Treatment of patients with burn wound infections may become complicated by the presence of antibiotic resistant bacteria and biofilms. Herein, we demonstrate an in vitro thermal wound infection model using human skin equivalents (HSE) and biofilm-forming methicillin-resistant Staphylococcus aureus (MRSA) for the testing of agents to combat such infections. Application of a liquid nitrogen-cooled metal device on HSE produced reproducible wounds characterized by keratinocyte death, detachment of the epidermal layer from the dermis, and re-epithelialization. Thermal wounding was accompanied by up-regulation of markers for keratinocyte activation, inflammation, and antimicrobial responses. Exposure of thermal wounded HSEs to MRSA resulted in significant numbers of adherent MRSA/HSE after 1 hour, and multiplication of these bacteria over 24-48 hours. Exposure to MRSA enhanced expression of inflammatory mediators such as TLR2 (but not TLR3), IL-6 and IL-8, and antimicrobial proteins human β-defensin-2, -3 and RNAse7 by thermal wounded as compared to control HSEs. Moreover, locally applied mupirocin effectively reduced MRSA counts on (thermal wounded) HSEs by more than 99.9% within 24 hours. Together, these data indicate that this thermal wound infection model is a promising tool to study the initial phase of wound colonization and infection, and to assess local effects of candidate antimicrobial agents.
(See the editorial commentary by Bagni and Whitby, on pages 873–4.)
Background. Candidemia is a severe invasive fungal infection with high mortality. Recognition of Candida species is mediated through pattern recognition receptors such as Toll-like receptors (TLRs). This study assessed whether genetic variation in TLR signaling influences susceptibility to candidemia.
Methods. Thirteen mostly nonsynonymous single nucleotide polymorphisms (SNPs) in genes encoding TLRs and signaling adaptors MyD88 and Mal/TIRAP were genotyped in 338 patients (237 white, 93 African American, 8 other race) with candidemia and 351 noninfected controls (263 white, 88 African American). The SNPs significant in univariate analysis were further analyzed with multivariable logistic regression to determine association with clinical outcomes. Functional consequences of these polymorphisms were assessed via in vitro stimulation assays.
Results. Analyses of TLR SNPs revealed that 3 TLR1 SNPs (R80T, S248N, I602S) were significantly associated with candidemia susceptibility in whites. This association was not found in African Americans, likely due to lower power in this smaller study population. Furthermore, these TLR1 polymorphisms displayed impaired cytokine release by primary monocytes. No associations with susceptibility to candidemia were observed for SNPs in TLR2, TLR4, TLR6, TLR9, MyD88, or TIRAP.
Conclusions. Nonsynonymous SNPs in TLR1 are associated with impaired TLR1 function, decreased cytokine responses, and predisposition to candidemia in whites.
We assessed the role of genetic variation in cytokine and cytokine receptor genes in susceptibility and severity of bloodstream infections with Candida species, which revealed a major role for functional polymorphisms in interleukin-10 and interleukin-12p40 in predisposing to persistent fungemia.
Background. Candida bloodstream infections cause significant morbidity and mortality among hospitalized patients. Although clinical and microbiological factors affecting prognosis have been identified, the impact of genetic variation in the innate immune responses mediated by cytokines on outcomes of infection remains to be studied.
Methods. A cohort of 338 candidemia patients and 351 noninfected controls were genotyped for single-nucleotide polymorphisms (SNPs) in 6 cytokine genes (IFNG, IL10, IL12B, IL18, IL1β, IL8) and 1 cytokine receptor gene (IL12RB1). The association of SNPs with both candidemia susceptibility and outcome were assessed. Concentrations of pro- and antiinflammatory cytokines were measured in in vitro peripheral blood mononuclear cell stimulation assays and in serum from infected patients.
