in AML or high-risk MDS patients with febrile neutropenia during chemotherapy or SCT.

Improving early diagnosis of pulmonary infections in patients with febrile neutropenia using low-dose chest computed tomography.

We performed a prospective study in patients with chemotherapy induced febrile neutropenia to investigate the diagnostic value of low-dose computed tomography compared to standard chest radiography. The aim was to compare both modalities for detection of pulmonary infections and to explore performance of low-dose computed tomography for early detection of invasive fungal disease. The low-dose computed tomography remained blinded during the study. A consensus diagnosis of the fever episode made by an expert panel was used as reference standard. We included 67 consecutive patients on the first day of febrile neutropenia. According to the consensus diagnosis 11 patients (16.4%) had pulmonary infections. Sensitivity, specificity, positive predictive value and negative predictive value were 36%, 93%, 50% and 88% for radiography, and 73%, 91%, 62% and 94% for low-dose computed tomography, respectively. An uncorrected McNemar showed no statistical difference (p = 0.197). Mean radiation dose for low-dose computed tomography was 0.24 mSv. Four out of 5 included patients diagnosed with invasive fungal disease had radiographic abnormalities suspect for invasive fungal disease on the low-dose computed tomography scan made on day 1 of fever, compared to none of the chest radiographs. We conclude that chest radiography has little value in the initial assessment of febrile neutropenia on day 1 for detection of pulmonary abnormalities. Low-dose computed tomography improves detection of pulmonary infiltrates and seems capable of detecting invasive fungal disease at a very early stage with a low radiation dose.

[Rapid diagnosis of psittacosis using a recently developed real-time PCR technique]. / Snelle diagnostiek van psittacose met behulp van een recent ontwikkelde realtime-PCR.

A 37-year-old man was admitted with cough and fever. Three days after admission he was tested using a newly developed real-time PCR technique that detects the DNA of Chlamydophila psittaci. The result was positive; serological investigation was not positive until 14 days later. Psittacosis is a potentially life-threatening infectious disease. Laboratory diagnosis relies mainly on the assessment of paired sera, but this approach has obvious disadvantages in the acute setting. Routine use of the real-time PCR technique led to the rapid diagnosis of psittacosis in 6 other patients. All 7 patients recovered after antibiotic treatment. This PCR technique is a valuable adjuvant to serological testing for the rapid diagnosis of psittacosis.

Nocardiosis: a case series and a mini review of clinical and microbiological features.

Infections caused by Nocardia species are uncommon and have a wide variety of clinical manifestations in immunocompetent and immunocompromised patients. The diagnosis of nocardiosis can easily be missed because there are no characteristic symptoms. We present one case of a Nocardia infection in detail and give a brief description of eight other cases, including a relatively unique type of Nocardia veterana, diagnosed in our hospital during a five-year period. The diversity of clinical manifestations, microbiological identification and general principles of treatment of nocardiosis are reviewed.

[The Dutch MRSA policy should be retained]. / Het Nederlandse MRSA-beleid is succesvol.

The Dutch methicillin-resistant Staphylococcus aureus (MRSA) 'search and destroy' policy is effective. MRSA should be banned from hospitals: MRSA infections are associated with increased mortality and costs. In addition, widespread use of vancomycin for treating MRSA infections encourages the spread and development of vancomycin-resistant micro-organisms.

Iron chelation and hydroxyl radical scavenging reduce the inflammatory response of endothelial cells after infection with Chlamydia pneumoniae or influenza A.

