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Pneumonia is a common condition that frequently leads to hospitalization. In hospitalized adults Streptococcus pneumoniae, or pneumococcus, is the most prevalent bacterial pathogen causing community-acquired pneumonia (CAP). The first part of this thesis is about diagnostic testing in patients with CAP. It was found that large inter-hospital variation exists in resource utilization in CAP care, mainly in microbiological testing. Also, more microbiological diagnostic testing was associated with early alteration of antibiotic therapy. The focus of second part is on Streptococcus pneumoniae. As of 2006, pneumococcal vaccination is included in the Dutch national immunization program for children to protect them from severe pneumococcal infections. This thesis shows that the introduction of these vaccines was followed by a reduction in vaccine-type severe pneumococcal disease, but also in adults hospitalized with non-invasive pneumococcal CAP. Bangladesh introduced a pneumococcal vaccine in 2015. It is shown in this thesis that the coverage of vaccine-type serotypes causing non-bacteremic pneumococcal pneumonia by the current vaccine is low. The last part of this thesis describes the results of a study in which adults hospitalized with CAP received dexamethasone or a placebo, in addition to standard therapy. The length of hospital stay was shorter and there were less ICU admission in the dexamethasone group, but more side effects were observed in this group as well. It remains unclear for which patients the risk-benefit ratio is optimal. Lastly, it was shown that serum troponin T (an indicator of cardiac damage) at hospital admission with CAP predicts short- and long-term mortality.

Patients with cancer are at risk for post influenza complications due to their underlying disease as well as their (immunosuppressive) treatment; therefore influenza vaccination is recommended to prevent influenza infections. Adherence to vaccination recommendations is however low, most likely explained by the lack of evidence regarding efficacy/effectiveness and optimal timing of vaccination in this particular patient group. This thesis addresses the burden of influenza in adult patients with either haematological cancers or solid tumours and response to influenza virus vaccination as well as optimal timing of vaccination during the chemotherapy cycle in patients with malignancies treated with chemotherapy. To assess the burden of influenza, incidence of influenza and other respiratory virus infections in patients with acute myeloid leukemia (AML) was studied as well as excess mortality in cancer patients during the seasonal influenza period in the Netherlands. Forty-four percent of patients with AML presenting with fever and/or upper respiratory tract complaints tested positive for an influenza virus or other respiratory viruses, influenza viruses (A and B) accounted for most of the infections. Regarding excess mortality, a trend for higher number of cancer deaths was observed during influenza season in the Netherlands. Moreover, a trend was found for a positive association between influenza incidence and number of cancer deaths. The second part of this thesis addresses efficacy of vaccination; antibody responses and the broadness of the antibody response to influenza vaccination were studied in breast cancer and colorectal cancer patients. As a surrogate end-point of vaccine efficacy, the acquired antibody levels after vaccination are considered to be above threshold of protection (seroprotection) and increase in antibody titres (seroconversion) were presented. In breast cancer patients overall antibody responses were adequate. Statistically significant higher responses were observed in patients who received early vaccination (day 5 after chemotherapy) when compared to late vaccination (day 16). In colorectal cancer patients overall serologic responses were adequate with no statistically difference found between early and late vaccination. In addition to the determination of vaccine virus specific antibody titres in breast cancer patients, the broadness of the response was assessed by using a protein microarray consisting of 11 different influenza strains. A wide range in serum antibody response after vaccination was found, overall the response was broad because of the significant increase of antibodies against strains not represented in the vaccine. Higher age and previous influenza vaccination had a significant negative influence on the antibody response to the vaccine. Apart from vaccine efficacy, influenza vaccination coverage in the Netherlands was analyzed in breast cancer patients and colorectal cancer patients treated with chemotherapy. Fifty- nine percent of our study population were vaccinated against influenza. In case of non-adherence to the recommendations, lack of awareness, lack of optimal timing of the vaccination during the chemotherapy cycle, and concerns on efficacy of vaccination were given as reasons for not vaccinating. According to 48% of the general practitioners, the treating medical oncologist has the primary responsibility for vaccination. Better guidelines may increase the vaccination coverage of cancer patients.

