Interplay of Virulence Factors in Complement Resistance of Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) is a normal inhabitant of the human nasopharynx. Symptoms occur in only a small proportion of those who become carriers, but the ubiquity of the organism in the human population results in a large burden of disease. S. pneumoniae is the leading bacterial cause of pneumonia, sepsis, and meningitis worldwide, causing the death of a million children each year. Middle-ear infection is the most common clinical manifestation of mucosal pneumococcal infections. In invasive disease, S. pneumoniae gains access to the bloodstream and spreads to normally sterile parts of the body. The progression from asymptomatic colonization to disease depends on factors characteristic of specific pneumococcal strains as well as the status of host defenses. The polysaccharide capsule surrounding the bacterium is considered to be the most important factor affecting the virulence of pneumococci. It protects pneumococci from phagocytosis and also may determine its affinity to the respiratory epithelium. S. pneumoniae as a species comprises more than 90 different capsular serotypes, but not all of them are equally prevalent in human diseases. Invasive serotypes are rarely isolated from healthy carriers, but relatively often cause invasive disease. Serotypes that are carried asymptomatically for a long time behave like opportunistic pathogens, causing disease in patients who have impaired immune defenses. The complement system is a collection of blood and cell surface proteins that act as a major primary defense against invading microbes. Phagocytic cells with receptors for complement proteins can engulf and destroy pneumococcal cells opsonized with these proteins. S. pneumoniae has evolved a number of ways to subvert mechanisms of innate immunity, and this is likely to contribute to its pathogenicity. The capsular serotype, proteins essential for virulence, as well the genotype, may all influence the ability of pneumococcus to resist complement and its potential to cause disease. Immunization with conjugate vaccines produces opsonic antibodies, which enhance complement deposition and clearance of the bacteria. The pneumococcal vaccine included in the Finnish national immunization program in 2010 contains the most common serotypes causing invasive disease. Clinical data suggest that protection from middle-ear infection and possibly also from invasive disease depends largely on the capsular serotype, for reasons hitherto unknown. The general aim of this thesis is to assess the relative roles of the pneumococcal capsule and virulence proteins in complement evasion and subsequent opsonophagocytic killing. The main question is whether differences between serotypes to resist complement explain the different abilities of serotypes to cause disease. The importance of particular virulence factors to the complement resistance of a strain may vary depending on its genotype. Prior studies have evaluated the effect of the capsule and virulence proteins on complement resistance of S. pneumoniae by comparing only a few strains. In this thesis, the role of pneumococcal virulence factors in the complement resistance of the bacterium was studied in several genotypically different strains. The ability of pneumococci to inhibit deposition of the complement protein C3 on the bacterial surface was found to depend on the capsular serotype as well as on other features of the bacteria. The results suggest that pneumococcal histidine triad (Pht) proteins may play a role in complement inhibition, but their contribution depends on the bacterial genotype. The capsular serotype was found to influence complement resistance more than the bacterial genotype. A higher concentration of anticapsular antibodies was required for the opsonophagocytic killing of serotypes resistant to C3 deposition. The invasive serotypes were more resistant to C3 deposition than the opportunistic serotypes, suggesting that the former are better adapted to resist immune mechanisms controlling the development of invasive disease. The different susceptibilities of serotypes to complement deposition, opsonophagocytosis, and resultant antibody-mediated protection should be taken into account when guidelines for serological correlates for vaccine efficacy evaluations are made. The results of this thesis suggest that antibodies in higher quantity or quality are needed for efficient protection against the invasive serotypes.

Molecular methods for the epidemiological analysis of methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae

Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae are major health problems worldwide, both found in symptomless carriage but also causing even life-threatening infections. The aim of this thesis was to characterise MRSA and S. pneumoniae in detail by using several molecular typing methods for various epidemiological purposes: clonality analysis, epidemiological surveillance, outbreak investigation, and virulence factor analysis. The characteristics of MRSA isolates from the strain collection of the Finnish National Infectious Disease Register (NIDR) and pneumococcal isolates collected from military recruits and children with acute otitis media (AOM) were analysed using various typing techniques. Antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and the detection of Panton-Valentine leukocidin (PVL) genes were performed for MRSA isolates. Pneumococcal isolates were analysed using antimicrobial susceptibility testing, serotyping, MLST, and by detecting pilus islet 1 (PI-1) and 2 (PI-2) genes. Several international community- and hospital-associated MRSA clones were recognised in Finland. The genetic diversity among MRSA FIN-4 isolates and among FIN-16 isolates was low. Overall, MRSA blood isolates from 1997 to 2006 were genetically diverse. spa typing was found to be a highly discriminatory, rapid and accurate typing method and it also qualifies as the primary typing method in countries with a long history of PFGE-based MRSA strain nomenclature. However, additional typing by another method, e.g. PFGE, is needed in certain situations to be able to provide adequate discrimination for epidemiological surveillance and outbreak investigation. An outbreak of pneumonia was associated with one pneumococcal strain among military recruits, previously healthy young men living in a crowded setting. The pneumococcal carriage rate after the outbreak was found to be exceptionally high. PI-1 genes were detected at a rather low prevalence among pneumococcal isolates from children with AOM. However, the study demonstrated that PI-1 has existed among pneumococcal isolates prior to pneumococcal conjugate vaccine and the increased antimicrobial resistance era. Moreover, PI-1 was found to associate with the serotype rather than the genotype. This study adds to our understanding of the molecular epidemiology of MRSA strains in Finland and the importance of an appropriate genotyping method to be able to perform high-level laboratory-based surveillance of MRSA. Epidemiological and molecular analyses of S. pneumoniae add to our knowledge of the characteristics of pneumococcal strains in Finland.

Probiotics in the prevention of clinical manifestations of common infectious diseases in children and in the elderly

Infectious diseases put an enormous burden on both children and the elderly in the forms of respiratory, gastrointestinal and oral infections. There is evidence suggesting that specific probiotics may be antagonistic to pathogens and may enhance the immune system, but the clinical evidence is still too sparce to make general conclusions on the disease-preventive effects of probiotics. This thesis, consisting of four independent, double-blind, placebo-controlled clinical trials, investigated whether Lactobacillus GG (LGG) or a specific probiotic combination containing LGG would reduce the risk of common infections or the prevalence of pathogens in healthy and infection-prone children and in independent and institutionalised elderly people. In healthy day-care children, the 7-month consumption of probiotic milk containing Lactobacillus GG appeared to postpone the first acute respiratory infection (ARI) by one week (p=0.03, adjusted p=0.16), and to reduce complicated infections (39% vs. 47%, p<0.05, adjusted p=0.13), as well as the need for antibiotic treatment (44% vs. 54%, p=0.03, adjusted p=0.08) and day-care absences (4.9 vs. 5.8 days, p=0.03, adjusted p=0.09) compared to the placebo milk. In infection-prone children, the 6-month consumption of a combination of four probiotic bacteria (LGG, L. rhamnosus LC705, Propionibacterium freudenreichii JS, Bifidobacterium breve 99) taken in capsules appeared to reduce recurrent ARIs (72% vs. 82%, p<0.05; adjusted p=0.06), and the effect was particularly noticeable in a subgroup of children with allergic diseases (12% vs. 33%, p=0.03), although no effect on the presence of nasopharyngeal rhinovirus or enterovirus was seen. The 5-month consumption of the same probiotic combination did not show any beneficial effects on the respiratory infections in frail, institutionalised elderly subjects. In healthy children receiving Lactobacillus GG, the reduction in complications resulted in a marginal reduction in the occurrence of acute otitis media (AOM) (31% vs. 39%, p=0.08; adjusted p=0.19), and the postponement of the first AOM episode by 12 days (p=0.04; adjusted p=0.09). However, in otitis-prone children, a probiotic combination did not reduce the occurrence of AOM or the total prevalence of common AOM pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis), except in the case of children with allergic diseases, in whom probiotics reduced recurrent AOM episodes (0% vs. 14%, p=0.03). In addition, interaction between probiotics and bacterial carriage was seen: probiot-ics reduced AOM in children who did not carry any bacterial pathogens (63% vs. 83%), but the effect was the reverse in children carrying bacteria in the nasopharynx (74% vs 62%) (p<0.05). Long-term probiotic treatment, either LGG given in milk to healthy children for 7 months or a combination of probiotics given in capsules to institutionalised elderly subjects for 5 months, did not reduce the occurrence of acute diarrhoea. However, when the probiotic combination (LGG, L. rhamnosus LC705, Propionibacterium JS) was given in cheese to independent elderly subjects for 4 months, the oral carriage of high Candida counts was reduced in the probiotic group vs. the placebo group (21% vs. 34%, p=0.01, adjusted p=0.004). The risk of hyposalivation was also reduced in the probiotic group (p=0.05). In conclusion, probiotics appear to slightly alleviate the severity of infections by postponing their appearance, by reducing complications and the need for antimicrobial treatments. In addition, they appear to prevent recurrent infections in certain subgroups of children, such as in infection-prone children with allergic diseases. Alleviating ARI by probiotics may lead to a marginal reduction in the occurrence of AOM in healthy children but not in infection-prone children with disturbed nasopharyngeal microbiota. On the basis of these results it could be supposed that Lactobacillus GG or a specific combination containing LGG are effective against viral but not against bacterial otitis, and the mechanism is probably mediated through the stimulation of the immune system. A specific probiotic combination does not reduce respiratory infections in frail elderly subjects. Acute diarrhoea, either in children or in the elderly, is not prevented by the continuous, long-term consumption of probiotics, but the consumption of a specific probiotic combination in a food matrix is beneficial to the oral health of the elderly, through the reduction of the carriage of Candida.

