Febrile Infections in Children with Leukemia with Special Reference to Respiratory Viral Infections

Leukemiaa sairastavien lasten kuumeiset infektiot, erityisesti respiratoriset virusinfektiot Tausta: Hengitysteiden virusinfektiot ovat lasten tavallisimpia sairauksia. Infektiota aiheuttava virus voidaan uusilla menetelmillä löytää lähes kaikissa tapauksissa. Leukemiaa sairastavilla lapsilla on perustaudin ja leukemian hoitojen takia tavallista suurempi infektioalttius, ja kuumeilu on tavallista leukemiahoidon aikana. Suurin osa syöpähoidon aikaisten kuumejaksojen syistä jää kuitenkin selvittämättä. Tavoitteet: Prospektiivisen 5 vuotta kestäneen monikeskustutkimuksen tavoitteena oli etsiä uusimmilla mikrobiologisilla menetelmillä leukemiaa sairastavien lasten kuumeen syy. Tätä varten tutkittiin 16 virusta virusviljelyllä, antigeeniosoituksella ja nukleiinihappo-osoituksella. Näytteitä otettiin nenästä, ulosteesta, virtsasta ja verestä. Lisäksi tutkittiin MxA-proteiinin kykyä osoittaa virusinfektio syöpälapsella. Tulokset: Tutkimuksen aikana analysoitiin 138 kuumejaksoa 51 leukemialapsella. Kokonaisseuranta-aika oli 1.5 vuotta/lapsi. Kuumejaksojen ilmaantuvuus oli 2.1 jaksoa potilasta kohden suhteutettuna vuoden riskiaikaan. Hengistysteiden virusinfektio voitiin osoittaa 82 kuumejaksossa (59%). Kaksi tai useampi virus löydettiin 12 %:ssa kuumejaksoista. Tavallisimmat virukset olivat rhinovirus (22 %), respiratory syncytial virus eli RS-virus (11 %), human herpes virus 6 (7 %), human bocavirus (5 %), sytomegalovirus (5 %), parainfluenssavirukset (5 %) ja influenssa A -virus (4 %). Kahdelle potilaalle kehittyi pneumonia, muilla oireet olivat lievät. Veriviljely oli positiivinen 19 kuumejaksossa (14 %), ja puolessa tapauksista löydettiin samanaikaisesti respiratorinen virus. MxA proteini ilmeni veren lymfosyyteissä useimmilla virusinfektioon sairastuneilla syöpälapsilla. Päätelmät: Kuumeiset respiratoriset virusinfektiot ovat tavallisia leukemiaa sairastavilla lapsilla. Infektion oireet ovat tavallisesti vähäiset, mutta pienelle osalle voi kehittyä veriviljelypositiivinen sepsis tai pneumonia. Kuumeen syy jäi selvittämättä vain harvoissa tapauksissa.

Virus Infections in Early Childhood, Cow’s Milk Formula Exposure and Genetic Predisposition in the Development of Diabetes-Associated Autoimmunity

