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Background: Chronic Q fever is a zoonosis caused by the bacterium Coxiella burnetii which can manifest as infection of an abdominal aortic aneurysm (AAA). Antibiotic therapy often fails, resulting in severe morbidity and high mortality. Whereas previous studies have focused on inflammatory processes in blood, the aim of this study was to investigate local inflammation in aortic tissue., Methods: Multiplex immunohistochemistry was used to investigate local inflammation in Q fever AAAs compared to atherosclerotic AAAs in aorta tissue specimen. Two six-plex panels were used to study both the innate and adaptive immune systems., Results: Q fever AAAs and atherosclerotic AAAs contained similar numbers of CD68 + macrophages and CD3 + T cells. However, in Q fever AAAs, the number of CD68 + CD206 + M2 macrophages was increased, while expression of GM-CSF was decreased compared to atherosclerotic AAAs. Furthermore, Q fever AAAs showed an increase in both the number of CD8 + cytotoxic T cells and CD3 + CD8 - FoxP3 + regulatory T cells. Finally, Q fever AAAs did not contain any well-defined granulomas., Conclusions: These findings demonstrate that despite the presence of pro-inflammatory effector cells, persistent local infection with C. burnetii is associated with an immune-suppressed microenvironment., Funding: This work was supported by SCAN consortium: European Research Area - CardioVascualar Diseases (ERA-CVD) grant [JTC2017-044] and TTW-NWO open technology grant [STW-14716]., Competing Interests: KC, AS, TS, MG, LV, AJ, PP, RV, PW, CB, MS, KH, Mv, JD, RV No competing interests declared, JV Senior editor, eLife, (© 2022, Cortenbach et al.)

Objective: Inborn errors of immunity (IEI) are a heterogeneous group of disorders, affecting different components of the immune system. Over 450 IEI related genes have been identified, with new genes continually being recognized. This makes the early application of next-generation sequencing (NGS) as a diagnostic method in the evaluation of IEI a promising development. We aimed to provide an overview of the diagnostic yield and time to diagnosis in a cohort of patients suspected of IEI and evaluated by an NGS based IEI panel early in the diagnostic trajectory in a multicenter setting in the Netherlands., Study Design: We performed a prospective observational cohort study. We collected data of 165 patients with a clinical suspicion of IEI without prior NGS based panel evaluation that were referred for early NGS using a uniform IEI gene panel. The diagnostic yield was assessed in terms of definitive genetic diagnoses, inconclusive diagnoses and patients without abnormalities in the IEI gene panel. We also assessed time to diagnosis and clinical implications., Results: For children, the median time from first consultation to diagnosis was 119 days versus 124 days for adult patients (U=2323; p=0.644). The median turn-around time (TAT) of genetic testing was 56 days in pediatric patients and 60 days in adult patients (U=1892; p=0.191). A definitive molecular diagnosis was made in 25/65 (24.6%) of pediatric patients and 9/100 (9%) of adults. Most diagnosed disorders were identified in the categories of immune dysregulation (n=10/25; 40%), antibody deficiencies (n=5/25; 20%), and phagocyte diseases (n=5/25; 20%). Inconclusive outcomes were found in 76/165 (46.1%) patients. Within the patient group with a genetic diagnosis, a change in disease management occurred in 76% of patients., Conclusion: In this cohort, the highest yields of NGS based evaluation for IEI early in the diagnostic trajectory were found in pediatric patients, and in the disease categories immune dysregulation and phagocyte diseases. In cases where a definitive diagnosis was made, this led to important disease management implications in a large majority of patients. More research is needed to establish a uniform diagnostic pathway for cases with inconclusive diagnoses, including variants of unknown significance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Elsink, Huibers, Hollink, Simons, Zonneveld-Huijssoon, van der Veken, Leavis, Henriet, van Deuren, van de Veerdonk, Potjewijd, Berghuis, Dalm, Vermont, van de Ven, Lambeck, Abbott, van Hagen, de Bree, Kuijpers, Frederix, van Gijn, van Montfrans and the Genetics First for Primary Immunodeficiency Disorders Consortium.)

Bloom Syndrome (BS) is a genetic DNA repair disorder, caused by mutations in the BLM gene. The clinical phenotype includes growth retardation, immunodeficiency and a strong predisposition to different types of malignancies. Treatment of malignancies in BS patients with radiotherapy or chemotherapy is believed to be associated with increased toxicity, but clinical and laboratory data are lacking. We collected clinical data of two Dutch BS patients with solid tumors. Both were treated with radiotherapy before the diagnosis BS was made and tolerated this treatment well. In addition, we collected fibroblasts from BS patients to perform in vitro clonogenic survival assays to determine radiosensitivity. BS fibroblasts showed less radiosensitivity than the severely radiosensitive Artemis fibroblasts. Moreover, studies of double strand break kinetics by counting 53BP1 foci after irradiation showed similar patterns compared to healthy controls. In combination, the clinical cases and laboratory experiments are valuable information in the discussion whether radiotherapy is absolutely contraindicated in BS, which is the Case in other DNA repair syndromes like Ataxia Telangiectasia and Artemis., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The clinical spectrum of COVID-19 varies and the differences in host response characterizing this variation have not been fully elucidated. COVID-19 disease severity correlates with an excessive proinflammatory immune response and profound lymphopenia. Inflammatory responses according to disease severity were explored by plasma cytokine measurements and proteomics analysis in 147 COVID-19 patients. Furthermore, peripheral blood mononuclear cell cytokine production assays and whole blood flow cytometry were performed. Results confirm a hyperinflammatory innate immune state, while highlighting hepatocyte growth factor and stem cell factor as potential biomarkers for disease severity. Clustering analysis revealed no specific inflammatory endotypes in COVID-19 patients. Functional assays revealed abrogated adaptive cytokine production (interferon-γ, interleukin-17, and interleukin-22) and prominent T-cell exhaustion in critically ill patients, whereas innate immune responses were intact or hyperresponsive. Collectively, this extensive analysis provides a comprehensive insight into the pathobiology of severe to critical COVID-19 and highlights potential biomarkers of disease severity., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Objectives: Complete deficiency of alternative pathway (AP) complement factors, explained by homozygous mutations, is a well-known risk factor for invasive bacterial infections; however, this is less obvious for heterozygous mutations. We describe two siblings with a heterozygous NM&#95;001928.3(CFD):c.125C>A p.(Ser42*) mutation in the complement factor D (fD) gene having a history of recurrent bacterial infections. We determined the effect of heterozygous fD deficiency on AP complement activity., Methods: We determined the effect of fD levels on complement activation as measured by AP activity, complement C3 binding to the bacterial surface of Neisseria meningitidis (Nm), Streptococcus pneumoniae (Sp) and non-typeable Haemophilus influenzae (NTHi), and complement-mediated killing of Nm and NTHi. In addition, we measured the effect of vaccination of complement C3 binding to the bacterial surface and killing of Nm., Results: Reconstitution of fD-deficient serum with fD increased AP activity in a dose- and time-dependent way. Reconstitution of patient serum with fD to normal levels increased complement C3 binding to Sp, Nm and NTHi, as well as complement-mediated killing of Nm and NTHi. Vaccination increased complement C3 binding and resulted in complete killing of Nm without fD reconstitution., Conclusion: We conclude that low fD serum levels (< 0.5 μg mL -1 ) lead to a reduced speed of complement activation, which results in diminished bacterial killing, consistent with recurrent bacterial infections observed in our index patients. Specific antibodies induced by vaccination are able to overcome the diminished bacterial killing capacity in patients with low fD levels., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Background: Patients with an IgG subclass deficiency (IgSD) ± specific polysaccharide antibody deficiency (SPAD) often present with recurrent infections. Previous retrospective studies have shown that prophylactic antibiotics (PA) and immunoglobulin replacement therapy (IRT) can both be effective in preventing these infections; however, this has not been confirmed in a prospective study., Objective: To compare the efficacy of PA and IRT in a randomized crossover trial., Methods: A total of 64 patients (55 adults and 9 children) were randomized (2:2) between two treatment arms. Treatment arm A began with 12 months of PA, and treatment arm B began with 12 months of IRT. After a 3-month bridging period with cotrimoxazole, the treatment was switched to 12 months of IRT and PA, respectively. The efficacy (measured by the incidence of infections) and proportion of related adverse events in the two arms were compared., Results: The overall efficacy of the two regimens did not differ (p = 0.58, two-sided Wilcoxon signed-rank test). A smaller proportion of patients suffered a related adverse event while using PA (26.8% vs. 60.3%, p < 0.0003, chi-squared test). Patients with persistent infections while using PA suffered fewer infections per year after switching to IRT (2.63 vs. 0.64, p < 0.01)., Conclusion: We found comparable efficacy of IRT and PA in patients with IgSD ± SPAD. Patients with persistent infections during treatment with PA had less infections after switching to IRT., Clinical Implication: Given the costs and associated side-effects of IRT, it should be reserved for patients with persistent infections despite treatment with PA.

