Pekka Saukko

Is cellulose sponge degradable or stable as implantation material? An in vivo subcutaneous study in the rat.

The long-term behaviour of cellulose sponge implants, 10 x 10 x 5 mm in size, and tissue reactions in and around them were examined in the subcutaneous tissue of the rat from 1 to 60 weeks after implantation. The cellulose sponge used was filled up with connective tissue 4 to 8 weeks after implantation. Histologically, moderate foreign body tissue reaction inside the implant, the appearance of cracks and fissures, spotty colouration, and softening of the pore walls were observed up to 16 weeks after implantation. Later, the foreign body reaction inside the sponge became milder, the spotty colouration disappeared and micropores enlarged in the viscose cellulose matrix. Histomorphometrically, the cross-sectional area of the implants and the size of the pore wall fragments decreased, and the number of pore wall fragments increased significantly. The cellulose sponge used can be regarded as a slowly degradable implantation material. However, the time needed for the total disappearance of the cellulose sponge from subcutaneous tissue is longer than the 60 weeks.

Screening of some anti-androgenic endocrine disruptors using a recombinant cell-based in vitro bioassay

The present work describes the development and optimization of a cell-based androgen reporter assay using the Chinese hamster ovarian cell line (CHO K1) in the 96-well format. The recent reports on increasing exposure of humans and wild-life to environmental endocrine disrupting chemicals (ED) prompt the need for high throughput screening systems for such compounds in environmental and biological samples. To this end, CHO cells were cotransfected with plasmids encoding mouse mammary tumour virus-neomycin-luciferase and human androgen receptor (hAR), and a stable cell line was established. After selection with neomycin, a highly active clone was obtained which stably expressed both the hAR and the androgen-responsive luciferase reporter. Stimulation of the cells with androgens for 24 h resulted in about 15-fold stimulation of luciferase activity, with the minimum effective dose of testosterone being 0.1 nmol/l. Potent steroidal and non-steroidal anti-androgens, such as hydroxyflutamide and cyproterone acetate, significantly inhibited the androgen-induced transactivation. Non-androgenic steroids like estradiol, progesterone, dexamethasone and cortisol showed weak activity at high concentrations. RT-PCR and western blot confirmed proper transcription and translation as well as stable expression of the AR gene in the cells. About 60 different chemicals (mostly pesticides or their metabolites, and common industrial chemicals) were screened with the cell line for their ability to stimulate luciferase activity or inhibit that evoked by 0.1 nmol/l R1881, used as a positive androgenic control. About 10 highly potent anti-androgenic chemicals were identified. The most potent anti-androgenic compounds identified in our assay included bisphenol A, alpha-hexachlorocyclohexane, vinclozolin and 4,4-DDE. These compounds had alone either no effect or were weak agonists (with cytotoxic effects at very high concentrations), but none showed any significant agonistic activity. In conclusion, we demonstrate that the bioassay based on this cell line provides a reliable test for detecting androgenic and anti-androgenic compounds. The 96-well plate format makes the assay suitable for high throughput screening.

Biocompatibility of Cellulose Sponge with Bone

The purpose of this study was to investigate the biocompatibility of viscose cellulose sponge (VCS) with bone. Twenty-five Sprague-Dawley rats were used for the study. After curettage of the bone marrow from both femoral cavities, VCS (15 × 1 × 1 mm) was implanted into one femur, leaving the contralateral side empty as a control. The rats were killed 1–6 weeks after curettage, and bone formation inside the sponge was assessed by light-microscopic examination and histomorphometric assessment. Whereas normal bone formation in rat femoral cavity took place in 2 weeks after curettage, 4 weeks were needed for bone formation in the cellulose sponge. VCS is a compatible matrix for osseous tissue ingrowth and it may be useful as a scaffold for bone tissue engineering in experiments and possibly also in clinical practice.

