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1.  Expression and Function of S100A8/A9 (Calprotectin) in Human Typhoid Fever and the Murine Salmonella Model 
PLoS Neglected Tropical Diseases  2015;9(4):e0003663.
Typhoid fever, caused by the Gram-negative bacterium Salmonella enterica serovar Typhi, is a major cause of community-acquired bacteremia and death worldwide. S100A8 (MRP8) and S100A9 (MRP14) form bioactive antimicrobial heterodimers (calprotectin) that can activate Toll-like receptor 4, promoting lethal, endotoxin-induced shock and multi-organ failure. We aimed to characterize the expression and function of S100A8/A9 in patients with typhoid fever and in a murine invasive Salmonella model.
Methods and principal findings
S100A8/A9 protein levels were determined in acute phase plasma or feces from 28 Bangladeshi patients, and convalescent phase plasma from 60 Indonesian patients with blood culture or PCR-confirmed typhoid fever, and compared to 98 healthy control subjects. To functionally characterize the role of S100A8/A9, we challenged wildtype (WT) and S100A9-/- mice with S. Typhimurium and determined bacterial loads and inflammation 2- and 5- days post infection. We further assessed the antimicrobial function of recombinant S100A8/A9 on S. Typhimurium and S. Typhi replication in vitro. Typhoid fever patients demonstrated a marked increase of S100A8/A9 in acute phase plasma and feces and this increases correlated with duration of fever prior to admission. S100A8/A9 directly inhibited the growth of S. Typhimurium and S. Typhi in vitro in a dose and time dependent fashion. WT mice inoculated with S. Typhimurium showed increased levels of S100A8/A9 in both the liver and the systemic compartment but S100A9-/- mice were indistinguishable from WT mice with respect to bacterial growth, survival, and inflammatory responses, as determined by cytokine release, histopathology and organ injury.
S100A8/A9 is markedly elevated in human typhoid, correlates with duration of fever prior to admission and directly inhibits the growth of S. Typhimurium and S. Typhi in vitro. Despite elevated levels in the murine invasive Salmonella model, S100A8/A9 does not contribute to an effective host response against S. Typhimurium in mice.
Author Summary
Bacterial pathogens are recognized by the host upon infection through interactions between their virulence factors and host cell receptors leading to the activation and recruitment of innate immune cells. Salmonella Typhi, the etiologic agent for typhoid fever, however harbors a number of factors, such as a polysaccharide capsule, which prevent the detection of these virulence factors, and thereby dampens the innate host response. Besides bacterial virulence factors, the host can detect endogenous danger molecules which are released upon tissue damage. S100A8/A9, an extracellular protein complex, is such a danger signal that is able to further amplify the systemic inflammatory response upon infection. In the present study we investigated the role of S100A8/A9 during invasive Salmonella infection and observed a marked increase of this protein in patients with typhoid fever, which correlates with disease stage and severity. Furthermore we found that S100A8/A9 directly inhibited the growth of Salmonella species in vitro thereby functioning as an antimicrobial. When mice were infected with Salmonella, the levels of S100A8/A9 were also elevated but mice lacking this protein did not have an altered host response to infection. The role and importance of the elevated levels of S100A8/A9 in human typhoid fever requires further study.
PMCID: PMC4393299  PMID: 25860480
2.  Urinary Proteins, Vitamin D and Genetic Polymorphisms as Risk Factors for Febrile Urinary Tract Infection and Relation with Bacteremia: A Case Control Study 
PLoS ONE  2015;10(3):e0121302.
Febrile urinary tract infection (UTI) is a common bacterial disease that may lead to substantial morbidity and mortality especially among the elderly. Little is known about biomarkers that predict a complicated course. Our aim was to determine the role of certain urinary cytokines or antimicrobial proteins, plasma vitamin D level, and genetic variation in host defense of febrile UTI and its relation with bacteremia.
A case-control study. Out of a cohort of consecutive adults with febrile UTI (n = 787) included in a multi-center observational cohort study, 46 cases with bacteremic E.coli UTI and 45 cases with non-bacteremic E.coli UTI were randomly selected and compared to 46 controls. Urinary IL-6, IL-8, LL37, β-defensin 2 and uromodulin as well as plasma 25-hydroxyvitamin D were measured. In 440 controls and 707 UTI patients polymorphisms were genotyped in the genes CXCR1, DEFA4, DEFB1, IL6, IL8, MYD88, UMOD, TIRAP, TLR1, TLR2, TLR5 and TNF.