Results. None of the cytokine SNPs studied were associated with susceptibility to candidemia. Persistent fungemia occurred in 13% of cases. In the multivariable model, persistent candidemia was significantly associated with (odds ratio [95% confidence interval]): total parenteral nutrition (2.79 [1.26–6.17]), dialysis dependence (3.76 [1.46–8.64]), and the SNPs IL10 rs1800896 (3.45 [1.33–8.93]) and IL12B rs41292470 (5.36 [1.51–19.0]). In vitro production capacity of interleukin-10 and interferon-γ was influenced by these polymorphisms, and significantly lower proinflammatory cytokine concentrations were measured in serum from patients with persistent fungemia.
Conclusions. Polymorphisms in IL10 and IL12B that result in low production of proinflammatory cytokines are associated with persistent fungemia in candidemia patients. This provides insights for future targeted management strategies for patients with Candida bloodstream infections.
Palamaro et al. describe a child with recurrent bronchopneumonia and very high IgE levels in which a variation, R156H, was found in the IL12RB1 gene that encodes the IL-12Rβ1 chain. Based on the absence of this variation in 50 unrelated individuals they conclude it is a mutation. We (van de Vosse and van Dissel) feel there is no reason to suspect a defect in IL-12 signaling based on the clinical data, nor evidence for a functional defect in IL-12 signaling in this patient. In addition, the variation is not novel and known as a polymorphism. Without any functional evidence that R156H is a mutation, the current claim is not substantiated.
Palamaro et al. respond to argue that the amino acid substitution, R156H described in the described case exerts a summatory effect, as a genetic cofactor, along with an additional and still unidentified molecular alteration of the same pathway.
IL12RB1; IL-12Rβ1; Immunodeficiency; Mutation; Mycobacterial disease
Nine consecutive isogenic Aspergillus fumigatus isolates cultured from a patient with aspergilloma were investigated for azole resistance. The first cultured isolate showed a wild-type phenotype, but four azole-resistant phenotypes were observed in the subsequent eight isolates. Four mutations were found in the cyp51A gene of these isolates, leading to the substitutions A9T, G54E, P216L, and F219I. Only G54 substitutions were previously proved to be associated with azole resistance. Using a Cyp51A homology model and recombination experiments in which the mutations were introduced into a susceptible isolate, we show that the substitutions at codons P216 and F219 were both associated with resistance to itraconazole and posaconazole. A9T was also present in the wild-type isolate and thus considered a Cyp51A polymorphism. Isolates harboring F219I evolved further into a pan-azole-resistant phenotype, indicating an additional acquisition of a non-Cyp51A-mediated resistance mechanism. Review of the literature showed that in patients who develop azole resistance during therapy, multiple resistance mechanisms commonly emerge. Furthermore, the median time between the last cultured wild-type isolate and the first azole-resistant isolate was 4 months (range, 3 weeks to 23 months), indicating a rapid induction of resistance.
Measures had substantial effects on contacts and household members.
After an imported case of Marburg hemorrhagic fever was reported in 2008 in the Netherlands, control measures to prevent transmission were implemented. To evaluate consequences of these measures, we administered a structured questionnaire to 130 contacts classified as either having high-risk or low-risk exposure to body fluids of the case-patient; 77 (59.2%) of 130 contacts responded. A total of 67 (87.0%) of 77 respondents agreed that temperature monitoring and reporting was necessary, significantly more often among high-risk than low-risk contacts (p<0.001). Strict compliance with daily temperature monitoring decreased from 80.5% (62/77) during week 1 to 66.2% (51/77) during week 3. Contacts expressed concern about development of Marburg hemorrhagic fever (58.4%, 45/77) and infecting a family member (40.2%, 31/77). High-risk contacts had significantly higher scores on psychological impact scales (p<0.001) during and after the monitoring period. Public health authorities should specifically address consequences of control measures on the daily life of contacts.
retrospective evaluation; temperature monitoring; control measures; Marburg hemorrhagic fever; Marburg virus; viruses; contacts; daily life; the Netherlands
Background. Dectin-1 is the major receptor for fungal β-glucans on myeloid cells. We investigated whether defective Dectin-1 receptor function, because of the early stop codon polymorphism Y238X, enhances susceptibility to invasive aspergillosis (IA) in at-risk patients.