Background: Despite the availability of effective antibiotics and vaccines, community-acquired pneumonia (CAP) remains the cause of significant morbidity and mortality worldwide.A major problem is that in absence of full spectrum rapid microbiology diagnostics, the antibiotic treatment at start is empirical what could mean inappropriate treatment in some patients. This thesis adds knowledge on CAP by studying 1) patient characteristics and alternative biomarkers to identify atypical aetiology, 2) novel biomarkers to predict unwanted outcomes and 3) the merits of adjunctive corticosteroid treatment during CAP. Methods: In this thesis different research methods were used: individual patient data meta-analysis to investigate the merits of adjunctive corticosteroid therapy, a randomized controlled trial to investigate bioavailability of oral versus intravenous dexamethasone, post-hoc analysis to study incidence of atypical CAP, costs of CAP and different biomarkers in respect to outcome. Results: Atypical CAP pathogens Chlamydia and Legionella species, Coxiella burnetii and Mycoplasma pneumoniae were more often identified in CAPs occurring from early May to early October, especially in male patients younger than 60 years of age. In this time period two-thirds of CAPs were caused by these atypical pathogens in all patients below 60 years old. Furthermore, we showed that, by routinely performing diagnostics to search for Chlamydia psittaci, incidence of this pathogen in the Netherlands is much higher than previously thought. We investigated the predictive value of respectively proteinuria, cardiac troponin T and the mainly pulmonary markers YKL-40, CCL18, SP-D and CA 15-3 in hospitalised CAP. Proteinuria might reflect the extent of inflammation and thereby serve as a parameter of overall organ damage. Cardiac troponin T (cTNT) proved to be a strong predictor for both short-term and long-term mortality. In 45% of the CAP patients, cTNT levels were elevated. CAP can be seen as a cardiac stress test, in which systemic inflammation with catecholamine release, tachycardia and peripheral vasodilation in combination with low blood oxygenation causes an increased cardiac demand. Of the investigated pulmonary markers especially YKL-40 proved to be useful for prognosis on CAP. The area under the curve of the PSI score was higher, but since this score exist of twenty variables, YKL-40 is easier to use. Chapter 8, on the course of these pulmonary markers during CAP, shows that in CAP caused by atypical pathogens especially CCL18 and YKL-40 levels are lower compared to levels in CAPs caused by other or unknown aetiology. The final part of the thesis concerns the adjunctive therapy of corticosteroids in CAP. In an individual patient data meta-analysis no significant difference in 30-day mortality was found between the corticosteroid and placebo group. A shorter time to clinical stability and shorter length of hospital stay of both one day were found favouring the corticosteroid group. However, incidence of the known side-effect hyperglycaemia and re-hospitalisations were higher in the corticosteroid group. No significant subgroup effects were found.

Recurrent respiratory tract infections are a common problem, and can have various causes, including an underlying immunodeficiency. This thesis investigates the value of humoral immune status assessment in patients with respiratory tract infections, lung transplant candidates/recipients and the long-term treatment results of primary antibody deficiency with antibody replacement therapy. For the most part, interpretation of the results from humoral immune status assessment is clear. However, some parts of the immune status screening protocol, such as mannose-binding lectin (MBL) deficiency, are less easily interpretable. As shown in Chapter 2, MBL-deficiency does not seem to be a contributing factor in patients presenting with recurrent respiratory tract infections. Therefore, the value of including MBL in the clinical immune status protocol can be questioned. Another part of the immune status screening protocol is assessment of the response to pneumococcal polysaccharide vaccination. Usually only the antibody response of the IgG isotype is measured. In Chapter 3 and Chapter 4, it is shown that the antibody response to vaccination can also be impaired in other isotypes, and also in IgG subclasses. Impaired IgA and IgG2 antibody responses seem to be of clinical relevance, as they were associated with sinusitis (IgA), pneumonia (IgG2) and severity of bronchiectasis (IgA and IgG2). Lung transplant candidates and recipients are a special population. After transplantation, all patients receive strong immunosuppressive therapy in order to prevent rejection. As shown in Chapter 5 a majority of lung transplant candidates had one or more abnormalities in the immune status investigation. Further, in Chapter 6 it is shown that humoral immune status significantly declines after lung transplantation. This was most outspoken in the first-year post-transplantation. Therefore, it can be recommended that revaccination with pneumococcal vaccines should be considered one year post-transplantation. Secondly, periodic monitoring of immunoglobulins, IgG subclasses and anti-pneumococcal antibodies should be included in routine clinical care. Chapter 7 reviews the current literature on etiology, diagnosis, and