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Objective Despite new strategies in tissue engineering, cartilage repair remains a major challenge. Our aim is to treat patients with focal lesions of articular cartilage with autologous hyaline cartilage implants using a scaffold-free approach. In this article, we describe experiments to optimize production of scaffold-free cartilage discs. Design Articular chondrocytes were expanded in vitro, seeded in transwell inserts and redifferentiated using established chondrogenic components. Experimental variables included testing 2 different expansion media, adding bone morphogenetic protein 2 (BMP2), insulin-like growth factor 1 (IGF1), growth/differentiation factor 5 (GDF5), or fibroblast growth factor 18 (FGF18) to the differentiation medium and allowing the disc to float freely in large wells. Cartilage discs were analyzed by weight and thickness, real-time RT-qPCR (reverse transcriptase qualitative polymerase chain reaction), fluorescence immunostaining, transmission electron microscopy, second harmonic generation imaging, and measurement of Young’s modulus. Results Addition of BMP2 to the chondrogenic differentiation medium (CDM) was essential for stable disc formation, while IGF1, GDF5, and FGF18 were redundant. Allowing discs to float freely in CDM on a moving platform increased disc thickness compared with discs kept continuously in transwell inserts. Discs cultured for 6 weeks reached a thickness of almost 2 mm and Young’s modulus of >200 kPa. There was abundant type II collagen. Collagen fibrils were 25 nm thick, with a tendency to be organized perpendicular to the disc surface. Conclusion Scaffold-free engineering using BMP2 and providing free movement in CDM produced firm, elastic cartilage discs with abundant type II collagen. This approach may potentially be used in clinical trials.

Supplemental material, sj-pdf-1-car-10.1177_19476035211007923 for Scaffold-Free Engineering of Human Cartilage Implants by Nadine Frerker, Tommy A. Karlsen, Magnus Borstad Lilledahl, Sverre-Henning Brorson, John E. Tibballs and Jan E. Brinchmann in CARTILAGE

Summary Optic atrophy 1 (OPA1), a GTPase at the inner mitochondrial membrane involved in regulating mitochondrial fusion, stability, and energy output, is known to be crucial for neural development: Opa1 heterozygous mice show abnormal brain development, and inactivating mutations in OPA1 are linked to human neurological disorders. Here, we used genetically modified human embryonic and patient-derived induced pluripotent stem cells and reveal that OPA1 haploinsufficiency leads to aberrant nuclear DNA methylation and significantly alters the transcriptional circuitry in neural progenitor cells (NPCs). For instance, expression of the forkhead box G1 transcription factor, which is needed for GABAergic neuronal development, is repressed in OPA1+/− NPCs. Supporting this finding, OPA1+/− NPCs cannot give rise to GABAergic interneurons, whereas formation of glutamatergic neurons is not affected. Taken together, our data reveal that OPA1 controls nuclear DNA methylation and expression of key transcription factors needed for proper neural cell specification., Graphical Abstract, Highlights • OPA1 haploinsufficiency impairs formation of DLX1/2-positive GABAergic neurons • Reduced OPA1 levels significantly alter the transcriptional circuitry in neural cells • Expression of the pioneer factor FOXG1 is decreased in OPA1+/− neural progenitor cells • Impaired FOXG1 expression correlates with increased CpG methylation at its promoter, Neurogenetics; Developmental Neuroscience

Optic atrophy 1 (OPA1), a GTPase at the inner mitochondrial membrane involved in regulating mitochondrial fusion, stability and energy output, is known to be crucial for neural development: OPA1 heterozygous mice show abnormal brain development, and inactivating mutations in OPA1 are linked to human neurological disorders. Although it known that mitochondria are important for proper neural progenitor cell (NPC) fate decisions, surprisingly little is known about the underlying regulatory mechanisms. Here, using human embryonic and patient-derived induced pluripotent stem cells, we reveal that OPA1 haploinsufficiency causes aberrant nuclear DNA methylation and significantly alters the transcriptional circuitry in NPCs. For instance, expression of the forkhead box G1 transcription factor, which is needed for GABAergic neuronal development, is repressed in OPA1 haploinsufficient NPCs. Consequently, OPA1 +/- NPCs cannot give rise to GABAergic interneurons, while formation of glutamatergic neurons is not affected. Taken together, our data reveal that OPA1 controls nuclear DNA methylation and expression of key transcription factors that are needed for proper neural cell specification.