Methods. Association of Dectin-1 Y238X polymorphism with occurrence and clinical course of IA was evaluated in 71 patients who developed IA post hematopoietic stem cell transplantation (HSCT) and in another 21 non-HSCT patients with IA. The control group consisted of 108 patients who underwent HSCT. Functional studies were performed to investigate consequences of the Y238X Dectin-1 polymorphism.
Results. The Y238X allele frequency was higher in non-HSCT patients with IA (19.0% vs 6.9%–7.7%; P < .05). Heterozygosity for Y238X polymorphism in HSCT recipients showed a trend toward IA susceptibility (odds ratio, 1.79; 95% CI, .77–4.19; P = .17) but did not influence clinical course of IA. Functional assays revealed that although peripheral blood mononuclear cells with defective Dectin-1 function due to Y238X responded less efficiently to Aspergillus, corresponding macrophages showed adequate response to Aspergillus.
Conclusions. Dectin-1 Y238X heterozygosity has a limited influence on susceptibility to IA and may be important in susceptible non-HSCT patients. This is partly attributable to redundancy inherent in the innate immune system. Larger studies are needed to confirm these findings.
Urinary tract infection (UTI) encompasses a variety of clinical syndromes ranging from mild to life-threatening conditions. As such, it represents an interesting model for the development of an analytically based scoring system of disease severity and/or host response. Here we test the feasibility of this concept using 1H NMR based metabolomics as the analytical platform. Using an exhaustively clinically characterized cohort and taking advantage of the multi-level study design, which opens possibilities for case–control and longitudinal modeling, we were able to identify molecular discriminators that characterize UTI patients. Among those discriminators a number (e.g. acetate, trimethylamine and others) showed association with the degree of bacterial contamination of urine, whereas others, such as, for instance, scyllo-inositol and para-aminohippuric acid, are more likely to be the markers of morbidity.
Electronic supplementary material
The online version of this article (doi:10.1007/s11306-012-0411-y) contains supplementary material, which is available to authorized users.
Metabolomics; NMR; Data analysis; Clinical metabolomics; Urinary tract infection
Although febrile urinary tract infections (UTIs) are relatively common in adults, data on optimal treatment duration are limited. Randomized controlled trials specifically addressing the elderly and patients with comorbidities have not been performed. This review highlights current available evidence. Premenopausal, non-pregnant women without comorbidities can be treated with a 5–7 day regimen of fluoroquinolones in countries with low levels of fluoroquinolone resistance, or, if proven susceptible, with 14 days of trimethoprim-sulfamethoxazole. Oral β-lactams are less effective compared with fluoroquinolones and trimethoprim-sulfamethoxazole. In men with mild to moderate febrile UTI, a 2-week regimen of an oral fluoroquinolone is likely sufficient. Although data are limited, this possibly holds even in the elderly patients with comorbidities or bacteremia.
Febrile urinary tract infections; Acute pyelonephritis; Complicated urinary tract infection; Prostatitis; Antibiotics; Fluoroquinolones
We have analysed a family with nine congenital neutropenia patients in four generations, several of which we have studied in a long-term follow-up of over 25 years. The patients were mild to severe neutropenic and suffered from various recurrent bacterial infections. Mutations in the genes ELANE, CSF3R and GFI1 have been reported in patients with autosomal dominant congenital neutropenias. Using a small-scale linkage analysis with markers around the ELANE, CSF3R, CSF3 and GFI1 genes, we were able to determine that the disease segregated with markers around the ELANE gene. We identified a novel mutation in the ELANE gene in all of the affected family members that was not present in any of the healthy family members. The mutation leads to an A28S missense mutation in the mature protein. None of these patients developed leukaemia. This is the first truly multigenerational family with mutations in ELANE as unambiguous cause of severe congenital neutropenia SCN.