treatment of primary antibody deficiency (PAD) and iatrogenic hypogammaglobulinemia (IHG). In PAD there is compelling evidence for the use of antibody replacement therapy (ART), mainly for more severe forms of PAD. For milder forms of PAD the evidence is more limited. Based on limited evidence, ART seems to be effective in reducing the infection frequency in IHG patients. The study described in chapter 8 provides new data on the efficacy of antibody replacement therapy in patients with mild or severe humoral immunodeficiency. The results confirm the effectiveness of ART in mild humoral immunodeficiency and add to previously published literature that ART is also an effective therapy in patients with severe humoral immunodeficiency. As shown in this thesis an antibody deficiency (also a mild antibody deficiency) can be the cause of recurrent respiratory tract infections. Systematic investigation of the humoral immune status can lead to this diagnosis. Treatment with antibody replacement therapy can significantly decrease the burden of this disease and prevent future organ damage. A high index of suspicion is necessary for diagnosing an antibody deficiency in patients with recurrent respiratory tract infections.

This thesis addresses various aspects on pneumococcal disease, pneumococcal conjugate vaccines (PCVs) and the societal impact of PCV implementation in children. Implementation of 7-valent pneumococcal conjugate vaccine (PCV7) in the Dutch national immunization program for infants led to a shift from vaccine to non-vaccine serotypes (i.e. replacement disease) in invasive pneumococcal disease (IPD). This shift was observed in all age groups, including non-vaccinated persons, due to indirect effects.Indirect were caused by a reduction in nasopharyngeal colonization of vaccine serotypes in children and subsequent transmission (i.e. herd effects). Also after PCV7 was replaced by a 10-valent pneumococcal conjugate vaccine (PCV10) we observed herd effects for extra serotypes covered by PCV10.Replacement disease following PCVs was primarily caused by expansion of pre-existing non-vaccine serotypes (NVT) rather than capsular switch variants.Despite replacement disease IPD incidence has decreased dramatically in children

This thesis addresses the development of the intestinal microbiota in infancy, investigated by different molecular approaches (all based on rRNA gene analysis), and includes studies describing consequences of early life modulation of microbiota, by supplementation of probiotics, on composition and functionality of the microbiota. A bacterial population, weighing more than 1.5 kilogram inhabits the adult human intestinal tract: the gut microbiota, comprising approximately 1014 bacteria. The gut microbiota is the ecological community consisting of commensal, symbiotic and pathogenic microorganisms that resides in a defined environment. After birth, infants are rapidly colonized by microbes from different environments they are exposed to. The classical early colonizers of the infant gut are facultative anaerobes of the phylum Proteobacteria, such as Escherichia coli and other Enterobacteriaceae. These organisms pave the way for strictly anaerobic bacteria by depleting the initial available oxygen in a matter of days. The neonatal period comprises a critical period for microbiota development, also because this is the period during which the (mucosal) immune system matures. The long-term composition and function of the newborn’s gut microbiota is suggested to be programmed during this period, thereby laying the foundation for future health and influencing the risk of developing disease later in life. The development of the gut microbiota continues during the first years of life and its composition is considered to resemble the adult gastrointestinal tract by the age of three. Mode of delivery, gestational age and feeding mode especially affect the infant’s microbiota composition. Besides, administration of antibiotics in early life and supplementation with prebiotics or probiotics (live microorganisms that, when administered in adequate amounts, confer a health benefit on the host) can have a major effect on the intestinal microbiota composition. Studies strongly suggest that dysbiosis (disturbance of the microbial balance in the gut) contributes to the development of various kinds of disease, such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), obesity, allergy, asthma and diabetes. The development of the gut microbiota in early life is a complex process, regulated by endogenous as well as exogenous factors, which are being unraveled at the moment but also need additional in-depth investigations. Ultimately, everyone develops his or her own unique, individual microbiota. The vast majority of those microbiota are associated with maintenance of health. In all those cases it is important not to disturb this developmental process in early life (e.g. with antibiotics). When the microbiota develops in an aberrant way, associated with disease, early intervention (e.g. with pre- or probiotics) seems to be warranted. These interventions must be optimized by personalization and targeted application, an approach which now is within reach: the microbiome has the greatest potential to be translated into the clinic in the very short term. The challenge will be to bring this approach into clinical practice.