Transcriptional analysis of persistent Chlamydia pneumoniae infection in vitro

Chlamydia pneumoniae can cause acute respiratory infections including pneumonia. Repeated and persistent Chlamydia infections occur and persistent C. pneumoniae infection may have a role in the pathogenesis of atherosclerosis and coronary heart disease and may also contribute to the development of chronic inflammatory lung diseases like chronic obstructive pulmonary disease (COPD) and asthma. In this thesis in vitro models for persistent C. pneumonia infection were established in epithelial and monocyte/macrophage cell lines. Expression of host cell genes in the persistent C. pneumoniae infection model of epithelial cells was studied by microarray and RT-PCR. In the monocyte/macrophage infection model expression of selected C. pneumoniae genes were studied by RT-PCR and immunofluorescence microscopy. Chlamydia is able to modulate host cell gene expression and apoptosis of host cells, which may assist Chlamydia to evade the host cells' immune responses. This, in turn, may lead to extended survival of the organism inside epithelial cells and promote the development of persistent infection. To simulate persistent C. pneumoniae infection in vivo, we set up a persistent infection model exposing the HL cell cultures to IFN-gamma. When HL cell cultures were treated with moderate concentration of IFN-gamma, the replication of C. pneumoniae DNA was unaffected while differentiation into infectious elementary bodies (EB) was strongly inhibited. By transmission electron microscopy small atypical inclusions were identified in IFN-gamma treated cultures. No second cycle of infection was observed in cells exposed to IFN-gamma , whereas C. pneumoniae was able to undergo a second cycle of infection in unexposed HL cells. Although monocytic cells can naturally restrict chlamydial growth, IFN-gamma further reduced production of infectious C. pneumoniae in Mono Mac 6 cells. Under both studied conditions no second cycle of infection could be detected in monocytic cell line suggesting persistent infection in these cells. As a step toward understanding the role of host genes in the development and pathogenesis of persistent C. pneumoniae infection, modulation of host cell gene expression during IFN-gamma induced persistent infection was examined and compared to that seen during active C. pneumoniae infection or IFN-gamma treatment. Total RNA was collected at 6 to 150 h after infection of an epithelial cell line (HL) and analyzed by a cDNA array (available at that time) representing approximately 4000 human transcripts. In initial analysis 250 of the 4000 genes were identified as differentially expressed upon active and persistent chlamydial infection and IFN-gamma treatment. In persistent infection more potent up-regulation of many genes was observed in IFN-gamma induced persistent infection than in active infection or in IFN-gamma treated cell cultures. Also sustained up-regulation was observed for some genes. In addition, we could identify nine host cell genes whose transcription was specifically altered during the IFN-gamma induced persistent C. pneumoniae infection. Strongest up-regulation in persistent infection in relation to controls was identified for insulin like growth factor binding protein 6, interferon-stimulated protein 15 kDa, cyclin D1 and interleukin 7 receptor. These results suggest that during persistent infection, C. pneumoniae reprograms the host transcriptional machinery regulating a variety of cellular processes including adhesion, cell cycle regulation, growth and inflammatory response, all of which may play important roles in the pathogenesis of persistent C. pneumoniae infection. C. pneumoniae DNA can be detected in peripheral blood mononuclear cells indicating that the bacterium can also infect monocytic cells in vivo and thereby monocytes can assist the spread of infection from the lungs to other anatomical sites. Persistent infection established at these sites could promote inflammation and enhance pathology. Thus, the mononuclear cells are in a strategic position in the development of persistent infection. To investigate the intracellular replication and fate of C. pneumoniae in mononuclear cells we analyzed the transcription of 11 C. pneumoniae genes in Mono Mac 6 cells during infection by real time RT-PCR. Our results suggest that the transcriptional profile of the studied genes in monocytes is different from that seen in epithelial cells and that IFN-gamma has a less significant effect on C. pneumoniae transcription in monocytes. Furthermore, our study shows that type III secretion system (T3SS) related genes are transcribed and that Chlamydia possesses a functional T3SS during infection in monocytes. Since C. pneumoniae infection in monocytes has been implicated to have reduced antibiotic susceptibility, this creates opportunities for novel therapeutics targeting T3SS in the management of chlamydial infection in monocytes.