Severe congenital neutropenia; ELANE; Neutrophil elastase; Mutation; ELA2
The human lactoferrin-derived peptide hLF1-11 displays antimicrobial activities in vitro and is effective against infections with antibiotic-resistant bacteria and fluconazole-resistant Candida albicans in animals. However, the mechanisms underlying these activities remain largely unclear. Since hLF1-11 is ineffective in vitro at physiological salt concentrations, we suggested modulation of the immune system as an additional mechanism of action of the peptide. We investigated whether hLF1-11 affects human monocyte-macrophage differentiation and determined the antimicrobial activities of the resulting macrophages. Monocytes were cultured for 7 days with GM-CSF in the presence of hLF1-11, control peptide, or saline for various intervals. At day 6, the cells were stimulated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or heat-killed C. albicans for 24 h. Thereafter, the levels of cytokines in the culture supernatants, the expression of pathogen recognition receptors, and the antimicrobial activities of these macrophages were determined. The results showed that a short exposure of monocytes to hLF1-11 during GM-CSF-driven differentiation is sufficient to direct differentiation of monocytes toward a macrophage subset characterized by both pro- and anti-inflammatory cytokine production and increased responsiveness to microbial structures. Moreover, these macrophages are highly effective against C. albicans and Staphylococcus aureus. In conclusion, hLF1-11 directs GM-CSF-driven differentiation of monocytes toward macrophages with enhanced effector functions.
T-SPOT.TB is a specific assay for the diagnosis of tuberculosis. The assay needs to be performed with freshly isolated cells, and interpretation requires training. T-SPOT.TB has been used in various clinical-epidemiological settings, but so far no studies have evaluated the effect of interobserver variation in test reading. Our aim was to evaluate variation between different observers in reading T-SPOT.TB results. The study was nested within an ongoing cohort study, in which part of the T-SPOT.TB had been performed with frozen material. Culture plates were read visually by four different observers from two laboratories and by two automated readers. Of 313 T-SPOT.TB assays, 235 were performed with fresh cells and 78 were performed with frozen cells. No significant difference was found between results obtained with fresh cells and those obtained with frozen cells. The percentage of positive results varied between readers by maximally 15%; five/six raters were within a 6% difference in positive results. Analysis of the observed interrater differences showed that some individuals systematically counted more spots than others did. Because test interpretation includes subtraction of background values, this systematic variance had little influence on interindividual differences. The test result as positive or negative varied between independent raters, mainly due to samples with values around the cutoff. This warrants further study regarding determinants affecting the reading of T-SPOT.TB.
The type-1 cytokine pathway plays a pivotal role in immunity against intracellular bacterial pathogens such as Salmonellae and Mycobacteria. Bacterial stimulation of pattern recognition receptors on monocytes, macrophages and dendritic cells initiates this pathway, and results in the production of cytokines that activate lymphocytes to produce interferon (IFN)-γ. Interleukin (IL)-12 and IL-23 are thought to be the key cytokines required for initiating a type-1 cytokine immune response to Mycobacteria and Salmonellae. The relative contribution of IL-23 and IL-12 to this process is uncertain.
We show that various TLR agonists induce the production of IL-23 but not IL-12 in freshly isolated human monocytes and cultured human macrophages. In addition, type 1 pro-inflammatory macrophages (Mϕ1) differentiated in the presence of GM-CSF and infected with live Salmonella produce IL-23, IL-1β and IL-18, but not IL-12. Supernatants of Salmonella-infected Mϕ1 contained more IL-18 and IL-1β as compared with supernatants of Mϕ1 stimulated with isolated TLR agonists, and induced IFN-γ production in human CD56+ cells in an IL-23 and IL-1β-dependent but IL-12-independent manner. In addition, IL-23 together with IL-18 or IL-1β led to the production of GM-CSF in CD56+ cells. Both IFN-γ and GM-CSF enhanced IL-23 production by monocytes in response to TLR agonists, as well as induced IL-12 production.