Streptococcus pneumoniae, the pneumococcus, is an important human pathogen causing considerable morbidity and mortality worldwide. This thesis addresses several interactions between pneumococcus and man. The first part of the thesis deals with the host immune response against pneumococci. We studied the anticapsular antibody response during pneumonia and found that only 45% of adult pneumococcal pneumonia patients mount a significant serotype-specific antibody response. We showed that corticosteroid use during pneumonia does not affect anticapsular pneumococcal antibody formation. In the second part of the thesis diagnostic options for pneumococcal pneumonia are discussed. We investigated the measurement of antibodies as an alternative method to detect pneumococcal pneumonia as well as two additional newly developed techniques: a quantitative PCR followed by capsular sequence typing, and a serotype-specific urinary antigen assay. Using all three techniques we were able to detect the pneumococcus in samples of 56% more patients compared to conventional methods. Furthermore, we investigated whether a urinary antigen test (UAT) could serve as a method to diagnose invasive pneumococcal pneumonia, classically defined as pneumonia in which S. pneumoniae is cultured from a normally sterile body site, by comparing the outcome of bacteraemic and non-bacteraemic/UAT-positive patients. A non-significant association with mortality was found for bacteraemia. In the third part of the thesis various techniques for distinguishing and typing cultured pneumococci are described. Two molecular serotyping techniques, a real-time multiplex PCR (rmPCR) assay detecting 21 different serotypes/-groups and a sequetyping assay, based on the sequence of the cpsB gene within the pneumococcal capsulation locus, were compared. The rmPCR assay was shown to perform well for the 21 serotypes/-groups included in the assay and for serotype mixtures. The sequetyping assay performs well, with specific exceptions, and may be more useful for regions where vaccine serotypes are less common. To gain more insight in the genetic background of pneumococci, we developed a mixed genome microarray containing 470 DNA fragments and evaluated genetic differences between 445 invasive and non-invasive pneumococcal isolates. The microarray was able to genetically distinguish pneumococci at a deeper level than the typing techniques generally used. The fourth and last part of the thesis focuses on pneumococcal vaccination. We have studied the impact of the seven-valent pneumococcal conjugate vaccine (PCV7) introduction in the Netherlands on the incidence and presentation of invasive pneumococcal disease (IPD). Four years after introduction of the vaccine, significant declines in overall IPD incidence were observed in children

Contains fulltext : 132063.pdf (Publisher’s version ) (Open Access) Radboud Universiteit Nijmegen, 11 december 2014 Promotores : Gooszen, H.G., Rijkers, G.T. Co-promotor : Besselink, M.G.

