Borrelia burgdorferi evades the effects of ceftriaxone treatment in a mouse model of Lyme borreliosis

Borrelia burgdorferi infektoitujen hiirten antibioottihoidon jälkeinen oireilu Lymen borrelioosi on puutiaisten välittämä monimuotoinen infektiotauti, jonka tunnetuin oire on ns. vaeltava ihottuma eli erythema migrans. Muita tavallisia ilmentymiä ovat erityisesti nivel- ja hermosto-oireet sekä harvemmin sydän- ja silmäoireet. Suurin osa potilaista paranee täysin terveeksi antibioottihoidon avulla, mutta jopa 10 % borrelioosiin sairastuneista oireilee suositusten mukaisesta hoidosta huolimatta. Pitkittyneen oireilun on ajateltu johtuvan mm. infektion laukaisemasta autoimmuunitaudista tai kroonisesta infektiosta, mutta teorioiden tueksi ei ole kyetty esittämään kiistattomia todisteita. Onkin todennäköistä, että antibioottihoidon jälkeisen oireilun takana on useampia mekanismeja eikä yksi teoria selitä kaikkien potilaiden oireilua. Tässä väitöskirjatyössä on tutkittu hoidonjälkeistä borrelioosia hiirimallin avulla. Varhaisvaiheessa (2 viikkoa infektoinnin jälkeen) annettu antibiootti vähensi hiirten nivelturvotusta ja esti B. burgdorferi – bakteerin kasvun kudoksista otetuista näytteissä. Hoidettu¬jen hiirten B. burgdorferi -spesifiset IgG-luokan vasta-aineet pysyivät kuitenkin koholla ja osasta kudosnäytteistä löytyi B. burgdorferi:n DNA:ta PCR-tutkimuksen avulla. Mikäli hiiret hoidettiin myöhäisessä vaiheessa (yli 18 viikkoa infektoinnista) tulokset olivat muuten samanlaiset, mutta keftriaksoni ei vaikuttanut nivelturvotukseen. Näin hiirissä oli aikaansaatu tilanne, joka on hyvin samankaltainen ihmisen hoitoresistentin borrelia-artriitin kanssa: oireet jatkuvat, mutta taudinaiheuttajaa ei saada esiin. Inflammaatiota vaimentavaa anti-TNF-alphaa on käytetty nivelreuman hoidossa menestyksekkäästi huonosti muuhun hoitoon reagoivilla potilailla ja siitä syystä sen ajateltiin voivan vaikuttaa suotuisasti myös B. burgdorferi -infektoitujen hiirten hoidonjälkeiseen nivelturvotukseen. Sillä ei kuitenkaan ollut vaikutusta nivelturvotukseen, mutta yllättäen hoidon jälkeen osa hiirten kudosnäytteistä osoittautui viljelypositiivisiksi. On siis ilmeistä, että hiirimallissamme osa B. burgdorferi spirokeetoista pystyy välttämään keftriaksonihoidon vaikutuksen joko hakeutumalla elimistössä kudokseen, jossa antibiootin pitoisuus ei nouse riittävän korkeaksi, tai ne kykenevät muuntautumaan metabolisesti inaktiiviin tilaan eikä mikrobilääke yhdessä immuunipuolustuksen kanssa onnistu tappamaan niitä. Jatkotutkimuksissa selvitimme B. burgdorferi - spirokeetan mahdollista piilopaikkaa tutkimalla antibioottihoidon jälkeen useita eri kudoksia PCR-menetelmällä. Tulosten perusteella spirokeetta näyttää suosivan nivelkudosta tai soluja, joita esiintyy nivelessä runsaasti. On kuitenkin edelleen epäselvää, missä muodossa B. burgdorferi –spirokeetat säilyvät kudoksessa antibioottihoidon jälkeen.

Tartrate-Resistant Acid Phosphatase 5b: a Serum Marker of Bone Resorption

Bone is a physiologically dynamic tissue being constantly regenerated throughout life as a consequence of bone turnover by bone-resorbing osteoclasts and bone-forming osteoblasts. In certain bone diseases, such as osteoporosis, the imbalance in bone turnover leads to bone loss and increased fracture risk. Measurement of bone mineral density (BMD) predicts the risk of fracture, but also biochemical markers of bone metabolism have been suggested to be suitable for prediction of fractures and monitoring the efficacy of antiresorptive treatment. Tartrate-resistant acid phosphatase 5b (TRACP 5b) is an enzyme released from osteoclasts into the circulation, from where it can be detected kinetically or immunologically. Conventional assays for serum total TRACP were spectrophotometric and suffered from interference by other acid phosphatases and non-osteoclastic TRACP 5a isoform. Our aim was to develop novel immunoassays for osteoclastic TRACP 5b. Serum TRACP 5b levels were elevated in individuals with high bone turnover, such as children, postmenopausal women, patients with osteoporosis, Paget’s disease and breast cancer patients with bone metastases. As expected, hormone replacement therapy (HRT) in postmenopausal women decreased the levels of serum TRACP 5b. Surprisingly, the highest TRACP 5b levels were observed in individuals with rare autosomal dominant osteopetrosis type II (ADO2), which is characterized by high BMD and fracture risk with simultaneously elevated levels of deficient osteoclasts. In ADO2 patients, elevated levels of serum TRACP 5b were associated with high fracture frequency. It is likely that serum TRACP 5b reflects the number of inactive osteoclasts in ADO2. Similar results supporting the hypothesis that TRACP 5b would reflect the number of osteoclasts instead of their activity were observed with cultured osteoclasts and in animal models. Novel TRACP 5b immunoassays may prove to be of value either as independent or combinatory tools with other bone metabolic markers and BMD measurements in clinical practice and bone research.