The findings implicate a positive feedback loop in which IL-23 can enhance its release via induction of IFN-γ and GM-CSF. The IL-23 induced cytokines allow for the subsequent production of IL-12 and amplify the IFN-γ production in the type-1 cytokine pathway.
Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. Earlier we reported maggot secretions to inhibit pro-inflammatory responses of human monocytes. The aim of this study was to investigate the effect of maggot secretions on the differentiation of monocytes into pro-inflammatory (MØ-1) and anti-inflammatory/pro-angiogenic macrophages (MØ-2) as these cells play a central role in wound healing.
Freshly isolated monocytes were incubated with secretions and GM-CSF or M-CSF for 6 days and then stimulated with LPS or LTA for 18 h. The expression of cell surface molecules and the levels of cytokines, chemokines and growth factors in supernatants were measured. Our results showed secretions to affect monocyte-macrophage differentiation leading to MØ-1 with a partial MØ-2-like morphology but lacking CD163, which is characteristic for MØ-2. In response to LPS or LTA, secretions-differentiated MØ-1 produced less pro-inflammatory cytokines (TNF-α, IL-12p40 and MIF) than control cells. Similar results were observed for MØ-2 when stimulated with low concentrations of LPS. Furthermore, secretions dose-dependently led to MØ-1 and MØ-2 characterized by an altered chemokine production. Secretions led to MØ-2, but not MØ-1, producing enhanced levels of the growth factors bFGF and VEGF, as compared to control cells. The expression of cell-surface receptors involved in LPS/LTA was enhanced by secretions, that of CD86 and HLA-DR down-regulated, while receptors involved in phagocytosis remained largely unaffected.
Maggot secretions skew the differentiation of monocytes into macrophages away from a pro-inflammatory to a pro-angiogenic type.
Adventure tourism may bring this disease to Western countries.
On July 10, 2008, Marburg hemorrhagic fever was confirmed in a Dutch patient who had vacationed recently in Uganda. Exposure most likely occurred in the Python Cave (Maramagambo Forest), which harbors bat species that elsewhere in Africa have been found positive for Marburg virus. A multidisciplinary response team was convened to perform a structured risk assessment, perform risk classification of contacts, issue guidelines for follow-up, provide information, and monitor the crisis response. In total, 130 contacts were identified (66 classified as high risk and 64 as low risk) and monitored for 21 days after their last possible exposure. The case raised questions specific to international travel, postexposure prophylaxis for Marburg virus, and laboratory testing of contacts with fever. We present lessons learned and results of the follow-up serosurvey of contacts and focus on factors that prevented overreaction during an event with a high public health impact.
Marburg virus diseases; hemorrhagic fever; exposure; contacts; temperature monitoring; filovirus; viruses; perspective
To investigate whether protective immune responses can be induced in the absence of normal interleukin-12/23/gamma interferon (IL-12/23/IFN-γ) axis signaling, we vaccinated with the seasonal influenza virus subunit vaccine two patients with complete IL-12/23 receptor β1 (IL-12/23Rβ1) deficiencies, two patients with partial IFN-γ receptor I (pIFN-γRI) deficiencies, and five healthy controls. Blood samples were analyzed before, 7 days after, and 28 days after vaccination. In most cases, antibody titers reached protective levels. Moreover, although T-cell responses in patients were lower than those observed in controls, significant influenza virus-specific T-cell proliferation, IFN-γ production, and numbers of IFN-γ-producing cells were found in all patients 7 days after the vaccination. Interestingly, influenza virus-specific IFN-γ responses were IL-12/23 independent, in striking contrast to mycobacterium-induced IFN-γ production. In conclusion, influenza virus vaccination induces IL-12/23-independent IFN-γ production by T cells and can result in sufficient humoral protection in both IL-12/23Rβ1- and pIFN-γRI-deficient individuals.