Objectives: Chronic Q fever is a persistent infection with the intracellular bacterium Coxiella burnetii. Development of chronic Q fever is associated with single nucleotide polymorphisms (SNPs) in genes encoding for pattern recognition receptors, for phagolysosomal pathway components and for matrix metalloproteinases (MMPs). We evaluated the association of SNPs in these innate-immunity and MMP genes with clinical outcomes., Methods: SNPs were selected from previous association studies and analysed in a cohort of patients with chronic Q fever. The primary outcome was all-cause mortality; secondary outcomes were therapy failure and chronic Q fever-related complications. Subdistribution hazard ratios (SHR) were calculated., Results: Nineteen SNPs were analysed in 134 patients with proven and 29 with probable chronic Q fever. In multivariable analysis, none of the selected SNPs was associated with all-cause mortality. However, SNP rs3751143 located in P2RX7 appeared to be associated with therapy failure (SHR 2.42; 95% confidence interval, 1.16-5.05; p 0.02), which is in line with other reports, showing that a loss of function of the P2X7 receptor leads to inefficient killing of intracellular organisms. In addition, SNP rs7125062 located in MMP1, involved in the cleavage of extracellular matrix, was associated with fewer chronic Q fever-related complications such as acute aneurysms (SHR 0.49; 95% confidence interval, 0.29-0.83; p 0.008)., Conclusions: A polymorphism in P2RX7, known to lead to loss of function of the receptor and inefficient killing of intracellular organisms, and a polymorphism in MMP1 were respectively associated with more therapy failures and fewer complications such as acute aneurysms in patients with chronic Q fever., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

COVID-19 patients can present with pulmonary edema early in disease. We propose that this is due to a local vascular problem because of activation of bradykinin 1 receptor (B1R) and B2R on endothelial cells in the lungs. SARS-CoV-2 enters the cell via ACE2 that next to its role in RAAS is needed to inactivate des-Arg9 bradykinin, the potent ligand of the B1R. Without ACE2 acting as a guardian to inactivate the ligands of B1R, the lung environment is prone for local vascular leakage leading to angioedema. Here, we hypothesize that a kinin-dependent local lung angioedema via B1R and eventually B2R is an important feature of COVID-19. We propose that blocking the B2R and inhibiting plasma kallikrein activity might have an ameliorating effect on early disease caused by COVID-19 and might prevent acute respiratory distress syndrome (ARDS). In addition, this pathway might indirectly be responsive to anti-inflammatory agents., Competing Interests: Fv, MN, Mv, Qd, RB, Hv No competing interests declared, Jv Senior editor, eLife, (© 2020, van de Veerdonk et al.)

Objective: Patients with classic ataxia-telangiectasia (A-T) generally die in the second or third decade of life. Clinical descriptions of A-T tend to focus on the symptoms at presentation. However, during the course of the disease, other symptoms and complications emerge. As long-term survivors with classic A-T develop a complex multisystem disorder with a largely unknown extent and severity, we aimed to comprehensively assess their full clinical picture., Methods: Data from Dutch patients with classic A-T above the age of 30 years were retrospectively collected. In addition, we searched the literature for descriptions of classic A-T patients who survived beyond the age of 30 years., Results: In the Dutch cohort, seven classic A-T patients survived beyond 30 years of age. Fourteen additional patients were retrieved by the literature search. Common problems in older patients with classic A-T were linked to ageing. Most patients had pulmonary, endocrine, cardiovascular, and gastro-intestinal problems. All patients had a tetraparesis with contractures. This led to immobilization and frequent hospital admissions. Most patients expressed the wish to no longer undergo intensive medical treatments, and waived follow-up programs., Conclusions: Paucity of descriptions in the literature, and withdrawal from medical care complicate the acquisition of follow-up data on the natural history of long-term survivors. Irrespective of these limitations, we have obtained impression of the many problems that these patients face when surviving beyond 30 years of age. Awareness of these problems is needed to guide follow-up, counselling, and (palliative) care; decisions about life-prolonging treatments should be well considered.

Objectives: To describe phenotypic and functional characteristics of patients with the homozygous c.973-2A>G splice site mutation in the adenosine deaminase 2 (ADA2) gene (rs139750129), resulting in deficiency of ADA2 (DADA2)., Methods: We present case synopses of six patients from three unrelated families. Clinical data were analysed and next-generation sequencing (NGS) was performed. We also tested for aberrant RNA splicing and measured ADA2 enzyme activity., Results: One family had common DADA2 symptoms, whereas Behçet's disease-like manifestations were observed in the other two families. We detected the homozygous c.973-2A>G splice site mutation in ADA2 in all patients tested. ADA2 enzyme activity was significantly lower in patients than in healthy controls, but no correlation between ADA2 activity levels and disease severity was observed. Aberrant splicing was detected in a minority of mRNA transcripts, but the formation of other, undetected, aberrant splicing products could not be excluded. Patients were treated with TNF-α inhibitors to prevent recurrence of inflammatory findings including cerebral vasculitis-associated stroke., Conclusions: We describe three families with the same homozygous splice site mutation in ADA2 and observed a novel combination of manifestations resembling Behçet's disease. This further expands the range of phenotypes caused by ADA2 mutations, although no complete genotype-phenotype association could be determined. Even without active disease, the risk of stroke should be addressed in making decisions regarding treatment of DADA2 patients.

Background: After the Q fever outbreak in the Netherlands between 2007 and 2010, more than 300 patients with chronic Q fever have been identified. Some patients were also diagnosed with systemic sclerosis, a rare immune-mediated disease. We aimed to increase awareness of concomitant chronic Q fever infection and systemic sclerosis and to give insight into the course of systemic sclerosis during persistent Q fever infection., Materials and Methods: Chronic Q fever patients were identified after the Dutch Q fever outbreak in 2007-2010. Systemic sclerosis was diagnosed by a scleroderma expert and patients fulfilled the 2013 Classification Criteria for Systemic Sclerosis., Results: Four cases presented with chronic Q fever, persistent Coxiella burnetii infection, shortly preceded or followed by the diagnosis of limited cutaneous systemic sclerosis. The three male patients of 60 years or older developed a relatively mild systemic sclerosis, which did not require immunosuppressive therapy during adequate treatment of the chronic Q fever infection. The 58-year-old female patient used immunosuppressives for her newly diagnosed systemic sclerosis at the time she likely developed a chronic Q fever infection., Conclusions: In this case series, chronic Q fever preceding systemic sclerosis was associated with a mild course of systemic sclerosis without the necessity of immunosuppressive drugs, while chronic Q fever development due to immunocompromised state was associated with a more deteriorating course of systemic sclerosis., (© 2019 The Authors. European Journal of Clinical Investigation published by John Wiley & Sons Ltd on behalf of Stichting European Society for Clinical Investigation Journal Foundation.)

Background: Diagnosis of primary immunodeficiencies (PIDs) is complex and cumbersome yet important for the clinical management of the disease. Exome sequencing may provide a genetic diagnosis in a significant number of patients in a single genetic test., Methods: In May 2013, we implemented exome sequencing in routine diagnostics for patients suffering from PIDs. This study reports the clinical utility and diagnostic yield for a heterogeneous group of 254 consecutively referred PID patients from 249 families. For the majority of patients, the clinical diagnosis was based on clinical criteria including rare and/or unusual severe bacterial, viral, or fungal infections, sometimes accompanied by autoimmune manifestations. Functional immune defects were interpreted in the context of aberrant immune cell populations, aberrant antibody levels, or combinations of these factors., Results: For 62 patients (24%), exome sequencing identified pathogenic variants in well-established PID genes. An exome-wide analysis diagnosed 10 additional patients (4%), providing diagnoses for 72 patients (28%) from 68 families altogether. The genetic diagnosis directly indicated novel treatment options for 25 patients that received a diagnosis (34%)., Conclusion: Exome sequencing as a first-tier test for PIDs granted a diagnosis for 28% of patients. Importantly, molecularly defined diagnoses indicated altered therapeutic options in 34% of cases. In addition, exome sequencing harbors advantages over gene panels as a truly generic test for all genetic diseases, including in silico extension of existing gene lists and re-analysis of existing data.