Vascular adhesion protein-1, intercellular adhesion molecule-1 and P-Selectin mediate leukocyte binding to ischemic heart in humans

OBJECTIVES The expression of endothelial adhesion molecules and their functional significance in leukocyte adhesion to human myocardial blood vessels in acute myocardial infarction (AMI) were studied. BACKGROUND Leukocyte extravasation, mediated by specific adhesion molecules, exacerbates tissue injury after restoration of blood supply to an ischemic tissue. Experimental myocardial reperfusion injury can be alleviated with antibodies that block the function of adhesion molecules involved in leukocyte emigration, but the relevant molecules remain poorly characterized in human AMI. METHODS Semiquantitative immunohistochemistry and in vitro adhesion assays were used to study the expression and granulocyte binding abilities of different endothelial adhesion molecules in human AMI. Changes in the molecular nature of vascular adhesion protein-1 (VAP-1) were evaluated using immunoblotting. RESULTS Certain endothelial adhesion molecules (intercellular adhesion molecule [ICAM-2], CD31 and CD73) were expressed in myocardial blood vessels homogeneously in normal and ischemic hearts, whereas others (E-selectin and peripheral lymph node addressin) were completely absent from all specimens. The synthesis of ICAM-1 was locally, and that of P-selectin regionally, upregulated in the infarcted hearts when compared with nonischemic controls. Vascular adhesion protein-1 showed ventricular preponderance in expression and alterations in posttranslational modifications during ischemia-reperfusion. Importantly, P-selectin, ICAM-1 and VAP-1 mediated granulocyte binding to blood vessels in the ischemic human heart. CONCLUSIONS Human P-selectin, ICAM-1 and VAP-1 appear to be the most promising targets when antiadhesive interventions preventing leukocyte-mediated tissue destruction after myocardial ischemia are planned.

Cytomegalovirus Infection of the Heart Is Common in Patients with Fatal Myocarditis

BACKGROUND Although enteroviruses and adenoviruses are considered to be the leading causes of the usually mild clinical myocarditis, little is known about the etiology of severe or fatal myocarditis. METHODS We collected all available clinical records and myocardial autopsy samples for patients who had myocarditis recorded as the underlying cause of death in Finland during the period of 1970-1998. Findings for all available patients (20 men and 20 women; median age, 49 years) with myocarditis that fulfilled the Dallas criteria and who had sufficient data were included in the study. Twelve subjects who had died accidentally served as control subjects. Polymerase chain reaction (PCR) and in situ hybridization assays were used for detection of viral genomes (adenovirus, cytomegalovirus, enterovirus, human herpesvirus 6, influenza A and B viruses, parvovirus B19, and rhinovirus) in heart samples. RESULTS Viral nucleic acids were found in the hearts of 17 patients (43%), including cytomegalovirus (15 patients), parvovirus B19 (4 patients), enterovirus (1 patient), and human herpesvirus 6 (1 patient). In 4 patients, cytomegalovirus DNA was found in addition to parvovirus B19 or enterovirus genomes. No adenoviruses, rhinoviruses, or influenza viruses were detected in this study of fatal myocarditis. In 67% of the patients for whom PCR was positive for cytomegalovirus, in situ hybridization revealed viral DNA in cardiomyocytes. Only 1 of these patients was immunocompromised. In the control group, only human herpesvirus 6 (1 subject) and parvovirus B19 (1 subject) DNA were detected. CONCLUSIONS In this population-based study, cytomegalovirus was found to be the most common specific finding in immunocompetent patients with fatal myocarditis. This may have important clinical implications for the treatment of severe acute myocarditis.

Indoors forensic entomology: Colonization of human remains in closed environments by specific species of sarcosaprophagous flies

Fly species that are commonly recovered on human corpses concealed in houses or other dwellings are often dependent on human created environments and might have special features in their biology that allow them to colonize indoor cadavers. In this study we describe nine typical cases involving forensically relevant flies on human remains found indoors in southern Finland. Eggs, larvae and puparia were reared to adult stage and determined to species. Of the five species found the most common were Lucilia sericata Meigen, Calliphora vicina Robineau-Desvoidy and Protophormia terraenovae Robineau-Desvoidy. The flesh fly Sarcophaga caerulescens Zetterstedt is reported for the first time to colonize human cadavers inside houses and a COI gene sequence based DNA barcode is provided for it to help facilitate identification in the future. Fly biology, colonization speed and the significance of indoors forensic entomological evidence are discussed.

Reduced cystatin B activity correlates with enhanced cathepsin activity in progressive myoclonus epilepsy.