High-throughput epidemiological typing systems that provide phylogenetic and genotypic information are beneficial for tracking bacterial pathogens in the field. The incidence of Salmonella enterica serovar Typhi infection in Indonesia is high and is associated with atypical phenotypic traits such as expression of the j and the z66 flagellum antigens. Utilizing a high-throughput genotyping platform to investigate known nucleotide polymorphisms dispersed around the genome, we determined the haplotypes of 140 serovar Typhi isolates associated with Indonesia. We identified nine distinct serovar Typhi haplotypes circulating in Indonesia for more than 30 years, with eight of these present in a single Jakarta suburb within a 2-year period. One dominant haplotype, H59, is associated with j and z66 flagellum expression, representing a potential pathotype unique to Indonesia. Phylogenetic analysis suggests that H59 z66+, j+ isolates emerged relatively recently in terms of the origin of serovar Typhi and are geographically restricted. These data demonstrate the potential of high-throughput genotyping platforms for analyzing serovar Typhi populations in the field. The study also provides insight into the evolution of serovar Typhi and demonstrates the value of a molecular epidemiological technique that is exchangeable, that is internet friendly, and that has global utility.
Experimental models show that apolipoprotein CI (apoCI) binds and enhances the inflammatory response to endotoxin. We studied in patients undergoing cardiopulmonary bypass surgery (CPB) and experiencing endotoxemia during reperfusion whether plasma apoCI levels correlate with the inflammatory response and perioperative cytokine release.
Prospective, observational, clinical cohort study.
Operating room (OR) and intensive care unit (ICU) of a university hospital.
One hundred fifty-nine consecutive patients > 18 years of age (66% males (n = 105), median age 65 and 67 years for males and females, respectively) undergoing elective cardiothoracic surgery with cardiopulmonary bypass.
Baseline apoCI, apoCIII, total cholesterol and triglyceride levels, and perioperative endotoxin and TNF-α levels were determined.
High preoperative plasma apoCI, but not apoCIII, levels were associated (p < 0.05) with increased perioperative levels of TNF-α in patients experiencing endotoxemia. This association was not observed in patients without endotoxemia.
High plasma apoCI is positively related to proinflammatory response in patients experiencing endotoxemia and confirms the observations in animal models.
Cardiopulmonary bypass; SIRS; Endotoxemia; Endotoxin; ApoCI; TNF-α
Implementation of yellow fever vaccination is currently hampered by limited supply of vaccine. An alternative route of administration with reduced amounts of vaccine but without loss of vaccine efficacy would boost vaccination programmes.
Methods and Findings
A randomized, controlled, non-inferiority trial was conducted in a Dutch university center between August 2005 and February 2007. A total of 155 primary vaccinated and 20 previously vaccinated volunteers participated. Participants were randomly assigned in a 1∶1 ratio to receive intradermal (i.d.) vaccination with live attenuated yellow fever 17D vaccine at a reduced dose (1/5th; 0·1 mL) or the conventional subcutaneous (s.c.) vaccination (0·5 mL). Antibody neutralization titers were determined at 2, 4 and 8 weeks and 1 year after vaccination by counting the reduction in virus-induced plaques in the presence of serial serum dilutions. Adverse events were documented in a 3-week dairy. Viraemia was measured 5 days after vaccination. From 2 weeks up to one year after vaccination, the maximum serum-dilution at which 80% of the virus plaques were neutralized, which indicates protection against yellow fever, did not differ between those given a reduced i.d. dose or standard s.c. dose of vaccine. In all cases the WHO standard of seroprotection (i.e. 80% virus neutralization) was reached (in 77/77 and 78/78, respectively). Similar results were found in the previously vaccinated individuals. Viraemia was detected in half of the primary vaccinated participants, which was not predictive of serological response. In revaccinees no viraemia was detected.
Intradermal administration of one fifth of the amount of yellow fever vaccine administered subcutaneously results in protective seroimmunity in all volunteers. Albeit this vaccination route should enable vaccination of five-times as many individuals at risk for disease, these results should now be confirmed in field studies in areas with potential yellow fever virus transmission to change vaccination policy.