BACKGROUND: Chronic low-grade inflammation is associated with increased risk of vascular diseases. The source of inflammation is unknown but may well be chronic and/or repetitive infections with microorganisms. Direct infection of endothelial cells (ECs) may also be a starting point for atherogenesis by initiating endothelial procoagulant activity, increased monocyte adherence and increased cytokine production. We hypothesized that iron-mediated intracellular hydroxyl radical formation after infection is a key event in triggering the production of interleukin-6 (IL-6) by ECs in vitro. METHODS: Cultured ECs were incubated with Fe(II) and Fe(III) or infected with Chlamydia pneumoniae or influenza A/H1N1/Taiwan/1/81 for 48 and 24 h, respectively. To determine the role of iron and reactive oxygen species, cells were coincubated with the H2O2 scavenger N-acetyl-l-cysteine, with the iron chelator deferoxamine (DFO) or with the intracellular hydroxyl radical scavenger dimethylthiourea (DMTU). After the incubation periods, supernatants were harvested for IL-6 determination. RESULTS: Incubating ECs with Fe(II) and Fe(III) resulted in increased IL-6 production. Similarly, infection with C. pneumoniae and influenza A also induced an IL-6 response. Coincubating ECs with DFO or DMTU blocked this response. Nuclear factor-kappaB activity was increased after infection and blocked by coincubation with DFO or DMTU. CONCLUSION: Cultured ECs respond to infection and iron incubation with increased production of IL-6. Iron, the generation of intracellular hydroxyl radical and NF-kappaB activity are essential in cellular activation, suggesting that reactive oxygen species generated in the Haber-Weiss reaction are essential in invoking an immunological response to infection by ECs.

Human immunodeficiency virus type 1 replication inhibition by the bidentate iron chelators CP502 and CP511 is caused by proliferation inhibition and the onset of apoptosis.

BACKGROUND: The iron chelators deferoxamine (DF) and deferiprone (CP20) have been shown to inhibit human immunodeficiency virus type 1 (HIV-1) replication in human peripheral blood lymphocytes (PBL). The orally active bidentate chelators CP502 and CP511, which also belong to the 3-hydroxypyridin-4-one family, but with higher affinities for iron than CP20, were monitored for their antiviral properties by checking for p24 antigen production and nuclear factor (NF)-kappaB activation, and their ability to induce apoptosis. MATERIALS AND METHODS: Human PBLs were isolated from HIV-1 seronegative donors and subsequently infected with HIV-1(Ba-L) for 2 h. After 5 days' incubation, HIV-1 replication was monitored by p24 antigen production. Cellular proliferation as well as caspase-3 activity were monitored in uninfected cells after a period of 5 days and after 1 day infection, respectively. NF-kappaB activity was also monitored by electromobility shift assays (EMSA) performed on nuclear extracts of Jurkat cells treated with the different chelators for 4 h. RESULTS: CP502 and CP511 decrease HIV-1 replication by decreasing cellular proliferation in a similar manner to DF and CP20. CP511 seemed to be more potent than either CP502 or CP20. Due to the reduction in cellular proliferation, there was an increase in caspase-3 activity after 24 h incubation. NF-kappaB activity was not affected by any of the chelators. CONCLUSIONS: Iron chelators with high affinities for iron, which are under development for the treatment of iron overload, could contribute to the reduction of HIV-1 replication in infected patients by cellular proliferation inhibition rather than by a direct antiviral action.

The chemotherapeutic agent bleomycin in a two-drug combination with zidovudine, ritonavir or indinavir synergistically inhibits HIV Type-1 replication in peripheral blood lymphocytes.

It has been suggested that the combination of cancer chemotherapy with antiviral therapy is helpful for the containment of lymphomas in HIV-infected patients. Since we have recently shown that the nucleic acid binding chemotherapeutic agent bleomycin in itself has antiviral properties, we looked to see if there was any possible synergy with current anti-HIV agents. Combinations of zidovudine, indinavir or ritonavir with bleomycin, synergistically inhibited HIV-1(AT) replication in stimulated peripheral blood lymphocytes (combination index at 50% virus inhibition was 0.427, 0.604 and 0.535, respectively) and this synergism was not accompanied by any synergistic effects on cytotoxicity. We conclude from these data that further studies to investigate the clinical efficacy of combinations of antiviral and cancer chemotherapeutic agents are warranted in relation to viral load improvement.

Activation of human macrophages by amyloid-beta is attenuated by astrocytes.

