B.J. Kullberg

FUNCTIONAL CONSEQUENCES OF TOLL-LIKE RECEPTOR 4 POLYMORPHISMS

Toll-like receptor 4 (TLR4) is an important pathogen recognition receptor that recognizes mainly lipopolysaccharide (LPS) of Gram-negative bacteria, but also structures from fungal and mycobacterial pathogens, as well as endogenous ligands. Two nonsynonymous polymorphisms of TLR4, Asp299Gly and Thr399lle, have been suggested to alter the function of the receptor. Some, but not all, studies have proposed that these polymorphisms lead to reduced cytokine response and increased susceptibility to Gram-negative infections. In this review, we compare studies that assessed the effect of the Asp299Gly and Thr399lle polymorphisms on susceptibility to Gram-negative infections and examine the phenotypic consequences of these polymorphisms. In addition, we review the geographical distribution of TLR4 polymorphisms and present a model for evolutionary pressures on the TLR4 genetic make-up.

Low-density lipoprotein receptor-deficient mice are protected against lethal endotoxemia and severe gram-negative infections.

Lipoproteins can bind lipopolysaccharide (LPS) and decrease the LPS-stimulated production of pro-inflammatory cytokines. We investigated the effect of increased plasma concentrations of low-density-lipoproteins (LDL) on survival and cytokine production after a lethal challenge with either LPS or live Gram-negative bacteria in LDL receptor deficient mice (LDLR-/-). The LDLR-/- mice challenged with LPS had an eightfold increased LD50 when compared with the wild type controls (C57Bl/6J), while tumor necrosis factor alpha (TNFalpha) and interleukin-1 alpha (IL-1 alpha) plasma concentrations were decreased twofold. LDLR-/- mice had significantly lower and delayed mortality than control mice after infection with Klebsiella pneumoniae. No differences in the outgrowth of bacteria in the organs were present between the two groups, while circulating cytokine concentrations were decreased twofold in LDLR-/- mice. In contrast, the LPS-stimulated in vitro production of cytokines by peritoneal macrophages of LDLR-/- mice was significantly increased compared with controls. This increase was associated with enhanced specific binding of LPS to the macrophages of LDLR-/- mice. In conclusion, endogenous LDL can protect against the lethal effects of endotoxin and Gram-negative infection. At least part of this protection is achieved through decreased in vivo production of pro-inflammatory cytokines, in spite of increased cytokine production capacity.

Recognition of fungal pathogens by toll-like receptors.

Toll-like receptors (TLRs) have been identified as a major class of pattern-recognition receptors. Recognition of pathogen-associated molecular patterns by TLRs, either alone or in heterodimerization with other TLR or non-TLR receptors, induces signals responsible for the activation of the innate immune response. Recent studies have demonstrated a crucial involvement of TLRs in the recognition of fungal pathogens such as Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans. Through the study of fungal infection in knock-out mice deficient in either TLRs or TLR-associated adaptor molecules, it became apparent that specific TLRs such as TLR2 and TLR4 play differential roles in the activation of the various arms of the innate immune response. Recent data also suggest that TLRs offer escape mechanisms to certain pathogenic microorganisms, especially through TLR2-driven induction of anti-inflammatory cytokines. These new data have substantially increased our knowledge of the recognition of fungal pathogens, and the study of TLRs remains one of the most active areas of research in the field of fungal infections.

Voriconazole Salvage Treatment of Invasive Candidiasis

Data on the salvage treatment of invasive candidiasis with voriconazole in 52 patients intolerant of other antifungal agents or with infection refractory to other antifungal agents were analyzed. Patients had received a mean of two previous antifungal agents (range, 1–4 agents), and 83% had received an azole. Manifestations of invasive candidiasis included candidemia (37%), disseminated disease (25%), and infection of other sites (38%). The median duration of voriconazole therapy was 60 days (range, 1–314 days). The overall rate of response was 56% (95%CI, 41–70), with the following response rates observed for individual Candida species: Candida albicans, 44% (20–70); Candida glabrata, 38% (14–68); Candida krusei, 70% (35–93); Candida tropicalis, 67% (30–93); and other Candida spp., 100% (40–100). The response rate in patients who had failed previous azole therapy was 58% (42–73). Common adverse events (~20%) included nausea and emesis, abnormal liver enzymes, and visual disturbances. Serious adverse events occurred in four patients, and nine patients died. Voriconazole has promise as a salvage agent for the treatment of invasive candidiasis, even in the settings of previous azole therapy and infection due to Candida krusei.

Pro-inflammatory cytokines in patients with essential hypertension

It has been suggested that abnormalities in the immune response play a role in the pathogenesis of essential hypertension (EH). The aim of this study was to assess circulating concentrations and ex vivo production of the pro‐inflammatory cytokines tumour necrosis factor‐α(TNF‐α) and interleukin‐1β (IL‐1β), and of the anti‐inflammatory cytokines IL‐1 receptor antagonist (IL‐1ra) and IL‐6 , in patients with EH and compare them with healthy volunteers.