Background: Patients with X-linked agammaglobulinemia (XLA) are protected against invasive bacterial infections due to IgG replacement therapy, but are still at higher risk for mucosal infections of the gut and respiratory tract. This might be explained by to the lack of IgA and IgM, as these antibodies are especially important for protection against invading bacterial pathogens on the mucosal surface., Methods: In an attempt to eliminate a chronic norovirus infection in a patient with X-linked agammaglobulinemia, fresh frozen plasma (FFP) was given two times a week for 3 weeks. At each visit, pre- and post-FFP infusion serum and saliva was collected to determine IgG-, IgA- and IgM-concentrations and serum half-life was calculated. Functionality of the immunoglobulins pre- and post-FFP infusion in both serum and saliva was tested by measuring complement activation, agglutination and killing of non-typeable Haemophilus influenzae (NTHi)., Results: Administration of FFP failed to eradicate the chronic norovirus infection. Serum IgA and IgM half-life was 4.2 ± 0.3 and 3.8 ± 0.3 days, respectively. The presence of serum IgM was associated with increased complement binding and complement-mediated killing of NTHi. IgA in saliva was detectable post-FFP and was associated with increased agglutination of NTHi. IgM in saliva was not detectable., Conclusions: We conclude that FFP treatment, although ineffective in clearing a chronic norovirus infection in this single patient, might be beneficial to prevent or eliminate bacterial infections in XLA patients by increasing IgM dependent complement-mediated killing in serum and IgA dependent bacterial agglutination on the mucosal surface.

Objectives: Chronic Q fever is a persistent infection, mostly of aortic aneurysms, vascular prostheses or damaged heart valves, caused by the intracellular bacterium Coxiella burnetii. Only a fraction of C. burnetii-infected individuals at risk develop chronic Q fever. In these individuals, a defective innate immune response may contribute to the development of chronic Q fever. We assessed whether genetic variations in genes involved in the killing machinery for C. burnetii by macrophages, contribute to the progression to chronic Q fever., Methods: The prevalence of 66 single nucleotide polymorphisms (SNPs) in 31 genes pivotal in phagolysosomal maturation, bacterial killing and autophagy, was determined in 173 chronic Q fever patients and 184 controls with risk factors for chronic Q fever and serological evidence of a C. burnetii infection. Associations were detected with univariate logistic regression models. To assess the effect of these SNPs on innate responses to C. burnetii, the C. burnetii-induced cytokine production and basal reactive oxygen species production of healthy volunteers was determined., Results: RAB7A (rs13081864) and P2RX7 loss-of-function SNP (rs3751143) were more common in chronic Q fever patients than in controls. RAB5A (rs8682), P2RX7 gain-of-function SNP (rs1718119), MAP1LC3A (rs1040747) and ATG5 (rs2245214) were more common in controls. In healthy volunteers, RAB7A (rs13081864) and MAP1LC3A (rs1040747) influenced the C. burnetii-induced cytokine production. RAB7A (rs13081864) modulated basal reactive oxygen species production., Conclusions: RAB7A (rs13081864) and P2RX7 (rs3751143) are associated with the development of chronic Q fever, whereas RAB5A (rs8682), P2RX7 (rs1718119), MAP1LC3A (rs1040747) and ATG5 (rs2245214) may have protective effects., (Copyright © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: Ataxia telangiectasia (A-T) is a neurodegenerative disorder. While patients with classic A-T generally die in their 20s, some patients with variant A-T, who have residual ataxia-telangiectasia mutated (ATM) kinase activity, have a milder phenotype. We noticed two commonly occurring ATM mutations that appeared to be associated with prolonged survival and decided to study patients carrying one of these mutations., Methods: Data were retrospectively collected from the Dutch, Italian, German and French A-T cohorts. To supplement these data, we searched the literature for patients with identical genotypes., Results: This study included 35 patients who were homozygous or compound heterozygous for the ATM c.3576G>A; p.(Ser1135&#95;Lys1192del58) mutation and 24 patients who were compound heterozygous for the ATM c.8147T>C; p.(Val2716Ala) mutation. Compared with 51 patients with classic A-T from the Dutch cohort, patients with ATM c.3576G>A had a longer survival and were less likely to develop cancer, respiratory disease or immunodeficiency. This was also true for patients with ATM c.8147T>C, who additionally became wheelchair users later in life and had fewer telangiectasias. The oldest patient with A-T reported so far was a 78-year-old patient who was compound heterozygous for ATM c.8147T>C. ATM kinase activity was demonstrated in cells from all patients tested with the ATM c.8147T>C mutant protein and only at a low level in some patients with ATM c.3576G>A., Conclusion: Compared with classic A-T, the presence of ATM c.3576G>A results in a milder classic phenotype. Patients with ATM c.8147T>C have a variant phenotype with prolonged survival, which in exceptional cases may approach a near-normal lifespan., Competing Interests: Competing interests: BPCvdW receives research support from ZonMW, Hersenstichting, Radboud University Medical Center, and Bioblast Pharma., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Good syndrome is an immunodeficiency presenting with thymoma, hypogammaglobulinemia and almost absent B cells. To investigate the origin of the B-cell lymphopenia in these patients, we studied B cell differentiation in the bone marrow of Good syndrome patients. We found very low numbers of precursor B cells in bone marrow of Good syndrome patients and a differentiation arrest after the pro-B-cell stage; this is different from other agammaglobulinemia patients with a defect in pre B-cell receptor signaling., (Copyright © 2018. Published by Elsevier Inc.)

Objective: To describe and classify the neurologic trajectories in patients with mild neurologic forms of ataxia telangiectasia (A-T) from the Dutch A-T cohort, combined with patients reported in the literature., Methods: Clinical, genetic, and laboratory data of 14 patients with mild neurologic phenotypes of A-T from the Dutch cohort were analyzed and combined with corresponding data from the literature. A mild neurologic phenotype was defined by a later onset, nonataxia presenting or dominant feature, or slower progression compared to the classic A-T phenotype. Neurologic trajectories were classified based on age at onset, presenting feature, and follow-up data., Results: One hundred five patients were included in the study. Neurologic trajectories were categorized into 6 groups: patients with childhood-onset extrapyramidal (EP) features with cerebellar symptoms developing later (group 1; 18 patients), childhood-onset cerebellar symptoms, with EP features developing later (group 2; 35 patients), childhood- to adolescence-onset dystonia, without cerebellar symptoms (group 3; 23 patients), childhood- to adolescence-onset isolated cerebellar symptoms (group 4; 22 patients), childhood- to adult-onset prominent muscle weakness (group 5; 2 patients), and patients with adult-onset EP features, with anterior horn cell disease arising subsequently (group 6; 5 patients)., Conclusions: This systematic study of the different motor abnormalities and their course over time in patients with mild phenotypes of A-T, enabled us to recognize 6 essentially different phenotypic patterns. Awareness of these different trajectories of motor abnormalities in milder forms of A-T will contribute to a reduction of diagnostic delay in this severe multisystem disorder., (© 2018 American Academy of Neurology.)