Mother-Infant Antibodies to Pneumococcal Polysaccharides and Proteins : Transfer and Persistence of Maternal Antibodies and Development of Vaccine-Induced and Naturally Acquired Antibodies in Infants

Symptomless nasopharyngeal carriage of Streptococcus pneumoniae (pneumococcus) is very common in young children. Occasionally the carriage proceeds into mild mucosal diseases, such as sinusitis or acute otitis media, or into serious life-threatening diseases, such as pneumonia, sepsis or meningitis. Each year, up to one million children less than five years of age worldwide die of invasive pneumococcal diseases (IPD). Especially in the low-income countries IPD is a leading health problem in infants; 75% of all IPD cases occur before one year of age. This stresses the need of increased protection against pneumococcus in infancy. Anti-pneumococcal antibodies form an important component in the defence against pneumococcal infection. Maternal immunisation and early infant immunisation are two possible ways by which potentially protective antibody concentrations against pneumococci could be achieved in early infancy. The aim of this thesis is to increase the knowledge of antibody mediated protection against pneumococcal disease in infants and young children. We investigated the transfer of maternal anti-pneumococcal antibodies from Filipino mothers to their infants, the persistence of the transferred antibodies in the infants, the immunogenicity of the 23-valent pneumococcal polysaccharide vaccine (PPV) in infants and the response of the children to a second dose of PPV at three years of age. We also investigated the development of antibodies to pneumococcal protein antigens in relation to culture-confirmed pneumococcal carriage in infants. Serum samples were collected from the mothers, the umbilical cords and from the infants at young age as well as at three years of age. The samples were used to determine the antibody concentrations to pneumococcal serotypes 1, 5, 6B, 14, 18C and 19F, as well as to the pneumococcal proteins PspA, PsaA, Ply, PspC, PhtD, PhtDC and LytC by the enzyme immunoassay. The findings of the present study confirm previously obtained results and add to the global knowledge of responses to PPV in young children. Immunising pregnant women with PPV provides the infants with increased concentrations of pneumococcal polysaccharide antibodies. Of the six serotypes examined, serotypes 1 and 5 were immunogenic already in infants. At three years of age, the children responded well to the second dose of PPV suggesting that maternal and early infant immunisations might not induce hyporesponsiveness to polysaccharide antigens after subsequent immunisations. The anti-protein antibody findings provide useful information for the development of pneumococcal protein vaccines. All six proteins studied were immunogenic in infancy and the development of anti-protein antibodies started early in life in relation to pneumococcal carriage.