Varhaislapsuuden virusinfektioiden, lehmänmaitopohjaisen äidinmaitovastikeen ja geneettisen alttiuden merkitys diabetekseen liittyvän autoimmuniteetin kehittymisessä Tyypin 1 diabetes on autoimmuunisairaus, joka syntyy haiman insuliinia tuottavien beta-solujen tuhouduttua elimistön oman immuunipuolustusjärjestelmän hyökkäyksen seurauksena. Sekä perimän että ympäristötekijöiden arvellaan vaikuttavan tautiprosessiin, mutta taudin tarkkaa syntymekanismia ei tunneta. Tutkimuksen tarkoituksena oli selvittää varhaislapsuuden ympäristötekijöiden vaikutusta beta-soluautoimmuniteetin syntyyn, erityispaino tutkimuksessa oli ympäristötekijöiden yhteisvaikutuksessa sekä geneettisten riskitekijöiden ja ympäristötekijöiden vuorovaikutuksessa. Varhaislapsuudessa sairastettu sytomegalovirus- tai enterovirusinfektio ei lisännyt beta-soluautoimmuniteetin riskiä lapsilla, joilla on geneettisesti kohonnut riski sairastua tyypin 1 diabetekseen. Ennen puolen vuoden ikää sairastettu rotavirusinfektio lisäsi hieman tyypin 1 diabetekseen liittyvän autoimmuniteetin riskiä. Tarkemmassa analyysissa varhaislapsuuden enterovirusinfektio osoittautui kuitenkin autovasta-aineiden muodostumisen riskitekijäksi niiden lasten joukossa, jotka olivat saaneet lehmänmaitopohjaista äidinmaidon vastiketta ensimmäisten elinkuukausien aikana. Tämä löydös viittaa enterovirusinfektion ja lehmänmaitopohjaisen vastikkeen yhteisvaikutukseen tyypin 1 diabetekseen liittyvän autoimmuniteetin synnyssä. Löydösten mukaan PTPN22 geenin C1858T polymorfismi vaikuttaa CD4+ T solujen aktivaatioon ja proliferaatiovasteeseen, 1858T alleeliin liittyy alentunut T-soluresepto-rivälitteinen aktivaatio. 1858T alleelin kantajuuteen liittyy lisäksi lisääntynyt autovasta-aineiden ja kliinisen diabeteksen ilmaantuvuus. Tämä yhteys rajoittui yksilöihin, jotka olivat altistuneet lehmänmaitopohjaiselle vastikkeelle ennen kuuden kuukauden ikää. Tulosten mukaan sekä ympäristötekijöiden väliset yhteisvaikutukset että perimä vaikuttavat yksittäisen ympäristötekijän merkitykseen tyypin 1 diabetekseen liittyvän autoimmuniteetin synnyssä. Nämä yhteisvaikutukset ympäristötekijöiden kesken ja perimän ja ympäristötekijöiden välillä selittävät aiemmin julkaistujen tulosten ristiriittaisuutta tutkimuksissa, joissa on analysoitu vain yhden ympäristötekijän vaikutusta diabeteksen ilmaantuvuuteen.

Genetic Basis and Diagnostics of Extended-Spectrum Beta-Lactamases among Enterobacteriaceae in Finland

Since the introduction of antibiotic agents, the amount and prevalence of Beta-lactam resistant enterobacteria has become an increasing problem. Many enterobacteria are opportunistic pathogens that easily acquire resistance mechanisms and genes, which make the situation menacing. These bacteria have acquired resistance and can hydrolyse extended spectrum cephalosporins and penicillins by producing enzymes called extended-spectrum Beta-lactamases (ESBLs). ESBL-producing bacteria are most commonly found in the gastro-intestinal tract of colonised patients. These resistant strains can be found in both health-care associated and community-acquired isolates. The detection and treatment of infections caused by bacteria producing ESBLs are problematic. This study investigated the genetic basis of extended-spectrum Beta-lactamases in Enterobacteriaceae, especially in Escherichia coli and Klebsiella pneumoniae isolates. A total of 994 Finnish Enterobacteriaceae strains, collected at 26 hospital laboratories, during 2000 and 2007 were analysed. For the genetic basis studies, PCR, sequencing and pyrosequencing methods were optimised. In addition, international standard methods, the agar dilution and disk diffusion methods were performed for the resistance studies, and the susceptibility of these strains was tested for antimicrobial agents that are used for treating patients. The genetic analysis showed that bla_CTX-M was the most prevalent gene among the E. coli isolates, while blaS_HV-12 was the most common Beta-lactamase gene in K. pneumoniae. The susceptibility testing results showed that about 60% of the strains were multidrug resistant. The prevalence of ESBL-producing isolates in Finland has been increasing since 2000. However, the situation in Finland is still much better than in many other European countries.