Cytokine responses of chronic Q fever patients to the intracellular bacterium Coxiella burnetii have mostly been studied using ex vivo stimulation of immune cells with heat-killed C. burnetii due to the extensive measures needed to work with viable biosafety level 3 agents. Whether research with heat-killed C. burnetii can be translated to immune responses to viable C. burnetii is imperative for the interpretation of previous and future studies with heat-killed C. burnetii Peripheral blood mononuclear cells (PBMCs) of chronic Q fever patients ( n = 10) and healthy controls ( n = 10) were stimulated with heat-killed or viable C. burnetii of two strains, Nine Mile and the Dutch outbreak strain 3262, for 24 h, 48 h, and 7 days in the absence or presence of serum containing anti- C. burnetii antibodies. When stimulated with viable C. burnetii , PBMCs of chronic Q fever patients and controls produced fewer proinflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha, and IL-1β) after 24 h than after stimulation with heat-killed C. burnetii In the presence of Q fever seronegative serum, IL-10 production was higher after stimulation with viable rather than heat-killed C. burnetii ; however, when incubating with anti- C. burnetii antibody serum, the effect on IL-10 production was reduced. Levels of adaptive, merely T-cell-derived cytokine (gamma interferon, IL-17, and IL-22) and CXCL9 production were not different between heat-killed and viable C. burnetii stimulatory conditions. Results from previous and future research with heat-killed C. burnetii should be interpreted with caution for innate cytokines, but heat-killed C. burnetii -induced adaptive cytokine production is representative of stimulation with viable bacteria., (Copyright © 2018 American Society for Microbiology.)

Q fever infection can lead to chronic Q fever, a potentially lethal disease occurring in 1-5% of patients infected with Coxiella burnetii, characterized by the persistence of this intracellular bacterium. It usually presents as endocarditis, infected vascular aneurysms, or infected vascular prostheses. This systematic review of the literature discusses the various autoimmune syndromes and B-cell dyscrasias in acute and chronic Q fever patients, that may interfere with or impede recognition and diagnosis of Q fever. Reportedly, high concentrations of anti-cardiolipin antibodies may be found in acute Q fever patients, while specifically cardiac muscle antibodies have been reported during chronic Q fever. Systemic lupus erythematosus and antiphospholipid syndrome are the most frequently reported autoimmune syndromes, followed by neuromuscular disorders and vasculitis. B-cell dyscrasia, mostly cryoglobulinaemia, is predominantly described in chronic Q fever patients with endocarditis. We conclude that immunological (epi)phenomena are not rare during Q fever and may obscure the infectious etiology of the disease., (Copyright © 2018 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.)

Approximately 20% of patients with acute Q fever develop Q fever fatigue syndrome (QFS), a debilitating fatigue syndrome. This study further investigates the role of C. burnetii-specific IFNγ, but also IL-2, CXCL9, CXCL10, and CXLC11 production in QFS patients. C. burnetii-specific IFNy, IL-2, CXCL9, CXCL10, and CXCL11 production were tested in ex vivo stimulated whole blood of QFS patients who recovered from their complaints (n = 8), QFS patients with persisting complaints (n = 27), and asymptomatic Q fever seropositive controls (n = 10). With the exclusion of one outlier, stimulation with C. burnetii revealed significantly higher IFNy and CXCL10 production in QFS patients with persisting complaints (medians 288.0 and 176.0 pg/mL, respectively) than in QFS patients who recovered from their complaints (medians 93.0 and 85.5 pg/mL, respectively) (p = 0.041 and 0.045, respectively). No significant differences between groups were found for C. burnetii-specific IL-2, CXCL9, and CXCL11 production. These findings point towards a difference in cell-mediated immunity in QFS patients with persisting complaints compared to those who recovered from their complaints. Such a difference may aid to eventually diagnose QFS more objectively and might serve as an indicator of its underlying etiology.

Ataxia Telangiectasia (AT) is named after the two key clinical features that characterize its classical phenotype, namely a progressive cerebellar gait disorder (ataxia) and vascular anomalies (telangiectasias) visible in the conjunctivae and skin. AT is an autosomal recessively inherited disorder, caused by mutations in the ATM gene that encodes the ATM protein. While the ataxia is subject of many publications, the telangiectasias are under emphasised. We here describe the observation that the absence or presence of ATM protein and the level of residual ATM kinase activity are related to the occurrence of telangiectasias and describe the clinical consequences of these vascular malformations. Finally, we hypothesize that ATM dysfunction dysregulates angiogenesis., (Copyright © 2017. Published by Elsevier Masson SAS.)

Bloom's syndrome (BS) is an autosomal recessive disease, caused by mutations in the BLM gene. This gene codes for BLM protein, which is a helicase involved in DNA repair. DNA repair is especially important for the development and maturation of the T and B cells. Since BLM is involved in DNA repair, we aimed to study if BLM deficiency affects T and B cell development and especially somatic hypermutation (SHM) and class switch recombination (CSR) processes. Clinical data of six BS patients was collected, and immunoglobulin serum levels were measured at different time points. In addition, we performed immune phenotyping of the B and T cells and analyzed the SHM and CSR in detail by analyzing IGHA and IGHG transcripts using next-generation sequencing. The serum immunoglobulin levels were relatively low, and patients had an increased number of infections. The absolute number of T, B, and NK cells were low but still in the normal range. Remarkably, all BS patients studied had a high percentage (20-80%) of CD4+ and CD8+ effector memory T cells. The process of SHM seems normal; however, the Ig subclass distribution was not normal, since the BS patients had more IGHG1 and IGHG3 transcripts. In conclusion, BS patients have low number of lymphocytes, but the immunodeficiency seems relatively mild since they have no severe or opportunistic infections. Most changes in the B cell development were seen in the CSR process; however, further studies are necessary to elucidate the exact role of BLM in CSR.

Telangiectasias are prominent small vessels (venules, capillaries or arterioles) that are visible as small red-purple focal lesions in the skin and mucous membranes. They can serve as a cutaneous marker for a number of primary (mostly hereditary) disorders and they can be secondary to other (systemic) diseases. Patients with telangiectasias are seen by general health practitioners, pediatricians, (pediatric) neurologists, dermatologists, and ophthalmologists. In this article we give an overview of the different disorders in which telangiectasias are a prominent feature, focusing on neurocutaneous disorders in which they serve as a marker for establishing the right diagnosis. The pattern of distribution of the telangiectasias, their age of onset and associated features are helpful to distinguish between the different disorders., (Copyright © 2017 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.)

Background: In the aftermath of the largest Q fever outbreak in the world, diagnosing the potentially lethal complication chronic Q fever remains challenging. PCR, Coxiella burnetii IgG phase I antibodies, CRP and 18 F-FDG-PET/CT scan are used for diagnosis and monitoring in clinical practice. We aimed to identify and test biomarkers in order to improve discriminative power of the diagnostic tests and monitoring of chronic Q fever., Methods: We performed a transcriptome analysis on C. burnetii stimulated PBMCs of 4 healthy controls and 6 chronic Q fever patients and identified genes that were most differentially expressed. The gene products were determined using Luminex technology in whole blood samples stimulated with heat-killed C. burnetii and serum samples from chronic Q fever patients and control subjects., Results: Gene expression of the chemokines CXCL9, CXCL10, CXCL11 and CCL8 was strongly up-regulated in C. burnetii stimulated PBMCs of chronic Q fever patients, in contrast to healthy controls. In whole blood cultures of chronic Q fever patients, production of all four chemokines was increased upon C. burnetii stimulation, but also healthy controls and past Q fever individuals showed increased production of CXCL9, CXCL10 and CCL8. However, CXCL9 and CXCL11 production was significantly higher for chronic Q fever patients compared to past Q fever individuals. In addition, CXCL9 serum concentrations in chronic Q fever patients were higher than in past Q fever individuals., Conclusion: CXCL9 protein, measured in serum or as C. burnetii stimulated production, is a promising biomarker for the diagnosis of chronic Q fever.

Ataxia-telangiectasia is a rare, neurodegenerative, and multisystem disease, characterized by cerebellar ataxia, oculocutaneous telangiectasia, immunodeficiency, progressive respiratory failure, and an increased risk of malignancies. It demands specialized care tailored to the individual patient's needs. Besides the classic ataxia-telangiectasia phenotype, a variant phenotype exists with partly overlapping but some distinctive disease characteristics. This guideline summarizes frequently encountered medical problems in the disease course of patients with classic and variant ataxia-telangiectasia, in the domains of neurology, immunology and infectious diseases, pulmonology, anaesthetic and perioperative risk, oncology, endocrinology, and nutrition. Furthermore, it provides a practical guide with evidence- and expert-based recommendations for the follow-up and treatment of all these different clinical topics., (© 2017 Mac Keith Press.)