Pain in juvenile idiopathic arthritis : parents and children as agents of disease management

Juvenile idiopathic arthritis (JIA) is a severe childhood disease usually characterized by long-term morbidity, unpredictable course, pain, and limitations in daily activities and social participation. The disease affects not only the child but also the whole family. The family is expected to adhere to an often very laborious regimen over a long period of time. However, the parental role is incoherently conceptualized in the research field. Pain in JIA is of somatic origin, but psychosocial factors, such as mood and self-efficacy, are critical in the perception of pain and in its impact on functioning. This study examined the factors correlating and possibly explaining pain in JIA, with a special emphasis on the mutual relations between parent- and patient-driven variables. In this patient series pain was not associated with the disease activity. The degree of pain was on average fairly low in children with JIA. When the children were clustered according to age, anxiety and depression, four distinguishable cluster groups significantly associated with pain emerged. One of the groups was described by concept vulnerability because of unfavorable variable associations. Parental depressive and anxiety symptoms accompanied by illness management had a predictive power in discriminating groups of children with varying distress levels. The parent’s and child’s perception of a child’s functional capability, distress, and somatic self-efficacy had independent explanatory power predicting the child’s pain. Of special interest in the current study was self-efficacy, which refers to the belief of an individual that he/she has the ability to engage in the behavior required for tackling the disease. In children with JIA, strong self-efficacy was related to lower levels of pain, depressive symptoms and trait anxiety. This suggests strengthening a child’s sense of self-efficacy, when helping the child to cope with his or her disease. Pain experienced by a child with JIA needs to be viewed in a multidimensional bio-psycho-social context that covers biological, environmental and cognitive behavioral mechanisms. The relations between the parent-child variables are complex and affect pain both directly and indirectly. Developing pain-treatment modalities that recognize the family as a system is also warranted.

Language in Acquisition : Early Lexical Development and Associations between Lexicon and Grammar Findings from Full-Term and Very-Low-Birth-Weight Finnish Children

The aim was to analyse the growth and compositional development of the receptive and expressive lexicons between the ages 0,9 and 2;0 in the full-term (FT) and the very-low-birth-weight (VLBW) children who are acquiring Finnish. The associations between the expressive lexicon and grammar at 1;6 and 2;0 in the FT children were also studied. In addition, the language skills of the VLBW children at 2;0 were analysed, as well as the predictive value of early lexicon to the later language performance. Four groups took part in the studies: the longitudinal (N = 35) and cross-sectional (N = 146) samples of the FT children, and the longitudinal (N = 32) and cross-sectional (N = 66) samples of VLBW children. The data was gathered by applying of the structured parental rating method (the Finnish version of the Communicative Development Inventory), through analysis of the children´s spontaneous speech and by administering a a formal test (Reynell Developmental Language Scales). The FT children acquired their receptive lexicons earlier, at a faster rate and with larger individual variation than their expressive lexicons. The acquisition rate of the expressive lexicon increased from slow to faster in most children (91%). Highly parallel developmental paths for lexical semantic categories were detected in the receptive and expressive lexicons of the Finnish children when they were analysed in relation to the growth of the lexicon size, as described in the literature for children acquiring other languages. The emergence of grammar was closely associated with expressive lexical growth. The VLBW children acquired their receptive lexicons at a slower rate and had weaker language skills at 2;0 than the full-term children. The compositional development of both lexicons happened at a slower rate in the VLBW children when compared to the FT controls. However, when the compositional development was analysed in relation to the growth of lexicon size, this development occurred qualitatively in a nearly parallel manner in the VLBW children as in the FT children. Early receptive and expressive lexicon sizes were significantly associated with later language skills in both groups. The effect of the background variables (gender, length of the mother s basic education, birth weight) on the language development in the FT and the VLBW children differed. The results provide new information of early language acquisition by the Finnish FT and VLBW children. The results support the view that the early acquisition of the semantic lexical categories is related to lexicon growth. The current findings also propose that the early grammatical acquisition is closely related to the growth of expressive vocabulary size. The language development of the VLBW children should be followed in clinical work.

Intergenerational Transfer of Sabbath Observance : How is Religious Tradition Transferred to Children among Finnish Seventh-day Adventists?