Aims Diastolic dysfunction is central to the development of heart failure. To date, there is no effective treatment and only limited understanding of its molecular basis. Recently, we showed that the transmembrane proteoglycan syndecan-4 increases in the left ventricle after pressure overload in mice and man, and that syndecan-4 via calcineurin/nuclear factor of activated T-cells (NFAT) promotes myofibroblast differentiation and collagen production upon mechanical stress. The aim of this study was to investigate whether syndecan-4 affects collagen cross-linking and myocardial stiffening in the pressure-overloaded heart. Methods and results Aortic banding (AB) caused concentric hypertrophy and increased passive tension of left ventricular muscle strips, responses that were blunted in syndecan-4−/− mice. Disruption of titin anchoring by salt extraction of actin and myosin filaments revealed that the effect of syndecan-4 on passive tension was due to extracellular matrix remodelling. Expression and activity of the cross-linking enzyme lysyl oxidase (LOX) increased with mechanical stress and was lower in left ventricles and cardiac fibroblasts from syndecan-4−/− mice, which exhibited less collagen cross-linking after AB. Expression of osteopontin (OPN), a matricellular protein able to induce LOX in cardiac fibroblasts, was up-regulated in hearts after AB, in mechanically stressed fibroblasts and in fibroblasts overexpressing syndecan-4, calcineurin, or NFAT, but down-regulated in fibroblasts lacking syndecan-4 or after NFAT inhibition. Interestingly, the extracellular domain of syndecan-4 facilitated LOX-mediated collagen cross-linking. Conclusions Syndecan-4 exerts a dual role in collagen cross-linking, one involving its cytosolic domain and NFAT signalling leading to collagen, OPN, and LOX induction in cardiac fibroblasts; the other involving the extracellular domain promoting LOX-dependent cross-linking.

Myocardial infarction (MI) triggers a reparative response involving fibroblast proliferation and differentiation driving extracellular matrix modulation necessary to form a stabilizing scar. Recently, it was shown that a genetic variant of the base excision repair enzyme NEIL3 was associated with increased risk of MI in humans. Here, we report elevated myocardial NEIL3 expression in heart failure patients and marked myocardial upregulation of Neil3 after MI in mice, especially in a fibroblast-enriched cell fraction. Neil3−/− mice show increased mortality after MI caused by myocardial rupture. Genome-wide analysis of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) reveals changes in the cardiac epigenome, including in genes related to the post-MI transcriptional response. Differentially methylated genes are enriched in pathways related to proliferation and myofibroblast differentiation. Accordingly, Neil3−/− ruptured hearts show increased proliferation of fibroblasts and myofibroblasts. We propose that NEIL3-dependent modulation of DNA methylation regulates cardiac fibroblast proliferation and thereby affects extracellular matrix modulation after MI. © 2017 The Authors. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Meningococcal disease is caused mainly by serogroups A, B, C, Y, W of N. meningitidis. However, numerous cases of meningitis caused by serogroup X N. meningitidis (MenX) have recently been reported in several African countries. Currently, there are no licensed vaccines against this pathogen and most of the MenX cases have been caused by meningococci from clonal complex (c.c) 181. Detergent extracted meningococcal outer membrane vesicle (dOMV) vaccines have previously shown to be safe and effective against epidemics of serogroup B meningococcal disease in all age groups. The aim of this work is therefore to obtain, characterize and evaluate the vaccine potential of dOMVs derived from a MenX strain (OMVx). Three experimental lots of OMVx were prepared by deoxycholate extraction from the MenX strain BF 2/97. Size and morphology of the vesicles was determined by Dynamic Light Scattering and electron microscopy, whereas the antigenic composition was characterized by gel electrophoresis and immunoblotting. OMVx were thereafter adsorbed to aluminium hydroxide (OMVx/AL) and two doses of OMVx were administered s.c. to groups of Balb/c mice three weeks apart. The immunogenicity and functional antibody activities in sera were evaluated by ELISA (anti-OMVx specific IgG responses) and serum bactericidal activity (SBA) assay. The size range of OMVx was shown to be between 90 and 120nm, whereas some of the antigens detected were the outer membrane proteins PorA, OpcA and RmpM. The OMVx/AL elicited high anti-OMVx antibody responses with bactericidal activity and no bactericidal activity was observed in the control group of no immunised mice. The results demonstrate that OMVx are immunogenic and could form part of a future vaccine to prevent the majority of meningococcal disease in the African meningitis belt.

