Human Torque teno virus: epidemiology, cell biology and immunology

Torque teno virus (TTV) was discovered in 1997 in the serum of a Japanese patient who had a post-transfusion hepatitis of unknown etiology. It is a small virus containing a circular single-stranded DNA genome which is unique among human viruses. Within a few years after its discovery, the TTVs were noted to form a large family of viruses with numerous genotypes. TTV is highly prevalent among the general population throughout the world, and persistent infections and co-infections with several genotypes occur frequently. However, the pathogenicity and the mechanism for the sustained occurrence of the virus in blood are at present unclear. To determine the prevalence of TTV in Finland, we set up PCR methods and examined the sera of asymptomatic subjects for the presence of TTV DNA and for genotype-6 DNA. TTV was found to be highly prevalent also in Finland; 85% of adults harbored TTV in their blood, and 4% were infected with genotype-6. In addition, TTV DNA was detected in a number of different tissues, with no tissue-type or symptom specificity. Most cell-biological events during TTV infections are at the moment unknown. Replicating TTV DNA has, however, been detected in liver and the hematopoietic compartment, and three mRNAs are known to be generated. To characterize TTV cell biology in more detail, we cloned in full length the genome of TTV genotype 6. We showed that in human kidney-derived cells TTV produces altogether six proteins with distinct subcellular localizations. TTV mRNA transcription was detected in all cell lines transfected with the full-length clone, and TTV DNA replicated in several of them, including those of erythroid, kidney, and hepatic origin. Furthermore, the viral DNA replication was shown to utilize the cellular DNA polymerases. Diagnoses of TTV infections have been based almost solely on PCR, whereas serological tests, measuring antibody responses, would give more information on many aspects of these infections. To investigate the TTV immunology in more detail, we produced all six TTV proteins for use as antigens in serological tests. We detected in human sera IgM and IgG antibodies to occur simultaneously with TTV DNA, and observed appearance of TTV DNA regardless of pre-existing antibodies, and disappearance of TTV DNA after antibody appearance. The genotype-6 nucleotide sequence remained stable for years within the infected subjects, suggesting that some mechanism other than mutations is used by this minute virus to evade our immune system and to establish chronic infections in immunocompetent subjects.

Joint Regression and Association Models for Repeated Categorical Responses

The focus of this study is on statistical analysis of categorical responses, where the response values are dependent of each other. The most typical example of this kind of dependence is when repeated responses have been obtained from the same study unit. For example, in Paper I, the response of interest is the pneumococcal nasopharengyal carriage (yes/no) on 329 children. For each child, the carriage is measured nine times during the first 18 months of life, and thus repeated respones on each child cannot be assumed independent of each other. In the case of the above example, the interest typically lies in the carriage prevalence, and whether different risk factors affect the prevalence. Regression analysis is the established method for studying the effects of risk factors. In order to make correct inferences from the regression model, the associations between repeated responses need to be taken into account. The analysis of repeated categorical responses typically focus on regression modelling. However, further insights can also be gained by investigating the structure of the association. The central theme in this study is on the development of joint regression and association models. The analysis of repeated, or otherwise clustered, categorical responses is computationally difficult. Likelihood-based inference is often feasible only when the number of repeated responses for each study unit is small. In Paper IV, an algorithm is presented, which substantially facilitates maximum likelihood fitting, especially when the number of repeated responses increase. In addition, a notable result arising from this work is the freely available software for likelihood-based estimation of clustered categorical responses.

Bioabsorbable materials for osteosynthesis and small joint arthroplasty in the hand

Fractures and arthritic joint destruction are common in the hand. A reliable and stable fracture fixation can be achieved by metal implants, which however, become unnecessary or even harmful after consolidation. The silicone implant arthroplasty is the current method of choice for reconstruction of metacarpophalangeal joints in rheumatoid patients. However, the outcome tends to worsen with long-term follow-up and implant-related complications become frequent. To address these problems, bioabsorbable implants were designed for the hand area. Aims of the studies were: 1) to evaluate the biomechanical stabilities provided by self- reinforced (SR) bioabsorbable implants in a transverse and an oblique osteotomy of small tubular bones and to compare them with those provided by metal implants; 2) to evaluate the SR poly-L/DL-lactide 70/30 plate for osteosynthesis in a proof-of-principle type of experiment in three cases of hand injuries; and 3) to evaluate the poly-L/D-lactide (PLA) 96/4 joint scaffold, a composite joint implant with a supplementary intramedullary Polyactive® stem and Swanson silicone implant in an experimental small joint arthroplasty model. Methods used were: 1) 112 fresh frozen human cadaver and 160 pig metacarpal bones osteotomised transversally or obliquely, respectively, and tested ex vivo in three point bending and in torsion; 2) three patient cases of complex hand injuries; and 3) the fifth metacarpophalangeal joints reconstructed in 18 skeletally-mature minipigs and studied radiologically and histologically. The initial fixation stabilities provided by bioabsorbable implants in the tubular bones of the hand were comparable with currently-employed metal fixation techniques, and were sufficient for fracture stabilisation in three preliminary cases in the hand. However, in torsion the stabilities provided by bioabsorbable implants were lower than that provided by metal counterparts. The bioabsorbable plate enhanced the bending stability for the bioabsorbable fixation construct. PLA 96/4 joint scaffolds demonstrated good biocompatibility and enabled fibrous tissue in-growth in situ. After scaffold degradation, a functional, stable pseudarthrosis with dense fibrous connective tissue was formed. However, the supplementary Polyactive® stem caused a deleterious tissue reaction and therefore the stem can not be applied to the composite joint implant. The bioabsorbable implants have potential for use in clinical hand surgery, but have to await validation in clinical patient series and controlled trials.

Simplified Treatment of Childhood Acute Bone and Joint Infections

Acute childhood osteomyelitis (OM), septic arthritis (SA), and their combination osteomyelitis with adjacent septic arthritis (OM+SA), are treated with long courses of antimicrobials and immediate surgery. We conducted a prospective multi-center randomized trial among Finnish children at age 3 months to 15 years in 1983-2005. According to the two-by-two factorial study design, children with OM or OM+SA received 20 or 30 days of antimicrobials, whereas those with SA were treated for 10 or 30 days. In addition, the whole series was randomized to be treated with clindamycin or a first-generation cephalosporin. Cases were included only if the causative agent was isolated. The treatment was instituted intravenously, but only for the first 2-4 days. Percutaneous aspiration was done to obtain a representative sample for bacteriology, but all other surgical intervention was kept at a minimum. A total of 265 patients fulfilled our strict inclusion criteria and were analyzed; 106 children had OM, 134 SA, and 25 OM+SA. In the OM group, one child in the long and one child in the short-term treatment group developed sequelae. One child with SA twice developed a late re-infection of the same joint, but the causative agents differed. Regarding surgery, diagnostic arthrocentesis or corticotomy was the only surgical procedure performed in most cases. Routine arthrotomy was not required even in hip arthritis. Serum C-reactive protein (CRP) proved to be a reliable laboratory index in the diagnosis and monitoring of osteoarticular infections. The recovery rate was similar regardless of whether clindamycin or a first-generation cephalosporin was used. We conclude that a course of 20 days of these well-absorbing antimicrobials is sufficient for OM or OM+SA, and 10 days for SA in most cases beyond the neonatal age. A short intravenous phase of only 2-5 days often suffices. CRP gives valuable information in monitoring the course of illness. Besides diagnostic aspiration, surgery should be reserved for selected cases.