Community-acquired pneumonia (CAP) is a common disease with considerable morbidity and mortality, despite effective antibiotic treatment. In this thesis, we showed that the major causative microorganisms in CAP trigger distinct inflammatory response profiles in the host. While an inflammatory response as such is required to combat invading pathogens, an excessive inflammatory response may contribute to adverse clinical outcome. Modulation of the immune response could therefore offer promising new treatment options in CAP. In this thesis, we focused on the immunomodulatory properties of corticosteroids, macrolide antibiotics and vitamin D, and their potential role in prevention and treatment of CAP. A randomised placebo-controlled trial in hospitalised, non-immunocompromised patients with CAP showed that administration of dexamethasone adjuvant to antibiotics reduced length of hospital stay by one day. In addition, our data indicate that certain subgroups of patients might benefit in particular from dexamethasone treatment. Differences in dexamethasone effect were found for different microbial aetiologies. Next, in patients with a high pro-inflammatory cytokine response, but a discrepantly low cortisol, adjuvant dexamethasone was associated with a significant decrease in mortality/intensive care unit admission (combined endpoint). In a literature review, we provided an overview of the existing evidence from in vitro and in vivo studies on the immunomodulatory effects of macrolides in CAP. Macrolides have been shown to change the nature of the immune response during acute inflammation in three ways: by suppression of the cytokine response, by changing the behaviour of inflammatory cells to a more anti-inflammatory nature and by affecting structural cells of the respiratory tract. However, experimental and clinical studies on the immunomodulatory effects of macrolides when given adjuvant to β-lactam antibiotics, are lacking. In order to further elucidate the mechanisms of immunomodulation by macrolides during acute inflammation, in particular when given in combination with β-lactam antibiotics, we designed an in vitro model of acute infection with Streptococcus pneumoniae. We found that macrolides, alone or adjuvant to β-lactam antibiotics, attenuated the pro-inflammatory cytokine response in whole blood stimulated with heat-killed S. pneumoniae. This suggests an immunomodulatory effect. However, this effect was not observed in subsequent series of experiments with viable S. pneumoniae (either macrolide-susceptible or -resistant). Vitamin D has pleiotropic immunomodulatory properties, apart from its function in calcium and bone homeostasis. Vitamin D deficiency is common worldwide. In our cohort of patients with CAP, 53% of the patients was vitamin D deficient. We showed that vitamin D deficiency is associated with adverse clinical outcome in CAP. Vitamin D status at the time of hospital admission appeared to be an independent predictor for 30-day mortality, adding prognostic value to other biomarkers and prognostic scores, in particular the Pneumonia Severity Index (PSI) score. In former studies, vitamin D deficiency has been associated with an increased susceptibility to respiratory tract infections. Whether this association is based on a causal relationship is unknown.Three parallel large independent case-control studies showed no preventive association between vitamin D supplementation and pneumonia in adults. This suggests that there is no causal relation between vitamin D deficiency and the risk of pneumonia

Despite advances during the last few decades in the prevention, diagnosis and treatment of community-acquired pneumonia (CAP), its incidence remains high. In this thesis, strategies are described for improvement of the quality of clinical management in patients with pneumonia and for the reduction of the healthcare burden. The focus is on the prevention of pneumonia, a better understanding of the immune response in CAP, further development of biomarkers, and adjunctive therapies. Recent studies have suggested that gastric acid-suppressive medication may increase the risk of CAP. In Chapter 2, a population-based case-control study is presented on the relationship between proton pump inhibitor (PPI) treatment and CAP. In Chapter 3, the cytokine response in a cohort of 201 non-immunocompromised patients with CAP is described. In addition to the inflammatory response as discussed in Chapter 3, the complement system is an important component of the innate immune system. In Chapter 4, the role of polymorphisms in the genes of these two lectins in patients with S. aureus peritonitis on continuous ambulatory peritoneal dialysis (CAPD) is studied. This led to the study described in Chapter 5 in which binding to the pneumococci of MBL, ficolin-2 and subsequently MASP-2 was explored. This study showed that pneumococci can bind MASP-2 and that ficolin-2 seems to be the overriding MASP-2 activator.In Chapter 6, a quantitative multiplex immunoassay for the measurement of serotype-specific antibody concentrations