The aim of the present study was to investigate leucocyte markers, CD11b, CD16, CD66b, CD68, myeloperoxidase and neutrophil elastase on skeletal muscle biopsies from biceps brachii after unaccustomed eccentric exercise followed by the second bout of exercise 3 weeks later. The subjects (10 subjects received COX-2 inhibitor (Celecoxib) and 13 subjects received placebo) were divided into three categories: mild, moderate and severe effect of eccentric exercise, according to the reduction and recovery of muscle force-generating capacity after performing 70 maximal eccentric actions with elbow flexors on an isokinetic dynamometer. The results showed that the CD66b antibody was applicable for localization of neutrophils in human skeletal muscle, whereas the other studied neutrophil markers recognized also other leucocytes than neutrophils. The number of CD66b positive cells in skeletal muscle was very low and was not affected by the exercise. The macrophage marker CD68 showed reactivity also against satellite cells and fibroblast-like cells in skeletal muscle and therefore cannot be applied as a quantitative value for inflammatory cells. Skeletal muscle fibre injury, shown as dystrophin negative fibres, was observed approximately in half of the biopsies at 4 and 7 days after the first exercise bout in the categories moderate and severe effect of eccentric exercise. These subjects represent the most prominent loss in muscle force-generating capacity both at the category and the individual levels. Furthermore, deformed skeletal muscle fibres were observed in five subjects in these categories after the second bout of exercise. The present results suggest that neutrophils are not involved in skeletal muscle fibre injury and the reduction in muscle force-generating capacity after a single bout of eccentric exercise is a good indirect indicator of muscle damage in humans. Furthermore, prolonged regeneration process could be one of the reasons for impaired peripheral muscle function after high-force eccentric exercise.

Numerous studies have addressed the association between the apolipoprotein E polymorphism and cardiovascular disease, but only a few reports are based on findings at autopsy. In the present retrospective study, we have used autopsy findings from a general hospital population to further investigate this issue.We collected information from 1522 consecutive autopsy reports (886 men, mean age 65.7 years; 636 women, mean age 69.7 years) conducted at Oslo University Hospital, Norway, in the period from 1996 to 2000. Cause of death and signs related to cardiovascular disease including the degree of atherosclerosis in the aorta and the coronary arteries, signs of myocardial infarction, heart weight, and signs of cerebrovascular disease were recorded. The patients were genotyped, and the apolipoprotein E allele frequencies (ɛ2, 8.0%; ɛ3, 72.6%; and ɛ4, 19.4%) were not statistically different from a group of healthy controls. Approximately 35% of the patients died from a cardiovascular disease. Genotypes differed significantly (P.05), with more ɛ4-carriers (34.3% vs. 29.6%) and fewer ɛ2-carriers (11.8% vs. 13.9%) among patients who died from cardiovascular disease compared to those who died from other causes. A similar distribution of genotypes was seen in patients recorded with myocardial infarction or cerebrovascular disease. There was an association between the presence of ɛ4 and atherosclerosis in the aorta and coronary arteries, but this did not reach statistical significance. Among patients with signs of coronary heart disease, standardized heart weights were significantly higher in ɛ2-carriers compared to ɛ4-carriers.The present autopsy study suggests that the risk of developing and dying from cardiovascular disease, including coronary heart disease and cerebrovascular disease, is influenced by the apolipoprotein E polymorphism.

