Negative impact of linezolid on human neutrophil functions in vitro.

BACKGROUND/AIMS: In a recent phase III clinical trial on linezolid, more patients in the linezolid treatment arm acquired Gram-negative catheter-related bloodstream infections despite the adequate therapy of infections caused by Gram-negative bacteria. We tested our hypothesis that linezolid impairs phagocytosis and the killing of Gram-negative bacteria by polymorphonuclear leukocytes (PMN). METHODS: The influence of clinically relevant concentrations (5, 20 and 50 mg/l) of linezolid on granulocyte function in vitro was tested. Phagocytosis was determined by flow cytometry, and killing of bacteria was evaluated by plate counting. Chemotaxis was examined by an under-agarose cell migration assay. Gram-positive and Gram-negative bacteria were used. RESULTS: Linezolid significantly impaired phagocytosis of a specific Escherichia coli strain in a concentration-dependent manner, whereas the effect on Pseudomonas aeruginosa was less prominent. No such effects were observed with a different E. coli strain or Staphylococcus aureus. Neither killing nor the chemotactic behaviour of PMN was significantly affected by linezolid. CONCLUSIONS: The observed concentration-dependent impairment of the phagocytic function might contribute to the higher frequency of catheter-related Gram-negative bloodstream infections in patients treated with linezolid. Individual patient risk may also depend on the causative Gram-negative strain.

Garenoxacin-induced increase of CD11b expression on human polymorphonuclear neutrophils does not affect phagocytosis and killing of Staphylococcus aureus.

Garenoxacin is considered to be the most active quinolone against Staphylococcus aureus. Quinolones are believed to alter the function of human polymorphonuclear leukocytes (PMN) and garenoxacin is known to be the only quinolone which alters the expression of the beta-chain (CD11b) of the complement receptor 3 (CR3) which is known to be important in the phagocytosis of S. aureus by PMN. Therefore, the effect of this altered CD11b expression on phagocytosis, oxidative burst, and killing of S. aureus was addressed and compared with that of standard quinolones. Phagocytosis and oxidative burst were determined by flow cytometry, and killing was measured by a colony-count method. Garenoxacin at therapeutic concentrations affected neither phagocytosis nor killing of Staphylococcus aureus NMS54. At supratherapeutic concentrations (1,500 mg/l) garenoxacin reduced and delayed phagocytosis like all other quinolones tested except norfloxacin. This decrease seems to be a result of inhibition of the oxidative burst of PMN and reduced CD11b expression at this supratherapeutic concentration. In conclusion, the alteration of CD11b expression of PMN caused by garenoxacin at 0.5, 5.0, and 100.0 mg/l is not considered to hamper the function of these first-line-defense phagocytes.

Citalopram-induced pathways regulation and tentative treatment-outcome-predicting biomarkers in lymphoblastoid cell lines from depression patients.

Antidepressant therapy is still associated with delays in symptomatic improvement and low response rates. Incomplete understanding of molecular mechanisms underlying antidepressant effects hampered the identification of objective biomarkers for antidepressant response. In this work, we studied transcriptome-wide expression followed by pathway analysis in lymphoblastoid cell lines (LCLs) derived from 17 patients documented for response to SSRI antidepressants from the Munich Antidepressant Response Signatures (MARS) study upon short-term incubation (24 and 48 h) with citalopram. Candidate transcripts were further validated with qPCR in MARS LCLs from responders (n = 33) vs. non-responders (n = 36) and afterward in an independent cohort of treatment-resistant patients (n = 20) vs. first-line responders (n = 24) from the STAR*D study. In MARS cohort we observed significant associations of GAD1 (glutamate decarboxylase 1; p = 0.045), TBC1D9 (TBC1 Domain Family Member 9; p = 0.014-0.021) and NFIB (nuclear factor I B; p = 0.015-0.025) expression with response status, remission status and improvement in depression scale, respectively. Pathway analysis of citalopram-altered gene expression indicated response-status-dependent transcriptional reactions. Whereas in clinical responders neural function pathways were primarily up- or downregulated after incubation with citalopram, deregulated pathways in non-responders LCLs mainly involved cell adhesion and immune response. Results from the STAR*D study showed a marginal association of treatment-resistant depression with NFIB (p = 0.068) but not with GAD1 (p = 0.23) and TBC1D9 (p = 0.27). Our results propose the existence of distinct pathway regulation mechanisms in responders vs. non-responders and suggest GAD1, TBC1D9, and NFIB as tentative predictors for clinical response, full remission, and improvement in depression scale, respectively, with only a weak overlap in predictors of different therapy outcome phenotypes.