In Alzheimer's disease, neuritic amyloid-beta plaques along with surrounding activated microglia and astrocytes are thought to play an important role in the inflammatory events leading to neurodegeneration. Studies have indicated that amyloid-beta can be directly neurotoxic by activating these glial cells to produce oxygen radicals and proinflammatory cytokines. This report shows that, using primary human monocyte-derived macrophages as model cells for microglia, amyloid-beta(1-42) stimulate these macrophages to the production of superoxide anions and TNF-alpha. In contrast, astrocytes do not produce both inflammatory mediators when stimulated with amyloid-beta(1-42). In cocultures with astrocytes and amyloid-beta(1-42)-stimulated macrophages, decreased levels of both superoxide anion and TNF-alpha were detected. These decreased levels of potential neurotoxins were due to binding of amyloid-beta(1-42) to astrocytes since FACScan analysis demonstrated binding of FITC-labeled amyloid-beta(1-42) to astrocytoma cells and pretreatment of astrocytes with amyloid-beta(1-16) prevented the decrease of superoxide anion in cocultures of human astrocytes and amyloid-beta(1-42)-stimulated macrophages. To elucidate an intracellular pathway involved in TNF-alpha secretion, the activation state of NF-kappaB was investigated in macrophages and astrocytoma cells after amyloid-beta(1-42) treatment. Interestingly, although activation of NF-kappaB could not be detected in amyloid-beta-stimulated macrophages, it was readily detected in astrocytoma cells. These results not only demonstrate that amyloid-beta stimulation of astrocytes and macrophages result in different intracellular pathway activation but also indicate that astrocytes attenuate the immune response of macrophages to amyloid-beta(1-42) by interfering with amyloid-beta(1-42) binding to macrophages.

Effect of corticosteroids on nuclear factor-kappaB activation and hemodynamics in late septic shock.

OBJECTIVE: To describe the underlying pathophysiologic mechanisms of the effect of corticosteroids in a patient with late septic shock. DESIGN: Case report. SETTING: The medical intensive care unit at University Medical Center Utrecht. PATIENT: An 86-yr-old female patient with late septic shock requiring mechanical ventilation and vasopressive agents. INTERVENTIONS: Administration of hydrocortisone, 300 mg daily. MEASUREMENTS AND MAIN RESULTS: Within 3 days of corticosteroid treatment, the patient could be weaned of vasopressive agents and mechanical ventilation. Serum C-reactive protein levels normalized. Nuclear factor-kappaB activation in unstimulated and in vitro lipopolysaccharide-stimulated peripheral blood mononuclear cells decreased to background level within 5 days. Repeated functional tests of the hypothalamic-pituitary-adrenal axis were normal. CONCLUSION: Our data suggest that the pathophysiologic mechanism behind the clinical effects of supraphysiologic doses of corticosteroids in late septic shock is directly related to the inhibition of nuclear factor-kappaB in peripheral blood mononuclear cells.

Intracellular pathways involved in TNF-alpha and superoxide anion release by Abeta(1-42)-stimulated primary human macrophages.

In this study, the intracellular signal transduction pathways leading to the production of TNF-alpha and superoxide anions by amyloid-beta-stimulated primary human monocyte-derived macrophages was investigated. Using Western blotting and specific inhibitors it is shown that both ERK 1/2 and p38 MAPK signal transduction pathways as well as PKC are involved in the amyloid-beta-stimulated superoxide anion production. In contrast, only ERK 1/2 MAPK seems to be involved in TNF-alpha production: questioning the connection between PKC and ERK 1/2 activation. Our results suggest the use of ERK 1/2 MAPK inhibitors in the prevention of macrophage activation in the context of Alzheimer's disease.

Anti-HIV effect of iron chelators: different mechanisms involved.

BACKGROUND: Drugs for the treatment of AIDS have been directed to specific events in the human immunodeficiency virus (HIV-1) life cycle, aimed to stop viral replication by inhibition of reverse transcriptase or protease activity. Studies showing that oxidative stress and iron may be important in the activation of HIV-1 have focused attention on the potential therapeutic use of iron chelators. OBJECTIVES: The goal of this review is to describe several possibilities as to how iron is involved in the replication of HIV and how iron chelation may interfere in this process. STUDY DESIGN: First some physico-chemical properties of iron concerning solubility, oxidation-reduction potential, catalysis, and chelation will be discussed. In the second part, the role of iron in various biochemical systems is explained. RESULTS: Nuclear factor kappa B (NF-kappaB) activation, regulating proviral transcription, can be influenced by iron through the production of reactive oxygen species. A second route by which iron chelation could influence HIV replication, is by inhibition of DNA synthesis through inactivation of iron-dependent ribonucleotide reductase. Another strategy which can be employed in targeting iron chelators against HIV-1, is direct oxidative viral RNA/DNA attack. This could be achieved by bleomycin, a cytostatic agent with the ability to form a complex with DNA and RNA. CONCLUSION: Chelation may withhold iron from viral metabolism but on the other hand may also favor catalysis of reactive oxygen species directed to viral constituents. In combination with existing antivirals, iron chelation could add to improve the treatment of HIV-disease.