after the onset of CAP is described. The estimated contribution of the pneumococcus in patients with an unidentified causative agent of CAP was calculated to be 57%. In Chapter 7, the usefulness of serum angiotensin I-converting enzyme (ACE) activity at the time of hospital admission as a prognostic marker is assessed. Serum ACE activity can become useful for the identification of bacteremia before the outcome of blood cultures is known. In Chapter 8, the incidence and predictive value of proteinuria as a biomarker for outcome in patients with CAP is presented. Proteinuria on admission was independently associated with increased length of hospital stay in CAP patients.In Chapter 9, the current literature concerning the various options for supportive therapy of patients treated with otherwise effective antibiotics is reviewed. In Chapter 10, a randomised placebo-controlled trial (RCT) using corticosteroids as an adjunctive treatment to antibiotics in patients with CAP is described. The median length of stay was 6.5 days for patients in the dexamethasone group and 7.5 days in the placebo group (p= 0.048).To further elucidate which patients benefit most from corticosteroids, the cytokine response of 304 patients in an RCT was evaluated in Chapter 11. The overall decrease in cytokine levels was more rapid in patients with CAP treated with dexamethasone in addition to antibiotic treatment than in patients without dexamethasone. In Chapter 12, it is shown that in patients with CAP, a cortisol level

Intriguing advances have been made in recent years in identifying the mechanisms and consequences of genetic diversity and immune defects of pattern recognition receptors in innate immunity. The subject of this thesis is the interplay between the gene, protein and disease for two of these receptors: mannose-binding lectin (MBL) and L-ficolin. By studying genotypes, protein levels and functionality in a cohort of hospitalized patients with community-acquired pneumonia and in an outbreak of legionellosis, we aimed to obtain more insight into the reciprocal effect of these proteins and pneumonia on each other, in the light of the genetic background of the patients. Both MBL and L-ficolin are part of the complement system of innate immunity, sharing similarities in structure and function. Both proteins have trimeric collagen-like subunits assembled into higher order oligomers, held together by disulfide bridges at the cysteine-rich N-terminal domain. The multimeric structure exposes multiple ligand-binding C-terminal heads: carbohydrate-recognition domains for MBL and fibrinogen-like domains for L-ficolin. This enables the binding of repeating molecular patterns on pathogens with high avidity, resulting in complement activation and in opsonization for phagocytosis. Multiple genetic polymorphisms in the gene encoding MBL lead to impaired oligomerization, resulting in deficiency of functional MBL. The gene encoding L-ficolin revealed to be highly polymorphic as well, with two single nucleotide polymorphisms (SNPs) leading to an altered substrate binding affinity of the protein. Both genes harbor promoter SNPs that influence plasma protein levels. MBL deficiency has been associated with an increased risk in infection. Conflicting results have been published on the effect of MBL deficiency on pneumococcal infections. In our cohort of community-acquired pneumonia, we did not find an association between MBL genotypes and pneumococcal infection. MBL-sufficient genotypes were more frequently found in a small group of patients with atypical pneumonia. In a large outbreak of legionellosis at a Dutch flower show, MBL genotypes were not a risk factor for legionellosis. However, patients with legionellosis displayed deficient MBL-mediated complement activation even with MBL-sufficient genotypes. Together, these genotypical and functional data suggest that the observed deficiency of lectin pathway activation is an effect of legionellosis rather than a risk factor for acquiring it. MBL and L-ficolin both revealed to act as acute-phase reactants in community-acquired pneumonia. However, the effect of genetic variants on this response differed between the two proteins. With MBL, the acute phase responsiveness was highly dependent upon the genotype. With L-ficolin, the genotype only determined variations in the protein levels, not in the acute-phase responsiveness itself. At the first glance MBL and L-ficolin look very much alike, with similar protein domains, multimeric structure, pattern recognition and complement activating properties. Yet the small differences in exactly these characteristics may give rise to essential differences in biological function, and this might have driven the different evolutionary strategies in shaping polymorphisms of the genes. In their similarities and differences, MBL and L-ficolin are an excellent example of one of nature’s successful strategies: creating necessary redundancy, without unnecessary abundance