This study examined religious home education in educational, psychological, and sociological context. Growing up within a religious denomination is a process of learning the rules, norms, opinions, and attitudes, which serve to make the individual an active member of the group. It is a process of transferring the cultural inheritance between generations. Sabbathkeeping can be regarded as a strong indicator of the Seventh-day Adventist value system, which is also why I have concentrated on this specific issue in my study. The purpose of the study was to find out, how the Sabbath is transferred from parents to children among Finnish Adventists. It was also examined how parents could make the day of rest positively exceptional for children, and how the parental authoritativeness affects the process of transference. According to Bull & Lockhart s (1989) theory, the amount of Adventist generations in family history influences the transfer of religious tradition. This study aimed to find out whether or not this theory would apply to the present-day Finland. The nature of religious development among Adventist young people was also one of the interests of the research. The methods used in the study were in-depth interviews (n = 10) and a survey (n = 106). The majority of the interviewees was young adults (age 15-30) grown up in Adventist families. The interviews were taped and transcribed for the study, and survey answers were analysed with SPSS-data analysis program. The amount of survey questionnaires evaluated was 106, whole population of 15-30 year-old Finnish Adventists being about one thousand. Democratic relationship between parents and children, parents' example, encouragement to own thinking, and positive experiences of Sabbath and the whole religion, including the social dimension of the Adventism, seem to be some of the most significant factors in transference of religious tradition. Both too severe and too permissive education were considered to lead to similar results: unsuccessful transfer of values, or even rebellion and adopting a totally opposite way of life than that of the parents. In this study the amount of Adventist generations in family history does not correlate significantly with the end results of value transference. Keywords: Sabbath, intergenerational, value transference, religious home education Avainsanat: sapatti, arvojen siirtyminen vanhemmilta lapsille, uskonnollinen kotikasvatus

Characterization of cytokines, matrix metalloproteinases and toll-like receptors in human periodontal tissue destruction

Periodontal Disease affects the supporting structures of the teeth and is initiated by a microbial biofilm called dental plaque. Severity ranges from superficial inflammation of the gingiva (gingivitis) to extensive destruction of connective tissue and bone leading to tooth loss (periodontitis). In periodontitis the destruction of tissue is caused by a cascade of microbial and host factors together with proteolytic enzymes. Matrix metalloproteinases (MMPs) are known to be central mediators of the pathologic destruction in periodontitis. Initially plaque bacteria provide pathogen-associated molecular patterns (PAMPs) which are sensed by Toll-like receptors (TLRs), and initiate intracellular signaling cascades leading to host inflammation. Our aim was to characterize TNF-α (tumor necrosis factor-alpha) and its type I and II receptors in periodontal tissues, as well as, the effects of TNF-α, IL-1β (interleukin-1beta) and IL-17 on the production and/or activation of MMP-3, MMP-8 and MMP-9. Furthermore we mapped the TLRs in periodontal tissues and assessed how some of the PAMPs binding to the key TLRs found in periodontal tissues affect production of TNF-α and IL-1β by gingival epithelial cells with or without combination of IL-17. TNF-α and its receptors were detected in pericoronitis. Furthermore, increased expression of interleukin-1β and vascular cell adhesion molecule-1 was found as a biological indicator of TNF-α ligand-receptor interaction. MMP-3, -8, and 9 were investigated in periodontitis affected human gingival crevicular fluid and gingival fibroblasts produced pro-MMP-3. Following that, the effect of IL-17 was studied on MMP and pro-inflammatory cytokine production. IL-17 was increased in periodontitis and up-regulated IL-1β, TNF-α, MMP-1 and MMP-3. We continued by demonstrating TLRs in gingival tissues, in which significant differences between patients with periodontitis and healthy controls were found. Finally, enzyme-linked immunosorbent assays were performed to show that the gingival cells response to inflammatory responses in a TLR-dependent manner. Briefly, this thesis demonstrates that TLRs are present in periodontal tissues and present differences in periodontitis compared to healthy controls. The cells of gingival tissues respond to inflammatory process in a TLR-dependent manner by producing pro-inflammatory cytokines. During the destruction of periodontal tissues, the release (IL-1β and TNF-α) and co-operation with other pro-inflammatory cytokines (IL-17), which in turn increase the inflammation and thus be more harmful to the host with the increased presence of MMPs (MMP-1, MMP-3, MMP-8, MMP-9) in diseased over healthy sites.