Regenerated cellulose ultrafiltration membranes in the treatment of pulp and paper mill process waters

Ultrafiltration (UF) is widely applied in different separation processes in the pulp and paper industry. The growing need to protect the environment, a lack of pure water and an interest in producing high-value chemicals from compounds present in process waters will probably lead to an increase in the use of UF in the pulp and paper industry. The efficiency and cost-effectiveness of a UF process depends on the applied membrane. The membrane should have a high and stable filtration capacity, a particular selectivity and a long operational lifetime. To meet these requirements a membrane should have a low fouling tendency. In addition, it should withstand the prevailing operational and chemical conditions. This thesis evaluates the performance and applicability of the regenerated cellulose (RC) membranes 00030T and C2 in the treatment of pulp and paper mill process waters based on the requirements above. The results demonstrated that both the tested RC membranes fulfilled well the requirement of high filtration capacity. In addition, in the filtration of a paper mill clear filtrate (CF) the RC membranes were not as greatly affected by variations in the CF quality as a polysulphone membrane. Furthermore, due to their extreme hydrophilicity and weak charge the fouling tendency of the membranes can be expected to be low in pulp and paper mill filtration applications. It is, however, known that fouling cannot be totally avoided even when the membrane is chosen very carefully. This study indicated that carbohydrates influenced negatively on permeability and caused fouling in the filtration of groundwood mill circulation water. Thus, a pre-treatment effectively reducing the amount of carbohydrates might help to maintain a stable capacity. However, the results of the thesis also showed that the removal of some of the possible foulants might just increase the harmful effect of others. Multivariate examination was useful in the understanding of the complicated factors causing the unstable capacity. The thesis also revealed that the 00030T and C2 membranes can be used at high pressure (max. tested pressure 12 bar). The C2 membrane, having a sponge-like substructure, was more pressure resistant, and its performance was more stable at high pressure compared to the UCO30T membrane containing macrovoids in its substructure. Both tested membranes can, according to the results, also be used at temperatures as high as 70°C in acidic, neutral and alkaline conditions. However, the use at extreme conditions might cause faster ageing of the membranes compared to ageing in neutral conditions. The thesis proved that both the tested RC membranes are very suitable for pulp and paper mill applications and that the membranes can be utilised in processes operating in challenging conditions. Thus, they could be used in more demanding applications than supposed earlier.

Osteoclastic tartrate-resistant acid phosphatase 5b. Diagnostic use and biological significance in bone physiology

The skeleton undergoes continuous turnover throughout life. In women, an increase in bone turnover is pronounced during childhood and puberty and after menopause. Bone turnover can be monitored by measuring biochemical markers of bone resorption and bone formation. Tartrate-resistant acid phosphatase (TRACP) is an enzyme secreted by osteoclasts, macrophages and dendritic cells. The secreted enzyme can be detected from the blood circulation by recently developed immunoassays. In blood circulation, the enzyme exists as two isoforms, TRACP 5a with an intact polypeptide chain and TRACP 5b in which the polypeptide chain consists of two subunits. The 5b form is predominantly secreted by osteoclasts and is thus associated with bone turnover. The secretion of TRACP 5b is not directly related to bone resorption; instead, the levels are shown to be proportional to the number of osteoclasts. Therefore, the combination of TRACP 5b and a marker reflecting bone degradation, such as C-terminal cross-linked telopeptides of type I collagen (CTX), enables a more profound analysis of the changes in bone turnover. In this study, recombinant TRACP 5a-like protein was proteolytically processed into TRACP 5b-like two subunit form. The 5b-like form was more active both as an acid phosphatase and in producing reactive oxygen species, suggesting a possible function for TRACP 5b in osteoclastic bone resorption. Even though both TRACP 5a and 5b were detected in osteoclasts, serum TRACP 5a levels demonstrated no change in response to alendronate treatment of postmenopausal women. However, TRACP 5b levels decreased substantially, demonstrating that alendronate decreases the number of osteoclasts. This was confirmed in human osteoclast cultures, showing that alendronate decreased the number of osteoclats by inducing osteoclast apoptosis, and TRACP 5b was not secreted as an active enzyme from the apoptotic osteoclasts. In peripubertal girls, the highest levels of TRACP 5b and other bone turnover markers were observed at the time of menarche, whereas at the same time the ratio of CTX to TRACP 5b was lowest, indicating the presence of a high number of osteoclasts with decreased resorptive activity. These results support the earlier findings that TRACP 5b is the predominant form of TRACP secreted by osteoclasts. The major source of circulating TRACP 5a remains to be established, but is most likely other cells of the macrophage-monocyte system. The results also suggest that bone turnover can be differentially affected by both osteoclast number and their resorptive activity, and provide further support for the possible clinical use of TRACP 5b as a marker of osteoclast number.