The purpose of this study was to compare the level of immunogold labeling of deplasticized acrylic sections and deplasticized epoxy sections. Pure protein gels of IgG, albumin and thyroglobulin were produced by glutaraldehyde fixation and embedded in non-crosslinked acrylic resin (Technovit 9100) and epoxy resin (Epon 812), respectively. Ultrathin sections of acrylic and epoxy resin were separately deplasticized in 2-methoxyethyl acetate (MEA) and sodium ethoxide. Quantitative immunogold labeling was performed with anti-IgG, anti-albumin and anti-thyroglobulin antibodies on sections of the corresponding protein gels. For all antibodies tested, the intensity of labeling for deplasticized acrylic sections was significantly higher (two to four times) than for the corresponding deplasticized epoxy sections. The results fit with a theoretically deduced relation: the quotient of the labeling of two deplasticized sections of different resins is equivalent to the square root of the quotient of the labeling of the similar sections not exposed to any kind of pre-treatment. The practical significance of the results is that immunolabeling of deplasticized non-crosslinked acrylic resin results in more intense immunogold labeling than deplasticized epoxy sections. Deplasticizing is most useful when the requirements for ultrastructural preservation according to conventional criteria are moderate. Our theoretically deduced results also indicate that deplasticized Technovit (or other non-crosslinked acrylic resins) sections will be significantly better suited for immunolabeling at the light microscopic level than deplasticized epoxy sections.

The main purpose of this study was to examine whether antigens can be retrieved by heating Lowicryl sections of paraformaldehyde-fixed (PFF) tissues. Thus the intensity of the immunogold signal for two bone proteins (Nucleobindin (Nuc) and osteoadherin (OSAD)) was compared in retrieved and non-retrieved sections of PFF rat bone. As an additional experiment, the effect of antigen retrieval (for Nuc) in sections of tissue primary stabilized by high pressure freezing with subsequent freeze substitution (HPF-FS) was studied. Finally, the tissue distribution patterns of Nuc labeling were compared in non-retrieved HPF-FS sections to that of retrieved and non-retrieved PFF sections. Antigen retrieval in Lowicryl sections of PFF tissues showed significantly enhanced labeling intensity for both proteins in all compartments where they are known to occur. Retrieved PFF Lowicryl sections showed only minor ultrastructural differences compared to non-retrieved ones. Retrieval of HPF-FS sections exhibited no enhancement of labeling but rather a slight reduction, which was significant in the cytoplasm and in cartilage. Furthermore, striking ultrastructural differences were observed in retrieved HPF-FS sections compared to non-retrieved ones with loss of coherence and structure in sections subjected to heating. Comparison of the distribution patterns of Nuc in the sections of PFF and HPF-FS tissues showed discrepancy in most compartments. Antigen retrieval by heating Lowicryl sections of PFF tissues significantly enhances immunogold labeling in all cell compartments where the bone proteins are known to occur. However, the procedure may distort the tissue distribution pattern of bone proteins.

In this work the susceptibility of mobile and cystic forms of Borrelia burgdorferi to hydroxychloroquine (HCQ) was studied. The minimal bactericidal concentration (MBC) of HCQ against the mobile spirochetes was32 microg/ml at 37 degrees C, and128 microg/ml at 30 degrees C. Incubation with HCQ significantly reduced the conversion of mobile spirochetes to cystic forms. When incubated at 37 degrees C, the MBC for young biologically active cysts (1-day old) was8 microg/ml, but it was32 microg/ml for old cysts (1-week old). Acridine orange staining, dark-field microscopy and transmission electron microscopy revealed that the contents of the cysts were partly degraded when the concentration of HCQ wasor = MBC. At high concentrations of HCQ (256 microg/ml) about 95% of the cysts were ruptured. When the concentration of HCQ wasor = MBC, core structures did not develop inside the cysts, and the amount of RNA in these cysts decreased significantly. Spirochetal structures inside the cysts dissolved in the presence of high concentrations of HCQ. When the concentration of HCQ wasor = MBC, the core structures inside the cysts were eliminated. These observations may be valuable in the treatment of resistant infections caused by B. burgdorferi, and suggest that a combination of HCQ and a macrolide antibiotic could eradicate both cystic and mobile forms of B. burgdorferi.