Inhibition of human immunodeficiency virus type 1 replication in human mononuclear blood cells by the iron chelators deferoxamine, deferiprone, and bleomycin.

Replication of human immunodeficiency virus type 1 (HIV-1) can be influenced by iron. Hence, decreasing the availability of iron may inhibit HIV-1 replication. Deferoxamine and deferiprone, both forming catalytically inactive iron-chelator complexes, and bleomycin, by use of which iron catalyzes oxidative nucleic acid destruction, were investigated. Expression of p24 antigen in human monocyte-derived macrophages and peripheral blood lymphocytes (PBL) was reduced by all 3 iron chelators. In PBL, p24 reduction was mirrored by a decrease in proliferation after incubation with deferoxamine or deferiprone, suggesting that viral inhibition is closely linked to a decrease in cellular proliferation. In contrast, clinically relevant bleomycin concentrations reduced p24 levels by approximately 50% without affecting proliferation. When deferoxamine and the nucleoside analogue dideoxyinosine were used in combination, they acted synergistically in inhibiting HIV-1 replication. These observations suggest that iron chelators with different mechanisms of action could be of additional benefit in antiretroviral combination therapy.

Potential role of CCR5 polymorphism in the development of AIDS dementia complex.

The chemokine receptor CCR5 and to a lesser extent CCR2b and CCR3 have been shown to serve as coreceptors for HIV-1 entry into macrophages. Individuals that are homozygous for a defective CCR5 allele (DeltaCCR5) are highly, but not fully, resistant to infection with HIV-1. Here, we want to emphasize the importance of DeltaCCR5 in in vitro as well as in vivo studies. We provide data that suggest that CCR5 polymorphism may affect the onset of AIDS dementia complex in vivo and data that show that HIV-1 replication is influenced by the DeltaCCR5 allele in vitro. Knowing the CCR5 genotype of an individual will help to better interpret research results and may even provide new information about mechanisms of disease.

Lipopolysaccharide-induced tumor necrosis factor alpha production by human monocytes involves the raf-1/MEK1-MEK2/ERK1-ERK2 pathway.

During gram-negative sepsis, human monocytes are triggered to produce large quantities of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) in response to endotoxin (lipopolysaccharide [LPS]). Several studies have identified signal transduction pathways that are activated by LPS, including activation of nuclear factor-kappaB (NF-kappaB) and activation of mitogen-activated protein kinases (MAPKs), including ERK1 and ERK2, c-Jun N-terminal kinase, and p38. In this study, the relevance of ERK1 and ERK2 activation for LPS-induced TNF-alpha production by primary human monocytes has been addressed with PD-098059, which specifically blocks activation of MAPK kinase (MEK) by Raf-1. TNF-alpha levels in the monocyte culture supernatant, induced by 10 ng of LPS/ml, were reduced by PD-098059 (50 microM). In addition, PD-098059 also reduced TNF-alpha mRNA expression when cells were stimulated for 1 h with LPS. On the other hand, LPS-induced interleukin-10 (IL-10) levels in the monocyte supernatant were only slightly inhibited by PD-098059. Ro 09-2210, a recently identified MEK inhibitor, completely abrogated TNF-alpha levels at nanomolar concentrations. IL-10 levels also were strongly reduced. To show the efficacy of PD-098059 and Ro 09-2210, ERK1 and -2 activation was monitored by Western blotting with an antiserum that recognizes the phosphorylated (i.e., activated) forms of ERK1 and ERK2. Addition of LPS to human monocytes resulted in activation of both ERK1 and ERK2 in a time- and concentration (50% effective concentration between 1 and 10 ng of LPS/ml)-dependent manner. Activation of ERK2 was blocked by PD-098059 (50 microM), whereas ERK1 seemed to be less affected. Ro 09-2210 completely prevented LPS-induced ERK1 and ERK2 activation. LPS-induced p38 activation also was prevented by Ro 09-2210. These data further support the view that the ERK signal transduction pathway is causally involved in the synthesis of TNF-alpha by human monocytes stimulated with LPS.