The Role of Integrins in Enterovirus Infections and in Metastasis of Cancer

Integrins are a family of transmembrane glycoproteins, composed of two different subunits (alpha and beta). Altered expression of integrins in tumor cells contributes to metastasis tendency by influencing on the cells‟ attachment to adjacent cells and their migration. Viral pathogens, including certain enteroviruses, use integrins as receptors. Enteroviruses have also been suggested to be involved in the etiopathogenesis of type 1 diabetes. The study focuses on the role of integrins in the pathogenesis of metastasis to cortical bone and on type 1 diabetes (T1D) and echovirus 1 infection. In the first part of the thesis, the role of different integrins in the initial attachment of MDA-MD-231 breast cancer cells to bovine cortical bone disks was studied. A close correlation between alpha2beta1 and alpha3beta1 integrin receptor expression and the capability of the tumor to attach to bone were observed. In the second part, a possible correlation between susceptibility to enterovirus infections in diabetic children and differences in enterovirus receptor genes, including certain integrins, was investigated. In parallel, virus-specific neutralizing antibodies and diabetic risk alleles were studied. In the diabetic group, an amino acid change was detected in the polio virus receptor and the neutralizing antibody titers against echovirus 30 were lower. However, to obtain statistically sustainable results, a larger number of individuals should be analyzed. Echovirus 1 (EV1) enters cells by attaching to the alpha2I domain of the alpha2beta1 integrin. In the third part EV1 was shown to attach to a chimeric receptor construct of the transferrin receptor and the alpha2I domain and to enter cells through clathrin-mediated endocytosis that is normally not used by the virus. The chimeric receptor was recycled to the plasma membrane, whereas the virus remained in intracellular vesicles. The virus replication cycle was initiated in these cells, suggesting that evolution pressure could possibly cause the virus to evolve to use a different entry mechanism. Moreover, a cDNA microarray analysis of host gene expression during EV1 replication showed that 0.53% of the total genes, including several immediate early genes, were differently expressed.

Outcomes of cervical human papillomavirus (HPV) infections among mothers in the Finnish Family HPV Study

To understand the natural history of cervical human papillomavirus (HPV)-infections, more information is needed on their genotype-specific prevalence, acquisition, clearance, persistence and progression. This thesis is part of the prospective Finnish Family HPV study. 329 pregnant women (mean age 25.5 years) were recruited during the third trimester of pregnancy and were followed up for 6 years. The outcomes of cervical HPV infections were evaluated among all the mothers participating in the study. Generalized estimating equation (GEE)-models and Poisson regression were used to estimate the risk factors of type-specific acquisition, clearance, persistence and progression of Species 7 and 9 HPV-genotypes. Independent protective factors against incident infections were higher number of life-time sexual partners, initiation of oral contraceptive use after age 20 years and becoming pregnant during FU. Older age and negative oral HR-HPV DNA status at baseline were associated with increased clearance, whereas higher number of current sexual partners decreased the probability of clearance. Early onset of smoking, practicing oral sex and older age increased the risk of type-specific persistence, while key predictors of CIN/SIL were persistent HR-HPV, abnormal Pap smear and new sexual partners. HPV16, together with multiple-type infections were the most frequent incident genotypes, most likely to remain persistent and least likely to clear. Collectively, LR-HPV types showed shorter incidence and clearance times than HR-HPV types. In multivariate models, different predictors were associated with these main viral outcomes, and there is some tentative evidence to suggest that oral mucosa might play a role in controlling some of these outcomes.

PET Imaging of Osteomyelitis. Feasibility of 18F-FDG, 68Ga-Chloride and 68Ga-DOTAVAP-P1 Tracers in Staphylococcal Bone Infections

Due to technical restrictions of the database system the title of the thesis does not show corretly on this page. Numbers in the title are in superscript. Please see the PDF-file for correct title. ---- Osteomyelitis is a progressive inflammatory disease of bone and bone marrow that results in bone destruction due to an infective microorganism, most frequently Staphylococcus aureus. Orthopaedic concern relates to the need for reconstructive and trauma-related surgical procedures in the fast grow¬ing population of fragile, aged patients, who have an increased susceptibility to surgical site infections. Depending on the type of osteomyelitis, infection may be acute or a slowly progressing, low-grade infection. Peri-implant infections lead to implant loosening. The emerging antibiotic resistance of com¬mon pathogens further complicates the situation. With current imaging methods, significant limitations exist in the diagnosing of osteomyelitis and implant-related infections. Positron emission tomography (PET) with a glucose analogue, 18F-fluoro¬deoxyglucose (18F-FDG), seems to facilitate a more accurate diagnosis of chronic osteomyelitis. The method is based on the increased glucose consumption of activated inflammatory cells. Unfortunately, 18F-FDG accumulates also in sterile inflammation regions and causes false-positive findings, for exam¬ple, due to post-operative healing processes. Therefore, there is a clinical need for new, more infection-specific tracers. In addition, it is still unknown why 18F-FDG PET imaging is less accurate in the detec¬tion of periprosthetic joint infections, most frequently due to Staphylococcus epidermidis. This doctoral thesis focused on testing novel PET tracers (68Ga-chloride and 68Ga-DOTAVAP-P1) for early detections of bone infections and evaluated the role of pathogen-related factors in the appli¬cations of 18F-FDG PET in the diagnostics of bone infections. For preclinical models of S. epidermidis and S. aureus bone/implant infections, the significance of the causative pathogen was studied with respect to 18F-FDG uptake. In a retrospective analysis of patients with confirmed bone infections, the significance of the presence or absence of positive bacterial cultures on 18F-FDG uptake was evalu¬ated. 18F-FDG and 68Ga-chloride resulted in a similar uptake in S. aureus osteomyelitic bones. However, 68Ga-chloride did not show uptake in healing bones, and therefore it may be a more-specific tracer in the early post-operative or post-traumatic phase. 68Ga-DOTAVAP-P1, a novel synthetic peptide bind¬ing to vascular adhesion protein 1 (VAP-1), was able to detect the phase of inflammation in healing bones, but the uptake of the tracer was elevated also in osteomyelitis. Low-grade peri-implant infec¬tions due to S. epidermidis were characterized by a low uptake of 18F-FDG, which reflects the virulence of the causative pathogen and the degree of leukocyte infiltration. In the clinical study, no relationship was found between the level of 18F-FDG uptake and the presence of positive or negative bacterial cul¬tures. Thus 18F-FDG PET may help to confirm metabolically active infection process in patients with culture-negative, histologically confirmed, low-grade osteomyelitis.