IL-6, IL-8, and LL37 are different between controls and UTI patients, although these proteins do not distinguish between patients with and without bacteremia. While uromodulin did not differ between groups, inability to produce uromodulin is more common in patients with bacteremia. Most participants in the study, including the controls, had insufficient vitamin D and, at least in winter, UTI patients have lower vitamin D than controls. Associations were found between the CC genotype of IL6 SNP rs1800795 and occurrence of bacteremia and between TLR5 SNP rs5744168 and protection from UTI. The rare GG genotype of IL6 SNP rs1800795 was associated with higher β-defensin 2 production.
Although no biomarker was able to distinguish between UTI with or without bacteremia, two risk factors for bacteremia were identified. These were inability to produce uromodulin and an IL6 rs1800795 genotype.
PMCID: PMC4373833  PMID: 25807366
3.  LL-37-Derived Peptides Eradicate Multidrug-Resistant Staphylococcus aureus from Thermally Wounded Human Skin Equivalents 
Burn wound infections are often difficult to treat due to the presence of multidrug-resistant bacterial strains and biofilms. Currently, mupirocin is used to eradicate methicillin-resistant Staphylococcus aureus (MRSA) from colonized persons; however, mupirocin resistance is also emerging. Since we consider antimicrobial peptides to be promising candidates for the development of novel anti-infective agents, we studied the antibacterial activities of a set of synthetic peptides against different strains of S. aureus, including mupirocin-resistant MRSA strains. The peptides were derived from P60.4Ac, a peptide based on the human cathelicidin LL-37. The results showed that peptide 10 (P10) was the only peptide more efficient than P60.4Ac, which is better than LL-37, in killing MRSA strain LUH14616. All three peptides displayed good antibiofilm activities. However, both P10 and P60.4Ac were more efficient than LL-37 in eliminating biofilm-associated bacteria. No toxic effects of these three peptides on human epidermal models were detected, as observed morphologically and by staining for mitochondrial activity. In addition, P60.4Ac and P10, but not LL-37, eradicated MRSA LUH14616 and the mupirocin-resistant MRSA strain LUH15051 from thermally wounded human skin equivalents (HSE). Interestingly, P60.4Ac and P10, but not mupirocin, eradicated LUH15051 from the HSEs. None of the peptides affected the excretion of interleukin 8 (IL-8) by thermally wounded HSEs upon MRSA exposure. In conclusion, the synthetic peptides P60.4Ac and P10 appear to be attractive candidates for the development of novel local therapies to treat patients with burn wounds infected with multidrug-resistant bacteria.
PMCID: PMC4136056  PMID: 24841266
4.  The Hd, Hj, and Hz66 flagella variants of Salmonella enterica serovar Typhi modify host responses and cellular interactions 
Scientific Reports  2015;5:7947.
Salmonella Typhi, the causative agent of typhoid fever, is a monophyletic, human-restricted bacterium that exhibits limited phenotypic variation. S. Typhi from Indonesia are a notable exception, with circulating strains expressing diverse flagella antigens including Hj, Hd and Hz66. Hypothesizing that S. Typhi flagella plays a key role during infection, we constructed an S. Typhi fliC mutant and otherwise isogenic S. Typhi strains expressing the Hj, Hd, Hz66 flagella antigens. Phenotyping revealed differences in flagellum structure, strain motility and immunogenicity, but not in the ability of flagellated isolates to induce TLR5 activity. Invasion assays using epithelial and macrophage cell lines revealed differences in the ability of these S. Typhi derivatives to invade cells or induce cellular restructuring in the form of ruffles. Notably, the Hj variant induced substantial ruffles that were not fully dependent on the GTPases that contribute to this process. These data highlight important differences in the phenotypic properties of S. Typhi flagella variation and how they impact on the pathogenesis of S. Typhi.
PMCID: PMC4302301  PMID: 25609312
6.  IL-12Rβ1 deficiency: mutation update and description of the IL12RB1 variation database 
Human mutation  2013;34(10):1329-1339.