We examined the increase in immunogold labeling of variably fixed, resin embedded tissue sections following antigen retrieval by heating in citrate solution. Fibrin clots and porcine renal tissue were fixed in glutaraldehyde, paraformaldehyde or ethanol, and specimens were embedded in LR-White or epoxy resin. Immunogold labeling was performed on ultra-thin sections with anti-fibrinogen for the fibrin clots and anti-IgG for the porcine renal tissue. Immunogold labeling increased greatly after heating epoxy sections regardless of the fixative used. The ratio labeling(retrieved)/labeling(nonretrieved) (Lr/Ln) was 2.8 or higher, and the largest increases were obtained for anti-IgG. Heating induced a large increase of immunolabeling for LR-White sections only when the specimens had been fixed in paraformaldehyde (Lr/Ln = 2.2 for anti-IgG and 1.4 for antifibrinogen). LR-White sections showed decreased, insignificant or weakly increased immunolabeling of ethanol or glutaraldehyde fixed tissues following antigen retrieval. Disruption of aldehyde cross-links is not the only mechanism for antigen retrieval when epoxy sections are heated in citrate solution since large increases in immunolabeling were obtained on ethanol fixed tissue. The large heat-induced increases in immunolabeling on epoxy sections are probably caused by the disruption of chemical bonds between the epoxy resin and side groups of proteins.

The purpose of this study was to examine how different incubation times with different concentrations of bovine serum albumin (BSA) affect the amount of non-specific immunogold labeling on epoxy sections and LR-White sections. Immunogold labeling was performed on epoxy sections and LR-White sections of renal tissue with IgG-deposits and fibrin clots, and the antibodies used were anti-IgG and anti-fibrinogen, respectively. The sections were incubated with different concentrations of BSA prior to application of primary antibodies, and the length of this pre-incubation step varied between 0 and 4 h. During the incubation with primary antibodies, BSA was added in the same concentration as in the pre-incubation step. The results showed that the non-specific labeling on the resin decreased significantly when the concentration of BSA or the length of the preincubation step was increased. The non-specific labeling was usually higher on the epoxy resin than on the LR-White resin when using the same conditions with respect to BSA. But, when the preincubation step with BSA lasted 4 h, the non-specific labeling was somewhat lower on epoxy resin than on the acrylic LR-White resin, without respect to the concentration of BSA. The specific labeling for both fibrinogen and IgG decreased slightly when the concentration of BSA and incubation time increased, probably due to the steric hindrance performed by BSA molecules on the section. Blocking procedures with at least 1 h incubation time for the blocking step with at least 5% BSA are recommended for both epoxy and LR-White sections.