Virulence properties of Aggregatibacter actinomycetemcomitans biofilm and characterisation of its putative cytokine exploitation

Biofilms are surface-attached multispecies microbial communities that are embedded by their self-produced extracellular polymeric substances. This lifestyle enhances the survival of the bacteria and plays a major role in many chronic bacterial infections. For instance, periodontitis is initiated by multispecies biofilms. The phases of active periodontal tissue destruction and notably increased levels of proinflammatory mediators, such as the key inflammatory mediator interleukin (IL)-1beta, are typical of the disease. The opportunistic periodontal pathogen Aggregatibacter actinomycetemcomitans is usually abundant at sites of aggressive periodontitis. Despite potent host immune system responses to subgingival invaders, A. actinomycetemcomitans is able to resist clearance attempts. Moreover, some strains of A. actinomycetemcomitans can generate genetic diversity through natural transformation, which may improve the species’ adjustment tothe subgingival environment in the long term. Some biofilm forming species are known to bind and sense human cytokines. As a response to cytokines, bacteria may increase biofilm formation and alter their expression of virulence genes. Specific outer membrane receptors for interferon-γ or IL-1β have been characterised in two Gram-negative pathogens. Because little is known about periodontal pathogens’ ability to sense cytokines, we used A. actinomycetemcomitans as a model organism to investigate how the species responds to IL-1beta. The main aims of this thesis were to explore cytokine binding on single-species A. actinomycetemcomitans biofilms and to determine the effects of cytokines on the biofilm formation and metabolic activity of the species. Additionally, the cytokine’s putative internalisation and interaction with A. actinomycetemcomitans proteins were studied. The possible impact of biofilm IL-1beta sequestering on the proliferation and apoptosis of gingival keratinocyte cells was evaluated in an organotypic mucosa co-culture model. Finally, the role of the extramembranous domain of the outer membrane protein HofQ (emHofQ) in DNA binding linked to DNA uptake in A. actinomycetemcomitans was examined. Our main finding revealed that viable A. actinomycetemcomitans biofilms can bind and take up the IL-1β produced by gingival cells. At the sites of pathogen-host interaction, the proliferation and apoptosis of gingival keratinocytes decreased slightly. Notably, the exposure of biofilms to IL-1beta caused their metabolic activity to drop, which may be linked to the observed interaction of IL-1beta with the conserved intracellular proteins DNA binding protein HU and the trimeric form of ATP synthase subunit beta. A Pasteurellaceaespecific lipoprotein, which had no previously determined function, was characterized as an IL-1beta interacting membrane protein that was expressed in the biofilm cultures of all tested A. actinomycetemcomitans strains. The use of a subcellular localisation tool combined with experimental analyses suggested that the identified lipoprotein, bacterial interleukin receptor I (BilRI), may be associated with the outer membrane with a portion of the protein oriented towards the external milieu. The results of the emHofQ study indicated that emHofQ has both the structural and functional capability to bind DNA. This result implies that emHofQ plays a role in DNA assimilation. The results from the current study also demonstrate that the Gram-negative oral species appears to sense the central proinflammatory mediator IL-1beta.