The effect of renin–angiotensin system inhibitors on pro- and anti-inflammatory cytokine production

The balance between pro‐ and anti‐inflammatory cytokines has been implicated in the pathogenesis of infectious and auto‐immune diseases, and its modulation has been proposed as a potential therapeutic target. The results reported in the present study show that modulators of the renin–angiotensin system, such as the angiotensin‐converting enzyme (ACE)‐inhibitor captopril and the angiotensin II receptor type I antagonist valsartan, have potent inhibitory effects on the lipopolysaccharide (LPS)‐stimulated production of pro‐inflammatory cytokines tumour necrosis factor (TNF) and interleukin‐1 (IL‐1) in vitro. The anti‐inflammatory cytokine IL‐1Ra is increased by captopril, whereas IL‐6 production is decreased by valsartan. These effects are exerted mainly at high concentrations of the drugs. Administration of one dose of captopril or valsartan in therapeutic dosages to patients with essential hypertension did not influence LPS‐stimulated production of cytokines by whole blood. In conclusion, despite inhibitory effects on pro‐inflammatory cytokine production in vitro, it is unlikely that captopril or valsartan could be used in anticytokine therapeutic strategies in vivo.

Barriers to optimal antibiotic use for community-acquired pneumonia at hospitals: a qualitative study

Background: Physician adherence to key recommendations of guidelines for community-acquired pneumonia (CAP) is often not optimal. A better understanding of factors influencing optimal performance is needed to plan effective change. Methods: The authors used semistructured interviews with care providers in three Dutch medium-sized hospitals to qualitatively study and understand barriers to appropriate antibiotic use in patients with CAP. They discussed recommendations about the prescription of empirical antibiotic therapy that adheres to the guidelines, timely administration of antibiotics, adjusting antibiotic dosage to accommodate decreased renal function, switching and streamlining therapy, and blood and sputum culturing. The authors then classified the barriers each recommendation faced into categories using a conceptual framework (Cabana). Results: Eighteen interviews were performed with residents and specialists in pulmonology and internal medicine, with medical microbiologists and a clinical pharmacist. Two additional multidisciplinary small group interviews which included nurses were performed. Each guideline recommendation elicited a different type of barrier. Regarding the choice of guideline-adherent empirical therapy, treating physicians said that they worried about patient outcome when prescribing narrow-spectrum antibiotic therapy. Regarding the timeliness of antibiotic administration, barriers such as conflicting guidelines and organisational factors (for example, delayed laboratory results, antibiotics not directly available, lack of time) were reported. Not streamlining therapy after culture results became available was thought to be due to the physicians’ attitude of “never change a winning team”. Conclusions: Efforts to improve the use of antibiotics for patients with CAP should consider the range of barriers that care providers face. Each recommendation meets its own barriers. Interventions to improve adherence should be tailored to these factors.

Crystals of monosodium urate monohydrate enhance lipopolysaccharide-induced release of interleukin 1 beta by mononuclear cells through a caspase 1-mediated process.

OBJECTIVE Recent studies suggest that crystals of monosodium urate (MSU), deposited in joints of patients with acute gouty arthritis, activate the NACHT domain, leucine-rich repeat and pyrin domain-containing protein (NALP)3 inflammasome. In the present study we have investigated whether production of proinflammatory cytokines by crystals was exacerbated during costimulation with Toll-like receptor (TLR) ligands. METHODS Mononuclear cells of 22 healthy donors were stimulated by various concentrations of MSU crystals in the absence or presence of lipopolysaccharide (LPS), Pam3Cys and flagellin. Production of tumour necrosis factor alpha (TNFalpha), interleukin (IL)1 beta and IL6, as well as the intracellular concentrations of proIL1 beta were measured by ELISA. mRNA transcripts of TNFalpha and IL1 beta were assessed by real-time PCR. Stimulation experiments were also performed with peripheral blood mononuclear cells (PBMCs) of one patient carrying a NALP3 mutation. RESULTS MSU induced a moderate release of IL1 beta and IL6, but not of TNFalpha. Urate crystals amplified IL1 beta production stimulated by the TLR4 ligand LPS, while no synergy was apparent for IL6 production. In addition, no synergy between urate crystals and Pam3Cys (TLR2 ligand) or flagellin (TLR5 ligand) was apparent. The synergy between urate crystals and LPS was directed at the level of the NALP3 inflammasome, as it was present only when active IL1 beta was measured, but not at the level of IL1 mRNA or proIL1 beta. The synergy between LPS and MSU crystals ceased to exist in the presence of a caspase 1 inhibitor. CONCLUSIONS MSU crystals act in synergy with LPS for the induction of enhanced release of IL1 beta. Increased cleavage of proIL1 beta by urate-activated caspase 1 is proposed as the underlying mechanism.

Toll‐like receptor‐4 Asp299Gly polymorphism does not influence progression of atherosclerosis in patients with familial hypercholesterolaemia

Background  Toll‐like receptor‐4 (TLR4) is a major receptor for inflammatory stimuli potentially involved in the pathogenesis of atherosclerosis, such as lipopolysaccharide (LPS) and heat‐shock proteins. The Asp299Gly polymorphism of the TLR4 gene has been associated with a reduced intima‐media thickness (IMT) of the common carotid artery in healthy individuals. We have investigated whether the presence of the Asp299Gly polymorphism in patients with familial hypercholesterolaemia (FH) has a similar protective effect, and whether it influences the effects of HMG‐CoA reductase treatment.

Apolipoprotein E-deficient mice have an impaired immune response to Klebsiella pneumoniae.

All lipoproteins are able to bind to bacterial lipopolysaccharide (LPS), thereby neutralizing its deleterious effects. However, we demonstrated, recently, that in the absence of apolipoprotein E (apoE), eight‐fold increased very‐low‐density lipoprotein levels were not sufficient to protect apoE‐deficient (apoE−/−) mice against LPS. During a live Gram‐negative infection, mechanisms other than LPS‐neutralization may play a role in the pathogenesis of the disease. In the present study we further examined the role of apoE in Gram‐negative sepsis.