Sarcoidosis is a systemic disorder of unknown etiology characterized by the formation of non-caseating granulomas, most often affecting the lungs, lymph nodes and skin. Sarcoidosis is characterized by a strong Thelper-1 driven immune reaction, making viruses or intracellular pathogens, such as Mycobacterium tuberculosis and Propionibacterium acnes, leading suspects as causative agents. The innate immune system is important in the first recognition of pathogens, mainly via recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs).The family of TLRs consists of 11 different receptors, each recognizing a distinct repertoire of microbial products. Innate immune responses can dictate the magnitude and direction of the subsequent adaptive immune response. The hypothesis of this thesis is that genetic and functional defects in TLRs are involved in the innate immune recognition of intracellular bacteria in sarcoidosis, and play a role in disease susceptibility and clinical outcome. Furthermore, it is hypothesized that mycobacteria and propionibacteria are possible antigenic triggers in sarcoidosis patients in The Netherlands. Genetic variation of 21 single nucleotide polymorphisms (SNPs) located in 7 genes were tested in sarcoidosis patients and healthy controls. The selected genes were TLR1, TLR2, TLR4, TLR6, TLR9, TLR10 and CD14 based on their capacity to recognize bacterial proteins. In vitro experiments on TLR-2 and TLR-9 function in sarcoidosis were also performed. Finally, using Interferon-Gamma Release Assays (IGRA), T-cell responses against mycobacteria and propionibacteria in sarcoidosis patients was assessed. The 4 genes associated with sarcoidosis are all involved in TLR-2 expression and/or function. A genetic association was found for TLR2 as well as for 3 different co-receptors of TLR-2, namely TLR1, TLR10 and CD14. Subjects carrying the TLR2 promoter genotype -16934 AA, which is suggested to be more prevalent in patients with chronic sarcoidosis, produce significantly more Tumor Necrosis Factor- alpha (TNF-?) upon stimulation with TLR-2 agonists compared to subjects without the AA-genotype. TNF-? is important in sarcoidosis inflammation and various clinical trials demonstrated that blocking TNF-? using antagonists such as infliximab or eternacept results in clinical improvement. In vitro experiments revealed that Peripheral Blood Mononuclear Cells (PBMCs) of sarcoidosis patients produce significantly less IL-23, important in the Thelper-17 pathway, upon stimulation with a TLR-9 agonist compared to PBMCs from healthy controls. Further studies are needed to clarify this observation. Latent tuberculosis infection was found in 5.3% of the sarcoidosis patients tested, which does not provide a strong links between previous M. tuberculosis infection and sarcoidosis in Dutch patients. Identification of a distinct subgroup of patients with propionibacteria as causative agents was not found based on the observation that these bacteria demonstrate mitogenic activity. In conclusion, genetic differences in TLR2 or its co-receptors are found in patients with a chronic form of sarcoidosis. This suggests that an aberrant TLR-2 function can predispose for developing chronic disease in patients already affected by sarcoidosis. Our in vitro data support this hypothesis. It seems that M. tuberculosis is not an important cause for sarcoidosis in Dutch patients. Further studies are needed to answer this question for propionibacteria.

Asplenic individuals are at increased risk for fulminant infections, especially with encapsulated bacteria such as Streptococcus pneumoniae, Haemophilus influenzae type b (Hib) and Neisseria meningitidis, and vaccination is indicated. An inventory was made of the awareness of the infectious risks and vaccination status of asplenic patients in the Netherlands. Less than 50% of the patients did receive adequate information about the infectious risks of their asplenic state. For S.pneumoniae, 88% of patients has received a vaccination. For Hib and Neisseria meningitidis, vaccination rates are considerably lower: 32% and 27% respectively are vaccinated. Only 17% of the patients had received all three vaccines (i.e. pneumococcal, meningococcal and Hib), in accordance with international recommendations. Patients were vaccinated with conjugated Hib, pneumococcal and meningococcal vaccines if they were not vaccinated according to international recommendations. In 92 patients, one dose of conjugated Hib vaccine was given. After a single dose of vaccine, 97% of the patients reached the chosen antibody threshold concentration of ? 1.0 g/mL. The responses to both pneumococcal 23-valent polysaccharide and 7-valent conjugated vaccines were analysed in 54 asplenic patients. After one dose of conjugate pneumococcal vaccine, 82% reached the chosen threshold of ? 1.0 g/mL for 5 out of 7 vaccine-serotypes. This percentage rose to 85% and 92% after a second dose of conjugate vaccine and one dose of polysaccharide vaccine, respectively. In 116 patients, the response to the meningococcal serogroup C (MenC) conjugate vaccine was studied. After a single dose of vaccine, 67% of the patients reached the chosen antibody threshold concentration of ? 