Low-dose N-acetylcysteine protects rats against endotoxin-mediated oxidative stress, but high-dose increases mortality.

We evaluated the effect of the antioxidant N-acetylcysteine (NAC) on oxidative stress, lung damage, and mortality induced by an endotoxin (lipopolysaccharide, or LPS) in the rat. Continuous intravenous infusion of 275 mg NAC/kg in 48 h, starting 24 h before LPS challenge, decreased hydrogen peroxide (H2O2) concentrations in whole blood (p < 0.01). This decrease was accompanied by fewer histologic abnormalities of the lung and decreased mortality (p < 0.025), compared with rats receiving LPS alone. N-Acetylserine, which has no sulfhydryl group, did not protect rats against LPS toxicity. Improved survival was not associated with an increase in pulmonary reduced glutathione, nor with inhibition of serum tumor necrosis factor (TNF) activity. In vitro, TNF production and DNA binding of nuclear factor kappa B (NF-kappaB) in human Mono Mac 6 cells was only inhibited at concentrations of NAC above 20 mM. High-dose NAC treatment (550 and 950 mg/kg in 48 h) decreased lung GSH (p < 0.05) and resulted in a significantly smaller number of surviving animals when compared with the low-dose NAC group (p < 0.025). In vitro, NAC increased hydroxyl radical generation in a system with Fe(III)-citrate and H2O2 by reducing ferric iron to its catalytic, active Fe2+ form. We conclude that low-dose NAC protects against LPS toxicity by scavenging H2O2, while higher doses may have the opposite effect.

Cytokine-induced protein tyrosine phosphorylation is essential for cytokine priming of human eosinophils.

BACKGROUND: Human eosinophils are strongly modulated by the eosinophilotrophic cytokines IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). A clear intracellular effect of these cytokines is the induction of tyrosine phosphorylation of multiple cellular substrates. However, the relevance of tyrosine phosphorylation for eosinophil functioning has not been established. OBJECTIVE: In this study we have investigated dose-response and time curves of IL-5-, IL-3-, and GM-CSF-induced tyrosine phosphorylation in eosinophils. Moreover, we have evaluated the importance of IL-5-induced tyrosine phosphorylation for priming of human eosinophils. METHODS: Cytokine-induced tyrosine phosphorylation was monitored on western blot with an antiphosphotyrosine antibody (4G10). To probe the relevance of tyrosine phosphorylation for priming, eosinophils were primed with IL-5 in the presence of the tyrosine kinase inhibitor herbimycin A. Platelet activating factor (PAF) was used as a control priming agent. Subsequently, the eosinophils were incubated with serum-treated zymosan (STZ) to activate the respiratory burst. Binding of STZ was determined by FACS analysis. RESULTS: IL-5-, IL-3-, and GM-CSF-induced tyrosine phosphorylation was found at concentrations that primed eosinophil effector mechanism (median effective dose values: approximately 5.10(-11) mol/L, approximately 5.10(-10) mol/L, and approximately 5.10(-12) mol/L for IL-5, IL-3, and GM-CSF, respectively). Cytokine-induced tyrosine phosphorylation was transient with an optimum value at 15 minutes. IL-5 priming of STZ-induced activation of the respiratory burst was blocked by herbimycin A, whereas PAF still primed this response. In fact, herbimycin A inhibited IL-5 priming of STZ binding to human eosinophils. On the other hand, PAF priming of STZ binding was not affected by herbimycin A. Both IL-5-induced and PAF-induced tyrosine phosphorylation were inhibited by herbimycin A. CONCLUSION: These data demonstrate for the first time that IL-5 priming of opsonized particle-induced responses is mediated by tyrosine kinase activity in human eosinophils.

Eosinophil priming by cytokines: from cellular signal to in vivo modulation.

Eosinophils play an important role in the effector phase of allergic inflammation. This review will focus on the conversion of the unprimed eosinophil phenotype in the peripheral blood of normal individuals to the primed phenotype found in the peripheral blood and tissues of allergic patients, a phenomenon called priming. Recent data on the signals initiated after cytokine receptor activation on eosinophils will be reviewed.