BACKGROUND: Loss-of-function mutations in the gene encoding cystatin B (CSTB) underlie an inherited neurodegenerative disorder, progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1). CSTB is an inhibitor of several papain-family cysteine proteases, the lysosomal cathepsins. Its physiological function and the molecular pathways leading to the clinical EPM1 phenotype are unknown. AIM: To elucidate the role of CSTB and different cathepsins in pathogenesis of EPM1. METHOD: We determined the total papain inhibitory (cystatin) and papain-like (cathepsin) activity as well as specific activities of cathepsins B, H, L and S in lymphoblastoid cells of EPM1 patients, carriers and controls. RESULTS: In EPM1 patients, who express reduced levels of CSTB mRNA, the papain inhibitory activity was significantly decreased or absent. This reduction was correlated with significant increase in general cathepsin activity. The increase in cathepsin B, L and S activities was highly significant, whereas the increase in cathepsin H activity was not. CONCLUSIONS: This is the first demonstration of cysteine protease activity being regulated by CSTB activity in a biological context. The effects of decreased CSTB activity in EPM1 pathogenesis may, at least in part, be mediated by cathepsins through increased activity of cathepsins S and L.

Loss of lysosomal association of cystatin B proteins representing progressive myoclonus epilepsy, EPM1, mutations

Loss-of-function mutations in the cystatin B (CSTB), a cysteine protease inhibitor, gene underlie progressive myoclonus epilepsy of Unverricht–Lundborg type (EPM1), characterized by myoclonic and tonic–clonic seizures, ataxia and a progressive course. A minisatellite repeat expansion in the promoter region of the CSTB gene is the most common mutation in EPM1 patients and leads to reduced mRNA levels. Seven other mutations altering the structure of CSTB, or predicting altered splicing, have been described. Using a novel monoclonal CSTB antibody and organelle-specific markers in human primary myoblasts, we show here that endogenous CSTB localizes not only to the nucleus and cytoplasm but also associates with lysosomes. Upon differentiation to myotubes, CSTB becomes excluded from the nucleus and lysosomes, suggesting that the subcellular distribution of CSTB is dependent on the differentiation status of the cell. Four patient mutations altering the CSTB polypeptide were transiently expressed in BHK-21 cells. The p.Lys73fsX2-truncated mutant protein shows diffuse cytoplasmic and nuclear distribution, whereas p.Arg68X is rapidly degraded. Two missense mutations, the previously described p.Gly4Arg affecting the highly conserved glycine, critical for cathepsin binding, and a novel mutation, p.Gln71Pro, fail to associate with lysosomes. These data imply an important lysosome-associated physiological function for CSTB and suggest that loss of this association contributes to the molecular pathogenesis of EPM1.

Compound Heterozygous Desmoplakin Mutations Result in a Phenotype with a Combination of Myocardial, Skin, Hair, and Enamel Abnormalities

Desmoplakin (DP) anchors the intermediate filament cytoskeleton to the desmosomal cadherins and thereby confers structural stability to tissues. In this study, we present a patient with extensive mucocutaneous blisters, epidermolytic palmoplantar keratoderma, nail dystrophy, enamel dysplasia, and sparse woolly hair. The patient died at the age of 14 years from undiagnosed cardiomyopathy. The skin showed hyperplasia and acantholysis in the mid- and lower epidermal layers, whereas the heart showed extensive fibrosis and fibrofatty replacement in both ventricles. Immunofluorescence microscopy showed a reduction in the C-terminal domain of DP in the skin and oral mucosa. Sequencing of the DP gene showed undescribed mutations in the maternal and paternal alleles. Both mutations affected exon 24 encoding the C-terminal domain. The paternal mutation, c.6310delA, leads to a premature stop codon. The maternal mutation, c.7964 C to A, results in a substitution of an aspartic acid for a conserved alanine residue at amino acid 2655 (A2655D). Structural modeling indicated that this mutation changes the electrostatic potential of the mutated region of DP, possibly altering functions that depend on intermolecular interactions. To conclude, we describe a combination of DP mutation phenotypes affecting the skin, heart, hair, and teeth. This patient case emphasizes the importance of heart examination of patients with desmosomal genodermatoses.

Molecular pathology in forensic medicine —Introduction

Techniques of molecular biology have improved diagnostic sensitivity, accuracy and validity in forensic medicine very much, especially in the field of identification (paternity testing, stain analysis). Since more than 10 years these techniques - meanwhile well established in clinical disciplines - are used also for other applications in forensic medicine: determination of cause and manner of death, tissue identification by mRNA and microRNA, examination of gene expression levels (survival time, time since death, cause of death), toxicogenetics.