Nederlands Trial Register ISRCTN46326316
We report a follow-up study of 29 subjects with negative tuberculin skin test (TST) results in association with positive gamma interferon release assay (IGRA) results, mainly due to responses to CFP-10 in the T-SPOT.TB assay, during a contact investigation. One year later, 12/29 subjects (41%) had converted to positive TST results in association with negative IGRA results.
The sensitivities of various gamma interferon release assays (IGRAs) for the detection of past latent Mycobacterium tuberculosis infection are not known. In this study, we aimed to assess the effects of various IGRA formats and in vitro incubation periods on test outcome. The results of the tuberculin skin test (TST) were compared with those of the QuantiFERON-TB Gold in-tube (QFT-GIT) test, an overnight enzyme-linked immunospot assay (ELISPOT), and a 6-day lymphocyte stimulation test (LST) by using the same M. tuberculosis-specific peptides and samples from 27 TST-positive persons with a history of exposure to M. tuberculosis, 4 patients cured of tuberculosis (TB), and 9 TST-negative controls. Among the TST-positive persons, the LST was more frequently positive (92%; P < 0.01) than either the QFT-GIT test (33%) or ELISPOT (46%). While good agreement was observed between the QFT-GIT test and ELISPOT (κ = 0.71) and between TST and LST (κ = 0.78), the agreement between TST or LST, on the one hand, and the QFT-GIT test or ELISPOT, on the other, was poor. These data indicate that the QFT-GIT test and overnight ELISPOT are less sensitive for the detection of past latent TB than the 6-day LST. The observed discrepancies between these IGRAs are most likely related to differences in incubation periods. Whether TST-positive persons with positive LST results but negative QFT-GIT and ELISPOT results are at risk for the development of TB needs to be elucidated before short-incubation IGRAs can be used for the screening of individuals for latent TB before immunosuppressive treatment.
The uptake and subsequent killing of Salmonella enterica serovar Typhimurium by human neutrophils was studied. In particular, two pattern recognition receptors, complement receptor 3 (CR3) and Toll-like receptor 4 (TLR4), were found to be essential for the efficient uptake and activation, respectively, of the NADPH oxidase. The uptake of Salmonella was almost completely inhibited by various monoclonal antibodies against CR3, and neutrophils from a patient with leukocyte adhesion deficiency type 1, which lack CR3, showed almost no uptake of Salmonella. A lipopolysaccharide (LPS) mutant strain of Salmonella was used to show that the expression of full-length, wild-type, or so-called smooth LPS is important for the efficient killing of intracellular Salmonella. Infection with wild-type-LPS-expressing Salmonella resulted in the generation of reactive oxygen species (ROS) in TLR4-decorated, Salmonella-containing vacuoles, whereas ROS were not induced by an LPS mutant strain. In addition, the recognition of Salmonella by neutrophils, leading to ROS production, was shown to be intracellular, as determined by priming experiments with intact bacteria under conditions where the bacterium is not taken up. Finally, the generation of ROS in the wild-type-Salmonella-infected neutrophils was largely inhibited by the action of a TLR4-blocking, cell-permeable peptide, showing that signaling by this receptor from the Salmonella-containing vacuole is essential for the activation of the NADPH oxidase. In sum, our data identify the sequential recognition of unopsonized Salmonella strains by CR3 and TLR4 as essential events in the efficient uptake and killing of this intracellular pathogen.
The tuberculin skin test (TST) was compared with QuantiFERON-TB Gold in-tube (QFT-GIT) test for the diagnosis of tuberculosis in non-Mycobacterium bovis BCG-vaccinated military personnel. Among subjects positive by TST, 44.4% of recruits were positive by QFT-GIT compared with 11.5% subjects tested after missions abroad, suggesting that most TST conversions in the latter group were caused by nontuberculous mycobacteria.