CD66b Overexpression and Loss of C5a Receptors as Surface Markers for Staphylococcus aureus-Induced Neutrophil Dysfunction.

Neutrophil granulocytes constitute the main component of innate immunity in the clearance of bacterial infections. However, during systemic inflammation, immunoparalysis may occur resulting in neutrophil dysfunction. This study presents a new in vitro model for analyzing the dysfunction of human peripheral blood neutrophils resulting from the interaction with Staphylococcus aureus components in whole blood. After induction of a massive complement activation by S. aureus supernatant, the neutrophils exhibit a reduced phagocytic capacity resulting in a dramatic reduction of the antibacterial activity similar to that of neutrophils isolated from septic patients. The number of phagocytozing neutrophils is drastically reduced as well as the phagocytic capacity designated by a significantly lower number of ingested microbes. This dysfunction correlates with the loss of complement component 5a receptor 1 from the neutrophil cell surface and can be further characterized by a C5a-induced CD66b overexpression. The presented in vitro model offers a new platform for preclinical testing of immunosuppressive drugs and delivers new information for the understanding of neutrophil dysfunctions under the conditions described.

CD66b overexpression and homotypic aggregation of human peripheral blood neutrophils after activation by a gram-positive stimulus.

Neutrophils represent the main component of innate immunity in the clearance of bacterial infections. To pass the tissue and to localize and reach the site of infection, the peripheral blood neutrophils have to pass through a complex receptor-mediated interaction with the endothelial layer. Under pathophysiological conditions, such as severe sepsis, this process is impaired and often characterized by neutrophil aggregation. In this study, we examined the impact of three different Staphylococcus aureus strains on the activation status of human peripheral blood neutrophils by coincubation of bacterial culture supernatant with whole blood. This complex interaction of a gram-positive stimulus with blood components leads to a special neutrophil activation phenotype, which is characterized by an overexpression of the cell-surface molecule CD66b. The process is accompanied by a strong increase of homotypic aggregates and seems to be initialized by a massive activation impulse caused by the interplay of plasma components. This maximum activation of neutrophils prior to the complex and highly regulated activation required for transmigration might play a key role in the neutrophil dysfunction in gram-positive sepsis.

Influence of fluoroquinolones on phagocytosis and killing of Candida albicans by human polymorphonuclear neutrophils.

Candida albicans infections often occur during or shortly after antibacterial treatment. Phagocytosis by polymorphonuclear neutrophil granulocytes (PMN) is the most important primarily defence mechanism against C. albicans. Certain antibiotics such as some fluoroquinolones (FQ) are known to influence phagocyte functions. Thus, we investigated the influence of older and newer FQ on the phagocytosis and killing of C. albicans by human PMN paying special attention to CD11b expression of these cells as an indicator of the degree of their activation. In order to obtain comprehensive and comparable results we tested 13 FQ over a wide range of concentrations and in a time dependent manner in a standardized approach. When used at therapeutic concentrations, the FQ tested did not influence to a clinically significant degree the phagocytosis or the killing of C. albicans by human PMN and also not their activation. However, at high concentrations those FQ with cyclopropyl-moiety at position N1 showed increase in CD11b expression and diminished phagocytosis and oxidative burst.