IL-12Rβ1 deficiency is an autosomal recessive disorder characterized by predisposition to recurrent and/or severe infections caused by otherwise poorly pathogenic mycobacteria and salmonella. IL-12Rβ1 is a receptor chain of both the IL-12 and the IL-23 receptor and deficiency of IL-12Rβ1 thus abolishes both IL-12 and IL-23 signaling. IL-12Rβ1 deficiency is caused by bi-allelic mutations in the IL12RB1 gene. Mutations resulting in premature stop codons, such as nonsense, frame shift, and splice site mutations, represent the majority of IL-12Rβ1 deficiency causing mutations (66%; 46/70). Also every other morbid mutation completely inactivates the IL-12Rβ1 protein. In addition to disease-causing mutations, rare and common variations with unknown functional effect have been reported in IL12RB1. All these variants have been deposited in the online IL12RB1 variation database ( In this article, we review the function of IL-12Rβ1 and molecular genetics of human IL12RB1.
PMCID: PMC4104692  PMID: 23864330
IL12RB1; IL-12Rβ1 deficiency; Mendelian Susceptibility to Mycobacterial Disease
7.  Limited role for ASC and NLRP3 during in vivo Salmonella Typhimurium infection 
BMC Immunology  2014;15:30.
The inflammasome is an intracellular protein complex triggered by exposure to intracellular pathogens, its components or other endogenous proteins. It leads to the activation of and subsequent release of proinflammatory cytokines such as IL-1β and IL-18. S. Typhimurium is a Gram-negative intracellular bacterium, which is known to trigger inflammasome assembly via recognition by the cytosolic receptors, NLRP3 and NLRC4 (which act via the adaptor protein, ASC) to induce cell death and cytokine release. We sought to characterize the role of ASC and NLRP3 in two different murine models (typhoid and colitis) of systemic Salmonella infection.
Release of the inflammasome cytokine IL-18 was hampered in Asc−/− but not Nlrp3−/− mice (background C57BL/6) during S. Typhimurium infection. Unexpectedly, neither ASC nor NLRP3 played a significant role in host defense against S. Typhimurium infection, as reflected by equal bacterial counts in WT, Asc−/− and Nlrp3−/− mice at all time points, in both the typhoid and colitis models. Proinflammatory cytokine levels (TNF-α, IL-6) and the extent of hepatic and splenic pathology did not differ between groups in the typhoid model. In the colitis model small differences were seen with regard to splenic and hepatic inflammation, although this was IL-18 independent.
IL-18 release was reduced in Asc−/− but not Nlrp3−/− mice during S. Typhimurium infection. Despite this reduction, bacterial counts, cytokine levels and histological inflammation did not differ between wild-type and knockout mice in either model. Our results reveal a limited role for ASC and NLRP3 during in vivo S. Typhimurium infection despite its role in cytokine maturation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12865-014-0030-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4243774  PMID: 25115174
Inflammasomes; Salmonella Typhimurium; Host-pathogen interactions
8.  A Novel Serine Protease Secreted by Medicinal Maggots Enhances Plasminogen Activator-Induced Fibrinolysis 
PLoS ONE  2014;9(3):e92096.
Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. As haemostatic processes play an important role in wound healing, this study focused on the effects of maggot secretions on coagulation and fibrinolysis. The results showed that maggot secretions enhance plasminogen activator-induced formation of plasmin and fibrinolysis in a dose- and time-dependent manner. By contrast, coagulation was not affected by secretions. Biochemical studies indicated that a novel serine protease within secretions, designated Sericase, cleaved plasminogen to several fragments. Recombinant Sericase degraded plasminogen leading amongst others to the formation of the mini-plasminogen like fragment Val454-plasminogen. In addition, the presence of a non-proteolytic cofactor in secretions was discovered, which plays a role in the enhancement of plasminogen activator-induced fibrinolysis by Sericase. We conclude from our in vitro studies that the novel serine protease Sericase, with the aid of a non-proteolytic cofactor, enhances plasminogen activator-induced fibrinolysis.
PMCID: PMC3960166  PMID: 24647546
9.  Inflammatory and Antimicrobial Responses to Methicillin-Resistant Staphylococcus aureus in an In Vitro Wound Infection Model 
PLoS ONE  2013;8(12):e82800.
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.
PMCID: PMC3858326  PMID: 24340061
10.  Toll-like Receptor 1 Polymorphisms Increase Susceptibility to Candidemia 
The Journal of Infectious Diseases  2012;205(6):934-943.
(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.