Objectives: Chronic Q fever is a persistent infection with the intracellular Gram-negative bacterium Coxiella burnetii, which can lead to complications of infected aneurysms. Matrix metalloproteinases (MMPs) cleave extracellular matrix and are involved in infections as well as aneurysms. We aimed to study the role of MMPs in the pathogenesis of chronic Q fever., Methods: We investigated gene expression of MMPs through microarray analysis and MMP production with ELISA in C. burnetii-stimulated peripheral blood mononuclear cells (PBMCs) of patients with chronic Q fever and healthy controls. Twenty single nucleotide polymorphisms (SNPs) of MMP and tissue inhibitor of MMP genes were genotyped in 139 patients with chronic Q fever and 220 controls with similar cardiovascular co-morbidity. Additionally, circulating MMPs levels in patients with chronic Q fever were compared with those in cardiovascular controls with and without a history of past Q fever., Results: In healthy controls, the MMP pathway involving four genes (MMP1, MMP7, MMP10, MMP19) was significantly up-regulated in C. burnetii-stimulated but not in Escherichia coli lipopolysaccharide -stimulated PBMCs. Coxiella burnetii induced MMP-1 and MMP-9 production in PBMCs of healthy individuals (both p<0.001), individuals with past Q fever (p<0.05, p<0.01, respectively) and of patients with chronic Q fever (both p<0.001). SNPs in MMP7 (rs11568810) (p<0.05) and MMP9 (rs17576) (p<0.05) were more common in patients with chronic Q fever. Circulating MMP-7 serum levels were higher in patients with chronic Q fever (median 33.5 ng/mL, interquartile range 22.3-45.7 ng/mL) than controls (20.6 ng/mL, 15.9-33.8 ng/mL)., Conclusion: Coxiella burnetii-induced MMP production may contribute to the development of chronic Q fever., (Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Ataxia-telangiectasia (AT) is a neurodegenerative disorder characterized by ataxia, telangiectasia, and immunodeficiency. An increased risk of malignancies and respiratory diseases dramatically reduce life expectancy. To better counsel families, develop individual follow-up programs, and select patients for therapeutic trials, more knowledge is needed on factors influencing survival. This retrospective cohort study of 61 AT patients shows that classical AT patients had a shorter survival than variant patients (HR 5.9, 95%CI 2.0-17.7), especially once a malignancy was diagnosed (HR 2.5, 95%CI 1.1-5.5, compared to classical AT patients without malignancy). Patients with the hyper IgM phenotype with hypogammaglobulinemia (AT-HIGM) and patients with an IgG 2 deficiency showed decreased survival compared to patients with normal IgG (HR 9.2, 95%CI 3.2-26.5) and patients with normal IgG 2 levels (HR 7.8, 95%CI 1.7-36.2), respectively. If high risk treatment trials will become available for AT, those patients with factors indicating the poorest prognosis might be considered for inclusion first., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objectives: Q fever is caused by Coxiella burnetii, an intracellular bacterium that infects phagocytes. The aim of the present study was to investigate whether the C. burnetii-induced IFN-γ response is defective in chronic Q fever patients., Methods: IFN-γ was measured in supernatants of C. burnetii-stimulated peripheral blood mononuclear cells (PBMCs) of 17 chronic Q fever patients and 17 healthy individuals. To assess IFN-γ responses, expression profiles of IFN-γ-induced genes in C. burnetii-stimulated PBMCs were studied in six patients and four healthy individuals. Neopterin was measured in PBMC supernatants (of eight patients and four healthy individuals) and in sera (of 21 patients and 11 healthy individuals). In a genetic association study, polymorphisms in genes involved in the Th1-cytokine response were analysed in a cohort of 139 chronic Q fever patients and a cohort of 220 control individuals with previous exposition to C. burnetii., Results: IFN-γ production by C. burnetii-stimulated PBMCs from chronic Q fever patients was significantly higher than in healthy controls. Many IFN-γ response genes were strongly upregulated in PBMCs of patients. Neopterin levels were significantly higher in PBMC supernatants and sera of patients. The IL12B polymorphisms rs3212227 and rs2853694 were associated with chronic Q fever., Conclusions: IFN-γ production, as well as the response to IFN-γ, is intact in chronic Q fever patients, and even higher than in healthy individuals. Polymorphisms in the IL-12p40 gene are associated with chronic Q fever. Thus, a deficiency in IFN-γ responses does not explain the failure to clear the infection. The genetic data suggest, however, that the IL-12/IFN-γ pathway does play a role., (Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

As part of the Human Functional Genomics Project, which aims to understand the factors that determine the variability of immune responses, we investigated genetic variants affecting cytokine production in response to ex vivo stimulation in two independent cohorts of 500 and 200 healthy individuals. We demonstrate a strong impact of genetic heritability on cytokine production capacity after challenge with bacterial, fungal, viral, and non-microbial stimuli. In addition to 17 novel genome-wide significant cytokine QTLs (cQTLs), our study provides a comprehensive picture of the genetic variants that influence six different cytokines in whole blood, blood mononuclear cells, and macrophages. Important biological pathways that contain cytokine QTLs map to pattern recognition receptors (TLR1-6-10 cluster), cytokine and complement inhibitors, and the kallikrein system. The cytokine QTLs show enrichment for monocyte-specific enhancers, are more often located in regions under positive selection, and are significantly enriched among SNPs associated with infections and immune-mediated diseases. PAPERCLIP., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Good syndrome (GS) or thymoma-associated immunodeficiency, is a rare condition that has only been studied in retrospective case series. General consensus was that GS has a worse prognosis than other humoral immunodeficiencies. In this study, physicians of GS patients completed two questionnaires with a two year interval with data on 47 patients, 499 patient years in total. Results on epidemiology, disease characteristics, and outcome are presented. Mean age at diagnosis was 60years and median follow-up from onset of symptoms was 9years. There was a high frequency of respiratory tract infections due to encapsulated bacteria. Median survival was 14years. Survival was reduced compared to age-matched population controls (5-year survival: 82% versus 95%, p=0.008). In this cohort survival was not associated with gender (HR 0.9, 95% CI 0.3-3.0), autoimmune diseases (HR 2.9, 95% CI 0.8-10.1) or immunosuppressive use (HR 0.3, 95% CI: 0.1-1.2)., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Whether immunological mechanisms underlie Q-fever fatigue syndrome (QFS) remains unclear. For acute Q-fever, the antigen-specific interferon-γ (IFNγ) response may be a useful tool for diagnosis, and the IFNγ/interleukin(IL)-2 production ratio may be a marker for chronic Q-fever and treatment monitoring. Here we explored the specific IFNγ production and IFNγ/IL-2 ratio in QFS patients., Methods: IFNγ and IL-2 production were tested in ex-vivo stimulated whole blood of QFS patients (n = 20), and compared to those previously determined in seropositive controls (n = 135), and chronic Q-fever patients (n = 28). Also, the correlation between patient characteristics and IFNγ, IL-2, and IFNγ/IL-2 ratio was determined., Results: QFS patients were younger (p < 0.001), but gender distribution was similar to seropositive controls and chronic Q-fever patients. Coxiella burnetii Nine Mile stimulation revealed a higher IFNγ production in QFS (median 319.5 pg/ml) than in seropositive controls (120 pg/ml, p < 0.01), but comparable to chronic Q-fever (2846 pg/ml). The IFNγ/IL-2 ratio was similar to that in seropositive controls, but lower than in chronic Q-fever patients (p < 0.01). Symptom duration was positively correlated with IL-2 production, and negatively correlated with the IFNγ/IL-2 ratio., Conclusions: These results point to an altered cell-mediated immunity in QFS, and suggest a different immune response than in chronic Q-fever., (Copyright © 2016 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Background: Q fever is an infection caused by Coxiella burnetii. Persistent infection (chronic Q fever) develops in 1%-5% of patients. We hypothesize that inefficient recognition of C. burnetii and/or activation of host-defense in individuals carrying genetic variants in pattern recognition receptors or adaptors would result in an increased likelihood to develop chronic Q fever., Methods: Twenty-four single-nucleotide polymorphisms in genes encoding Toll-like receptors, nucleotide-binding oligomerization domain-like receptor-2, αvβ3 integrin, CR3, and adaptors myeloid differentiation primary response protein 88 (MyD88), and Toll interleukin 1 receptor domain-containing adaptor protein (TIRAP) were genotyped in 139 patients with chronic Q fever and in 220 controls with cardiovascular risk-factors and previous exposure to C. burnetii. Associations between these single-nucleotide polymorphisms and chronic Q fever were assessed by means of univariate logistic regression models. Cytokine production in whole-blood stimulation assays was correlated with relevant genotypes., Results: Polymorphisms in TLR1 (R80T), NOD2 (1007fsX1), and MYD88 (-938C>A) were associated with chronic Q fever. No association was observed for polymorphisms in TLR2, TLR4, TLR6, TLR8, ITGAV, ITGB3, ITGAM, and TIRAP. No correction for multiple testing was performed because only genes with a known role in initial recognition of C. burnetii were included. In the whole-blood assays, individuals carrying the TLR1 80R-allele showed increased interleukin 10 production with C. burnetii exposure., Conclusions: Polymorphisms in TLR1 (R80T), NOD2 (L1007fsX1), and MYD88 (-938C>A) are associated with predisposition to development of chronic Q fever. For TLR1, increased interleukin 10 responses to C. burnetii in individuals carrying the risk allele may contribute to the increased risk of chronic Q fever., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: In 2011, a unique Q fever vaccination campaign targeted people at risk for chronic Q fever in the southeast of the Netherlands. General practitioners referred patients with defined cardiovascular risk-conditions (age >15 years). Prevalence rates of those risk-conditions were lacking, standing in the way of adequate planning and coverage estimation. We aimed to obtain prevalence rates retrospectively in order to estimate coverage of the Q fever vaccination campaign., Methods: With broad search terms for these predefined risk-conditions, we extracted patient-records from a large longitudinal general-practice research-database in the Netherlands (IPCI-database). After validation of these records, obtained prevalence rates (stratified for age and sex) extrapolated to the Q fever high-incidence area population, gave an approximation of the size of the targeted patient-group. Coverage calculation addressed people actually screened by a pre-vaccination Q fever skin test and serology (coverage) and patients referred by their general practitioners (adjusted-coverage) in the 2011 campaign., Results: Our prevalence estimate of any risk-condition was 3.1% (lower-upper limits 2.9-3.3%). For heart valve defects, aorta aneurysm/prosthesis, congenital anomalies and endocarditis, prevalence was 2.4%, 0.6%, 0.4% and 0.1%, respectively. Estimated number of eligible people in the Q fever high-incidence area was 11,724 (10,965-12,532). With 1330 people screened for vaccination, coverage of the vaccination campaign was 11%. For referred people, the adjusted coverage was 18%. Coverage was lowest among the very-old and highest for people aged 50-70 years., Conclusion: The estimated coverage of the vaccination campaign was limited. This should be interpreted in the light of the complexity of this target-group with much co-morbidity, and of the vaccine that required invasive pre-vaccination screening. Calculation of prevalence rates of risk-conditions based on the IPCI-database was feasible. This procedure proved an efficient tool for future use, when prevalence estimates for policy, implementation or surveillance of subgroup-vaccination or other health-care interventions are needed.