2.0 g/mL MenC IgG. The quality of the antibodies (expressed as avidity maturation) was poor. After revaccination of patients with a suboptimal response, the response to vaccination rose from 67% to 78%, but again, with only marginal rise in avidity maturation. In 38 patients with breast cancer, the response to influenza virus vaccination was determined at day 4 or at day 16 of a chemotherapy cycle of 21 days. Patients on chemotherapy have significant lower antibody responses than healthy adults. When comparing the two different moments of vaccination during a chemotherapy cycle, vaccination early during the chemotherapy cycle induces better responses than does vaccination in the last week of the cycle. Follow-up studies in larger patient groups are needed to confirm this effect. The response to pneumococcal, Hib, meningococcal and DTP (diphtheria, poliomyelitis and tetanus) vaccines was studied in 26 patients starting at 12 months after allogeneic stem cell transplantation with reduced intensity conditioning regimens (allo-RIST). After two doses of conjugated pneumococcal vaccines, > 73% of the patients developed antibody levels ? 0.35 g/mL for all pneumococcal serotypes included in the vaccine, except for pneumococcal serotype 6B. For Hib and tetanus toxoid (TT), protective antibody levels were found in 77% and 96% of the patients respectively, after one dose of Hib vaccine and two doses of DTP vaccine. Thus, vaccination of allo-RIST patients starting at approximately 12 months post-transplantation induces adequate antibody concentrations in the majority of patients

Atopic diseases such as (atopic) eczema, food allergy, asthma, and allergic rhinitis are common diseases. The cumulative incidence during childhood is estimated to be 20 to 30%. In countries with a so called ‘’Western lifestyle’’ an increase in the prevalence of atopic diseases has been observed during the last decades. This increase may be due to a reduced exposure to microbial antigens, which is supposed to be a prerequisite for a normal development of the immune system (the hygiene hypothesis). The gastrointestinal tract is the largest surface in direct contact with the outer microbial environment and the intestines harbour the intestinal microbiota with approximately 1014 microbes. Enhanced presence of typical probiotic bacteria in the intestinal microbiota has been associated with reduced prevalence of atopic disease. These differences are already present before the development of atopic diseases, which supports a causal relationship. We aimed to further investigate the possibilities of primary prevention of atopic diseases by perinatal administration of probiotic bacteria and their mechanism of action. In preparation of a clinical trial, probiotic bacteria for primary prevention of atopic diseases in children were selected by their capacity to modulate cytokine responses. The desired immunomodulatory effect of probiotic bacteria would be reduction of Th2 function, and stimulation of Th1 function, tolerance and regulatory immune responses. Specific strains were shown to produce large amounts of IL-10 which coincided with low levels of IL-12p70. Furthermore lactic acid bacteria decreased the production of Th2 cytokines IL-5 and IL-13 which was IL-10 dependent for specific strains since neutralizing IL-10 production resulted in partial to full restoration of Th2 cytokine production and concurred with an increase in IL-12p70 and TNF-α. In addition, the effect of lactic acid bacteria on neonatal immune cells and dendritic cells was investigated and resulted in comparable outcomes. Based on these in vitro studies, B. bifidum, B. lactis, and Lc. Lactis (Ecologic Panda?) were selected. This multi-species probiotics was used in the clinical trial on primary prevention of atopic diseases by probiotic bacteria, the PandA study. In a double-blind, randomized, placebo-controlled trial, probiotic bacteria were administered prenatally to mothers of high-risk children (i.e. positive family history of atopic diseases) and to their offspring during the first 12 months of life. This particular combination of probiotic bacteria was shown to have a preventive effect on the incidence of eczema in high-risk children, which seems to be sustained during the first two years of life. In addition to previous studies, the preventive effect appeared to be established within the first three months of life. Extensive analysis of the intestinal microbiota showed that probiotic supplementation resulted in a significantly different composition of the intestinal microbiota compared to placebo. In addition, probiotic bacteria may only be capable to exert their clinical beneficial effects if they induced a richer and more diverse microbiota, and stimulated or inhibit the presence of specific bacterial species. Furthermore, results from the Panda study suggest that probiotic bacteria modulate the developing immune system but long term effects should be investigated in future studies.

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