PMCID: PMC3282566  PMID: 22301633
11.  Cytokine Gene Polymorphisms and the Outcome of Invasive Candidiasis: A Prospective Cohort Study 
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.
PMCID: PMC3269308  PMID: 22144535
12.  The R156H variation in IL-12Rβ1 is not a mutation 
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.
PMCID: PMC3584738  PMID: 23410226
IL12RB1; IL-12Rβ1; Immunodeficiency; Mutation; Mycobacterial disease
13.  Rapid Induction of Multiple Resistance Mechanisms in Aspergillus fumigatus during Azole Therapy: a Case Study and Review of the Literature 
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.
PMCID: PMC3256077  PMID: 22005994
14.  Retrospective Evaluation of Control Measures for Contacts of Patient with Marburg Hemorrhagic Fever 
Emerging Infectious Diseases  2012;18(7):1107-1114.
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.
PMCID: PMC3376788  PMID: 22710186
retrospective evaluation; temperature monitoring; control measures; Marburg hemorrhagic fever; Marburg virus; viruses; contacts; daily life; the Netherlands
15.  The Y238X Stop Codon Polymorphism in the Human β-Glucan Receptor Dectin-1 and Susceptibility to Invasive Aspergillosis 
The Journal of Infectious Diseases  2011;203(5):736-743.
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.
PMCID: PMC3072717  PMID: 21242599
16.  1H NMR-based metabolic profiling of urinary tract infection: combining multiple statistical models and clinical data 
Metabolomics  2012;8(6):1227-1235.
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.
PMCID: PMC3483096  PMID: 23136561
Metabolomics; NMR; Data analysis; Clinical metabolomics; Urinary tract infection
17.  Elderly Subjects Have a Delayed Antibody Response and Prolonged Viraemia following Yellow Fever Vaccination: A Prospective Controlled Cohort Study 
PLoS ONE  2011;6(12):e27753.
Yellow fever vaccination (YF-17D) can cause serious adverse events (SAEs). The mechanism of these SAEs is poorly understood. Older age has been identified as a risk factor. We tested the hypothesis that the humoral immune response to yellow fever vaccine develops more slowly in elderly than in younger subjects.
We vaccinated young volunteers (18–28 yrs, N = 30) and elderly travelers (60–81 yrs, N = 28) with YF-17D and measured their neutralizing antibody titers and plasma YF-17D RNA copy numbers before vaccination and 3, 5, 10, 14 and 28 days after vaccination.
Ten days after vaccination seroprotection was attained by 77% (23/30) of the young participants and by 50% (14/28) of the elderly participants (p = 0.03). Accordingly, the Geometric Mean Titer of younger participants was higher than the GMT of the elderly participants. At day 10 the difference was +2.9 IU/ml (95% CI 1.8–4.7, p = 0.00004) and at day 14 +1.8 IU/ml (95% CI 1.1–2.9, p = 0.02, using a mixed linear model. Viraemia was more common in the elderly (86%, 24/28) than in the younger participants (60%, 14/30) (p = 0.03) with higher YF-17D RNA copy numbers in the elderly participants.
We found that elderly subjects had a delayed antibody response and higher viraemia levels after yellow fever primovaccination. We postulate that with older age, a weaker immune response to yellow fever vaccine allows the attenuated virus to cause higher viraemia levels which may increase the risk of developing SAEs. This may be one piece in the puzzle of the pathophysiology of YEL-AVD.
Trial Registration NTR1040
PMCID: PMC3233541  PMID: 22163273
18.  Treatment Duration of Febrile Urinary Tract Infections 
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.
PMCID: PMC3207126  PMID: 21882085
Febrile urinary tract infections; Acute pyelonephritis; Complicated urinary tract infection; Prostatitis; Antibiotics; Fluoroquinolones
19.  Influence of Polymorphisms in Innate Immunity Genes on Susceptibility to Invasive Aspergillosis after Stem Cell Transplantation 
PLoS ONE  2011;6(4):e18403.