Coxiella burnetii, a Gram-negative intracellular bacterium, can give rise to Q fever in humans and is transmitted mainly by inhalation of infected aerosols from animal reservoirs. Serology is commonly used to diagnose Q fever, but the early cellular immune response-i.e., C. burnetii-specific interferon γ (IFN-γ) production in response to antigen challenge-might be an additional diagnostic. Detection of IFN-γ responses has been used to identify past and chronic Q fever infections, but the IFN-γ response in acute Q fever has not been described. By challenging immunocompetent BALB/c mice with aerosols containing phase I C. burnetii, the timing and extent of IFN-γ recall responses were evaluated in an acute C. burnetii infection. Other cytokines were also measured in an effort to identify other potential diagnostic markers. The data show that after initial expansion of bacteria first in lungs and then in other tissues, the infection was cleared from day 10 onwards as reflected by the decreasing number of bacteria. The antigen-induced IFN-γ production by splenocytes coincided with emergence of IgM phase II antibodies at day 10 postinfection and preceded appearance of IgG antibodies. This was accompanied by the production of proinflammatory cytokines including interleukin (IL) 6, keratinocyte-derived cytokine, and IFN-γ-induced protein 10, followed by monocyte chemotactic protein 1, but not by IL-1β and tumor necrosis factor α, and only very low production of the anti-inflammatory cytokine IL-10. These data suggest that analysis of antigen-specific IFN-γ responses could be a useful tool for diagnosis of acute Q fever. Moreover, the current model of C. burnetii infection could be used to give new insights into immunological factors that predispose to development of persistent infection., (Published by Elsevier Inc.)

Background: Infection with Coxiella burnetii can lead to acute and chronic Q fever. Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR6, nucleotide-binding oligomerization domain receptor 1 (NOD1), NOD2, and the mitogen-activated protein kinases are central in the innate immune response against microorganisms, but little is known about their role in the recognition of C. burnetii in humans., Methods: Human peripheral blood mononuclear cells (PBMCs) were stimulated with C. burnetii Nine Mile and the Dutch outbreak isolate C. burnetii 3262. TLRs were inhibited using specific antibodies or antagonists. Additionally, the influence of human polymorphisms in TLRs and Nod-like receptors (NLRs) on C. burnetii-induced cytokine production was assessed., Results: Inhibition of TLR2, p38, JNK, and ERK led to decreased cytokine responses in C. burnetii-stimulated human PBMCs. Humans with polymorphisms in TLR1 and NOD2 had reduced cytokine production, compared with humans with wild-type genotypes, after stimulation. Interestingly, polymorphisms in TLR6 led to decreased cytokine production after C. burnetii 3262 stimulation but not after C. burnetii Nine Mile stimulation., Conclusions: The TLR1/TLR2 heterodimer and NOD2 are important recognition receptors for the induction of cytokine responses against C. burnetii in humans. Furthermore, an interesting finding was the divergent recognition of C. burnetii Nine Mile and C. burnetii 3262., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Antibiotic treatment of chronic Q fever is cumbersome and of long duration. To monitor treatment, there is a need for alternative biomarkers. Coxiella burnetii-specific interferon (IFN)-γ and interleukin (IL)-2 production reflect the type of effector and memory T-cell response. In chronic Q fever, C. burnetii-specific IFN-γ production is higher and IL-2 production is lower than in individuals with past Q fever. Here we explore whether C. burnetii-specific IFN-γ and IL-2 production correlate to treatment response., Methods: We studied the longitudinal C. burnetii-specific IFN-γ/IL-2 ratio in fifteen proven chronic Q fever patients. All patients were followed for at least 18 months during antibiotic treatment. Treatment was considered successful when clinical recovery was observed, a positive PCR for C. burnetii DNA in blood became persistently negative, anti-phase I IgG showed a fourfold decrease or more, and imaging techniques showed disappearance of infectious foci., Results: Overall, the IFN-γ/IL-2 ratio declined when patients experienced a successful treatment outcome. When treatment failed, IFN-γ/IL-2 ratios did not significantly decrease. The median (±IQR) slope of the longitudinal IFN-γ/IL-2 ratio with successful treatment was -2.10 (-7.02 to -0.06), and -0.15 (-1.13 to 0.25) with unsuccessful treatment (P = 0.19). Q fever endocarditis patients had higher IFN-γ/IL-2 ratios than patients with endovascular infections., Conclusion: We propose that the IFN-γ/IL-2 ratio can be used as an additional biomarker for monitoring chronic Q fever treatment, with declining ratios being indicative of successful treatment.