The innate immune system plays a pivotal role in the primary defence against invasive fungal infection. Genetic variation in genes that regulate this response, initiated by pulmonary macrophages, may influence susceptibility to invasive aspergillosis in patients at risk. We investigated in a clinical setting whether common polymorphisms in Toll-like receptor (TLR) and cytokine genes involved in macrophage regulation are associated with susceptibility to invasive aspergillosis. Forty-four allogeneic stem cell transplantation recipients diagnosed with probable or proven IA according to the criteria of the European Organization for Research and Treatment of Cancer/Mycoses Study Group, were enrolled. The control group consisted of 64 allogeneic stem cell transplantation recipients without invasive aspergillosis. The TLR4 1063A>G single nucleotide polymorphism was associated with invasive aspergillosis when present in donors of allogeneic stem cell transplantation recipients (unadjusted OR 3.77 95%CI 1.08–13.2, p = 0.03). In a multivariate analysis, adjusted for occurrence of graft-versus-host-disease, Cytomegalovirus serostatus and duration of neutropenia, paired presence of the TLR4 1063A>G and IFNG 874T>A single nucleotide polymorphisms showed a trend towards increased susceptibility to invasive aspergillosis (p = 0.04). These findings point to the relevant immunological pathway involved in resistance to invasive aspergillosis and warrant further study of the effects of TLR and cytokine polymorphisms and their interaction, which may occur on different levels of the complex biological interplay between the immunocompromised host and Aspergillus sp.
PMCID: PMC3070725  PMID: 21483748
20.  Procalcitonin reflects bacteremia and bacterial load in urosepsis syndrome: a prospective observational study 
Critical Care  2010;14(6):R206.
Guidelines recommend that two blood cultures be performed in patients with febrile urinary tract infection (UTI), to detect bacteremia and help diagnose urosepsis. The usefulness and cost-effectiveness of this practice have been criticized. This study aimed to evaluate clinical characteristics and the biomarker procalcitonin (PCT) as an aid in predicting bacteremia.
A prospective observational multicenter cohort study included consecutive adults with febrile UTI in 35 primary care units and 8 emergency departments of 7 regional hospitals. Clinical and microbiological data were collected and PCT and time to positivity (TTP) of blood culture were measured.
Of 581 evaluable patients, 136 (23%) had bacteremia. The median age was 66 years (interquartile range 46 to 78 years) and 219 (38%) were male. We evaluated three different models: a clinical model including seven bed-side characteristics, the clinical model plus PCT, and a PCT only model. The diagnostic abilities of these models as reflected by area under the curve of the receiver operating characteristic were 0.71 (95% confidence interval (CI): 0.66 to 0.76), 0.79 (95% CI: 0.75 to 0.83) and 0.73 (95% CI: 0.68 to 0.77) respectively. Calculating corresponding sensitivity and specificity for the presence of bacteremia after each step of adding a significant predictor in the model yielded that the PCT > 0.25 μg/l only model had the best diagnostic performance (sensitivity 0.95; 95% CI: 0.89 to 0.98, specificity 0.50; 95% CI: 0.46 to 0.55). Using PCT as a single decision tool, this would result in 40% fewer blood cultures being taken, while still identifying 94 to 99% of patients with bacteremia.
The TTP of E. coli positive blood cultures was linearly correlated with the PCT log value; the higher the PCT the shorter the TTP (R2 = 0.278, P = 0.007).
PCT accurately predicts the presence of bacteremia and bacterial load in patients with febrile UTI. This may be a helpful biomarker to limit use of blood culture resources.
PMCID: PMC3220019  PMID: 21083886
21.  Severe congenital neutropenia in a multigenerational family with a novel neutrophil elastase (ELANE) mutation 
Annals of Hematology  2010;90(2):151-158.
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.
PMCID: PMC3018258  PMID: 20803142
Severe congenital neutropenia; ELANE; Neutrophil elastase; Mutation; ELA2
22.  Antimicrobial Peptide hLF1-11 Directs Granulocyte-Macrophage Colony-Stimulating Factor-Driven Monocyte Differentiation toward Macrophages with Enhanced Recognition and Clearance of Pathogens ▿  
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.
PMCID: PMC2812139  PMID: 19933796
24.  Variation in T-SPOT.TB Spot Interpretation between Independent Observers from Different Laboratories▿  
Clinical and Vaccine Immunology : CVI  2009;16(10):1439-1442.
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.
PMCID: PMC2756839  PMID: 19710293
25.  Salmonella Induced IL-23 and IL-1β Allow for IL-12 Production by Monocytes and Mϕ1 through Induction of IFN-γ in CD56+ NK/NK-Like T Cells 
PLoS ONE  2009;4(12):e8396.
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.
Methodology/Principal Findings
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.
PMCID: PMC2791865  PMID: 20027291

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