Purpose: To analyze the data of the national registry of all Dutch primary immune deficiency (PID) patients, according to the European Society for Immunodeficiencies (ESID) definitions., Results: In the Netherlands, 745 patients had been registered between 2009 and 2012. An overall prevalence of 4.0 per 100,000 inhabitants was calculated. The most prevalent PID was 'predominantly antibody disorder (PAD)' (60.4%). In total, 118 transplantations were reported, mostly hematopoietic stem cell transplantations (HSCT). Almost 10% of the PID patients suffered from a malignancy, in particular 'lymphoma' and 'skin cancer'. Compared to the general Dutch population, the relative risk of developing any malignancy was 2.3-fold increased, with a >10-fold increase for some solid tumors (thymus, endocrine organs) and hematological disease (lymphoma, leukemia), varying per disease category., Conclusions: The incidence rate and characteristics of PID in the Netherlands are similar to those in other European countries. Compared to the general population, PID patients carry an increased risk to develop a malignancy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Following a large Q fever outbreak in the Netherlands, patients at risk for chronic Q fever received a whole-cell Q fever vaccine. Sensitized people were excluded based on pre-vaccination screening with skin test (ST) and serology. An investigational IFN-γ-production assay was added. No previous experience existed for Q fever vaccination in this patient risk-group with predefined cardiac valvular anomalies or aortic aneurysm/prosthesis and many co-morbidities. We studied the adverse events (AE) and their association with patient characteristics and immunological parameters., Methods: AE registration covered the week after skin test and 90 days following vaccination, with the use of diaries, interviews and spontaneous reports. Serious (S)AE were assessed immediately to ensure safety. We coded AE according to reported severity. Univariate and multivariate analysis addressed associations., Results: Pre-vaccination screening led to exclusion of 182 patients with positive serology and 207 patients with positive skin test-reading. The skin test did not lead to any causally related SAE. Subsequent vaccination of 1370 patients did not reveal unexpected AE; however, 80% of vaccinees reported local AE (in 26% of these pronounced or extensive). The two causally related SAE (0.1%) both concerned a persistent subcutaneous injection site mass. AE were more frequent in women, younger patients, and those without immunosuppressive co-morbidity/medication. The occurrence of local AE after skin test was associated with pre-vaccination positive serology and high IFN-γ production. This was also true for local AE following vaccination, with a strong association with local AE after skin test as well. The proportion of vaccinees with positive serology and positive IFN-γ values 6 months after vaccination was higher in those with local AE after skin test or after vaccination (non-significant, probably due to small numbers)., Conclusion: Q fever vaccination was safe but reactogenic in this high-risk patient-group. Rates of local AE were higher in women, younger age groups and in those with positive immunological parameters. Vaccinees with local AE after skin test or after vaccination appear to have more pronounced post-vaccination immune responses.

In humans, infection with Coxiella burnetii, the causative agent of Q fever, leads to acute or chronic infection, both associated with specific clinical symptoms. In contrast, no symptoms are observed in goats during C. burnetii infection, although infection of the placenta eventually leads to premature delivery, stillbirth and abortion. It is unknown whether these differences in clinical outcome are due to the early immune responses of the goats. Therefore, peripheral blood mononuclear cells (PBMCs) were isolated from pregnant goats. In total, 17 goats were included in the study. Six goats remained naive, while eleven goats were infected with C. burnetii. Toll-like receptor (TLR) and cytokine mRNA expression were measured after in vitro stimulation with heat-killed C. burnetii at different time points (prior infection, day 7, 35 and 56 after infection). In naive goats an increased expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-10 and interferon (IFN)-γ mRNA upon C. burnetii stimulation was detected. In addition, TLR2 expression was strongly up-regulated. In goats infected with C. burnetii, PBMCs re-stimulated in vitro with C. burnetii, expressed significantly more TNF-α mRNA and IFN-γ mRNA compared to naive goats. In contrast, IL-10 mRNA production capacity was down-regulated during C. burnetii infection. Interestingly, at day 7 after inoculation a decreased IFN-γ protein level was observed in stimulated leukocytes in whole blood from infected goats, whereas at other time-points increased production of IFN-γ protein was seen. Our study shows that goats initiate a robust pro-inflammatory immune response against C. burnetii in vitro. Furthermore, PBMCs from C. burnetii infected goats show augmented pro-inflammatory cytokine responses compared to PBMCs from non-infected goats. However, despite this pro-inflammatory response, goats are not capable of clearing the C. burnetii infection.

Cytokine production assays have been primarily used in research settings studying novel immunodeficiencies. We sought to determine the diagnostic value of cytokine production assays in patients with recurrent and/or severe infectious diseases (IDs) without known immunodeficiencies and unclassified noninfectious inflammatory disorders (NIIDs). We retrospectively examined cytokine production in whole-blood and peripheral blood mononuclear cell samples from 157 adult patients. A cytokine production rate of <5% of that of healthy controls was considered defective. While monocyte-derived cytokine (tumor necrosis factor alpha [TNF-α], interleukin-1β [IL-1β], and IL-6) production was rarely affected, 30% of all included patients had deficient production of interferon gamma (IFN-γ), IL-17A, or IL-22. Twenty-five percent of the NIID patients displayed defective IFN-γ production, whereas IL-17A production was generally unaffected. In the group of ID patients, defective IFN-γ production was found in 19% and 14% of the patients with viral and bacterial infections, respectively, and in 38%, 24%, and 50% of patients with mycobacterial, mucocutaneous, and invasive fungal infections, respectively. Defective IL-17A and IL-22 production was mainly confined to ID patients with mucocutaneous fungal infections. In conclusion, cytokine production assays frequently detect defective Th1 responses in patients with mycobacterial or fungal infections, in contrast to patients with respiratory tract infections or isolated bacterial infections. Defective IL-17A and IL-22 production was primarily found in patients with fungal infections, while monocyte-derived cytokine production was unaffected. Thus, lymphocyte-derived cytokine production assays are helpful in the diagnostic workup of patients with recurrent infections and suspected immunodeficiencies and have the potential to reveal immune defects that might guide adjunctive immunomodulatory therapy., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Objectives: The Q fever skin test is used to measure cell-mediated immunity to Coxiella burnetii in pre-vaccination screening to exclude individuals with pre-existing immunity. We investigated whether this in-vivo test influences subsequent measurements of immune response., Methods: We assessed the humoral and cellular immune responses before, and 6 and 12 months after skin testing in 63 individuals who were not vaccinated because of either a positive skin test or positive serology in screening. IgG anti-C. burnetii antibodies were measured using immune-fluorescence assay (IFA). The cellular immune response was assessed by measuring in-vitro C. burnetii-specific interferon (IFN)-γ production in blood., Results: Of the 35 subjects with a positive skin test and negative serology, 15/35 (43%) showed seroconversion at 6 months, and 7/32 (22%) seropositivity at 12 months. The mean ± SE specific IFN-γ production in this group increased from 185 ± 88 pg/mL (at baseline) to 422 ± 141 pg/mL at 6 months (P = 0.009) and 223 ± 91 pg/mL at 12 months (P = 0.17). Of the 28 subjects with positive serology (and unknown skin test results), 21/28 (75%) showed an increase in IgG anti-phase I titres at 6 months, and 11/25 (44%) at 12 months. The mean ± SE specific IFN-γ production was significantly increased at 6 months, but not at 12 months., Conclusions: Q fever skin testing causes higher antibody titres and higher in-vitro IFN-γ to C. burnetii, and therefore affects subsequent Q fever diagnostics., (Copyright © 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Diagnosis of ongoing or past infection with Coxiella burnetii, the causative agent of Q fever, relies heavily on serology: the measurement of C. burnetii-specific antibodies, reflecting the host's humoral immune response. However, cell-mediated immune responses play an important, probably even more relevant, role in infections caused by the intracellular C. burnetii bacterium. Recent studies have investigated interferon-gamma (IFN-γ) based assays, including a whole-blood IFN-γ production assay and a Coxiella enzyme-linked immunospot (Coxiella ELISPOT), as potential diagnostic tools for Q fever diagnosis. Both are in-house developed assays using stimulating antigens of different origin. The main objective of this study was to compare the test performance of the IFN-γ production assay and the Coxiella ELISPOT for detecting a cellular immune response to C. burnetii in Q fever patients, and to assess the correlation between both assays. To that end, both tests were performed in a well-defined patient group of chronic Q fever patients (n = 16) and a group of healthy seronegative individuals (n = 17). Among patients, both the Coxiella ELISPOT and the IFN-γ production assay detected positive response in 14/16. Among controls, none were positive in the Coxiella ELISPOT, whereas the IFN-γ production assay detected positive results in 1/17 and 3/17, when using Henzerling and Nine Mile as stimulating antigens, respectively. These results suggest the Coxiella ELISPOT has a somewhat higher specificity than the IFN-γ production assay when Nine Mile is used as antigen stimulus. The assays showed moderate correlation: the Spearman correlation coefficient r ranged between 0.37-0.60, depending on the antigens used. Further investigation of the diagnostic potential for C. burnetii infection of both assays is warranted.

Infection with Coxiella burnetii may lead to life-threatening chronic Q fever endocarditis or vascular infections, which are often difficult to diagnose. The present study aims to investigate whether measurement of in-vitro interferon-gamma (IFN-γ) production, a key cytokine in the immune response against C. burnetii, differentiates chronic from a past cleared infection, and whether measurement of other cytokines would improve the discriminative power. First, C. burnetii-specific IFN-γ production was measured in whole blood of 28 definite chronic Q fever patients and compared with 135 individuals with past Q fever (seropositive controls) and 908 seronegative controls. IFN-γ production was significantly higher in chronic Q fever patients than in controls, but with overlapping values between patients and seropositives. Secondly, the production of a series of other cytokines was measured in a subset of patients and controls, which showed that interleukin (IL)-2 production was significantly lower in patients than in seropositive controls. Subsequently, measuring IL-2 in all patients and all controls with substantial IFN-γ production showed that an IFN-γ/IL-2 ratio >11 had a sensitivity and specificity of 79% and 96%, respectively, to diagnose chronic Q fever. This indicates that a high IFN-γ/IL-2 ratio is highly suggestive for chronic Q fever. In an additional group of 25 individuals with persistent high anti-Coxiella phase I IgG titres without definite chronic infection, all but six showed an IFN-γ/IL-2 ratio <11. In conclusion, these findings hold promise for the often difficult diagnostic work-up of Q fever and the IFN-γ/IL-2 ratio may be used as an additional diagnostic marker., (© 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.)

Background: Q fever is caused by the intracellular bacterium Coxiella burnetii. Initial infection can present as acute Q fever, while a minority of infected individuals develops chronic Q fever endocarditis or vascular infection months to years after initial infection. Serology is an important diagnostic tool for both acute and chronic Q fever. However, since immunosuppressive drugs may hamper the humoral immune response, diagnosis of Q fever might be blurred when these drugs are used., Case Presentation: A 71-year-old Caucasian male was diagnosed with symptomatic acute Q fever (based on positive C. burnetii PCR followed by seroconversion) while using anti-tumor necrosis factor-α (anti-TNFα) drugs for rheumatoid arthritis (RA). He was treated for two weeks with moxifloxacin. After 24 months of follow-up, the diagnosis of probable chronic Q fever was established based on increasing anti-C. burnetii phase I IgG antibody titres in a immunocompromised patient combined with clinical suspicion of endocarditis. At the time of chronic Q fever diagnosis, he had been treated with anti B-cell therapy for 16 months. Antibiotic therapy consisting of 1.5 years doxycycline and hydroxychloroquine was started and successfully completed and no signs of relapse were seen after more than one year of follow-up., Conclusion: The use of anti-TNFα agents for RA in the acute phase of Q fever did not hamper the C. burnetii-specific serological response as measured by immunofluorescence assay. However, in the presented case, an intact humoral response did not prevent progression to probable chronic C. burnetii infection, most likely because essential cellular immune responses were suppressed during the acute phase of the infection. Despite the start of anti-B-cell therapy with rituximab after the acute Q fever episode, an increase in anti-C. burnetii phase I IgG antibodies was observed, supporting the notion that C. burnetii specific CD20-negative memory B-cells are responsible for this rise in antibody titres.

Alpha-fetoprotein (AFP) is present in fetal serum in concentrations up to 5,000,000 μg/l. After birth, AFP gene expression is turned down with a subsequent fall of the serum concentrations of this albumin-like protein to 'adult values' of circa 0.5-15 μg/l from the age of 2 years onwards. Irrespective of its assumed important functions, individuals with AFP deficiency appear fully healthy. The other way around, the presence of AFP in the circulation after the first years of life doesn't seem to harm, since individuals with 'hereditary persistence of AFP' are also without clinical abnormalities. During pregnancy, AFP (in maternal serum) has long been recognized as a marker for congenital anomalies of the fetus. Equally well known is AFP as biomarker for hepatocellular carcinoma and some other malignancies. There are at least four neurodegenerative disorders, all inherited as autosomal recessive traits and characterized by the presence of cerebellar ataxia, abnormal ocular movements, and neuropathy, for which an elevated concentration of serum AFP is an important diagnostic biomarker. The availability of a reliable biomarker is not only important during screening or diagnostic processes, but is also relevant for objective follow-up during (future) therapeutic interventions., (Copyright © 2013. Published by Elsevier Ltd.)

Objectives: Autoinflammatory disorders are disorders of the innate immune system. Standard genetic testing provided no correct diagnosis in a female patient from a non-consanguineous family of British descent with a colchicine-responsive autosomal dominant periodic fever syndrome. We aimed to unravel the genetic cause of the symptoms., Methods: Whole exome sequencing was used to screen for novel sequence variants, which were validated by direct Sanger sequencing. Ex vivo stimulation with peripheral blood mononuclear cells was performed to study the functional consequences of the mutation. mRNA and cytokine levels were measured by quantitative PCR and ELISA, respectively., Results: Whole exome sequencing revealed a novel missense sequence variant, not seen in around 6800 controls, mapping to exon 8 of the MEFV gene (c.1730C>A; p.T577N), co-segregating perfectly with disease in this family. Other mutations at the same amino acid (c.1730C>G; p.T577S and c.1729A>T; p.T577S) were found in a family of Turkish descent, with autosomal dominant inheritance of familial Mediterranean fever (FMF)-like phenotype, and a Dutch patient, respectively. Moreover, a mutation (c.1729A>G; p.T577A) was detected in two Dutch siblings, who had episodes of inflammation of varying severity not resembling FMF. Peripheral blood mononuclear cells from one patient of the index family showed increased basal interleukin 1β mRNA levels and cytokine responses after lipopolysaccharide stimulation. Responses normalised with colchicine treatment., Conclusions: Heterozygous mutations at amino acid position 577 of pyrin can induce an autosomal dominant autoinflammatory syndrome. This suggests that T577, located in front of the C-terminal B30.2/SPRY domain, is crucial for pyrin function.

Objectives: In the Netherlands, people at risk for chronic Q fever were vaccinated against Coxiella burnetii with the inactivated whole cell vaccine Q-vax®. We aimed to measure the immune responses to C. burnetii six and twelve months after vaccination in this relevant population., Methods: In 260 vaccinees, antibody responses were assessed by immunofluorescence assay (IFA), complement fixation test and ELISA. The cellular immune responses were assessed by measuring C. burnetii-specific interferon (IFN)-γ production in blood. Serological results of 200 individuals with past Q fever were used for comparison., Results: At six months, 46% of vaccinees showed low IFA antibody titres and 67% had a positive IFN-γ assay; At twelve months, both were 60%. In contrast, individuals with a past Q fever were seropositive in 99.5% at six and twelve months, with relatively higher IFA titres. Interestingly, vaccinees with positive IFN-γ assay pre-vaccination, showed a higher seroconversion rate than IFN-γ negative vaccinees: 74% vs. 41% (p < 0.001)., Conclusions: The immune response after Q-vax® vaccination is lower and restricted to a smaller proportion than found after past Q fever and than previously described after vaccination, suggesting decreased vaccine immunogenicity in this high-risk population. A positive IFN-γ assay before vaccination in seronegative vaccinees likely points to pre-existing immunity resulting in boosting by vaccination., (Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Nitric oxide (NO) is a key mediator in the pathophysiology of septic shock that can be measured in exhaled breath. To assess whether a pulmonary infection itself or systemic inflammation is responsible for NO production, we determined exhaled NO in ventilated patients with respiratory and non-respiratory septic shock and compared it with the concentration in ventilated intensive care patients without systemic inflammation. In addition, the change of NO production over time and correlations with haemodynamic instability were evaluated. The controls without systemic inflammation, as witnessed by the absence of systemic inflammatory response syndrome criteria and low levels of interleukin-6, had similar concentrations of NO as the patients with non-respiratory septic shock. The respiratory sepsis patients exhaled more NO than the non-respiratory sepsis patients (p = 0.05), and a time dependent decline in time in both groups (p = 0.04). Exhaled NO did not correlate with markers of disease severity, systemic inflammation and haemodynamic instability. These data indicate that the infected lungs are the source of exhaled NO.