The purpose of this study was to examine how the intensity of the immunogold labeling on epoxy sections was affected by the use of propylene oxide as an agent in addition to ethanol in the dehydration and infiltration, and also to examine the effect on the immunogold labeling by adding small amounts of propylene oxide to the embedding mixture. Increased knowledge of the mechanism for antigen detection on resin section was another aim. Thyroid tissue, kidney tissue, and fibrin were embedded in epoxy resin; some with ethanol as the only dehydration agent and others with propylene oxide as an additional agent in dehydration, infiltration or embedding steps in different ways. Immunogold labeling was performed with anti-thyroglobulin, anti-IgG, and anti-fibrinogen, respectively. A higher degree of immunogold labeling was achieved by increasing the concentration of accelerator during infiltration and embedding (Brorson and Skjorten, 1996a, Micron, 27, 211–217). The immunogold labeling of the sections that were based on additional dehydration and infiltration with propylene oxide showed significantly more intense labeling than the section of tissues that had only been exposed to ethanol in the dehydration and infiltration steps. The embedding of tissues in a mixture of epoxy resin and 5–10% propylene oxide gave higher yields of immunogold labeling than if pure epoxy resin was used for the embedding. The improved labeling is explained by higher amplitudes of protruding antigens on the surface of the sections because antigens are less tightly incorporated in the polymer network when using propylene oxide as additional agent in dehydration, infiltration or embedding. These results illustrate the advantage of using propylene oxide as an additional agent when preparing specimens for immunoelectron microscopy with epoxy resin embedding.

The purpose of this study was to predict the ratio of immunogold labeling of LR-White sections and epoxy sections using theoretical methods. Tissues used in the experiments were pancreas, pituitary, kidney, thyroid and fibrin. Antigens used as test proteins were glucagon, somatostatin, thyroglobulin, chromogranin A, ACTH (adrenocorticotropt hormone), amyloid A and fibrinogen. These are proteins of different sizes. The quotient labeling LR-White /labeling epoxy was deduced theoretically and compared to calculations based on practical immunogold experiments. The theoretically deduced formula showed acceptable correlation to these calculations. This study gives a theory—expressed mathematically—for what is happening on the molecular level at the surface of resin sections in immunoelectron microscopy. The theory explains why acrylic resins normally are better suited for immunoelectron microscopy than epoxy sections, and indicates increased usefulness of epoxy sections when the diameter of the protein carrying the epitope decreases.

The purpose of this study was to improve the immunogold labeling of epoxy sections and to increase our knowledge of the mechanism for how antigens become immunolabeled on resin sections. Tissues from pancreas, thyroid and fibrin clots were embedded in an epoxy resin and LR-White. The epoxy mixture was composed and treated in different ways, especially with respect to altered amounts of accelerator (DMP-30). Immunogold labeling was performed with anti-glucagon, anti-thyroglobulin and anti-fibrinogen respectively. By increasing the amount of DMP-30 in the infiltration steps and/or embedding step, we observed a significant rise in the immunogold labeling. For the largest proteins the labeling was up to 8 times more intense than the labeling achieve with epoxy sections produced by 'normal' amount of accelerator in the embedding mixture and without accelerator in the infiltration mixture. For the smallest protein, glucagon, the differences were almost absent. The labeling of thyroglobulin and fibrinogen on the high accelerator epoxy sections was up to 70% of the labeling of LR-White sections, while conventional epoxy sections showed a labeling of 5-10% of that obtained with acrylic labeling. The cutting qualities of the high-accelerator blocks were similar to that of conventional epoxy embedding. The ultrastructure of the sections from the high-accelerator epoxy blocks were good, and the contrast was improved when tannic acid was used as enhancer. Our theory to explain the improved labeling is that the antigens are less tightly incorporated in the polymer network when the concentration of the accelerator is increased. The method outlined significantly improves the detectability of antigens on epoxy sections, which is the embedding resin routinely used in many laboratories.

The name of the fifth author was given incorrectly. The correct name is: K. Elizabeth Tanner. The correct citation is: Hessle L, Stordalen GA, Wenglen C, Petzold C, Tanner KE, et al. (2013) The Skeletal Phenotype of Chondroadherin Deficient Mice. PLoS ONE 8(6): e63080. doi:10.1371/journal.pone.0063080. The correct abbreviation in Contributions is: KET.

The purpose of this investigation was to explain why deplasticizing of epoxy sections gives higher immunogold labeling than non-deplasticizing. The methods used were the following: (1) Comparison of the ratio of immunogold labeling of deplasticized and non-deplasticized sections with gold particles of different sizes and comparison of this ratio with respect to sections of different thickness, (2) the tilt method (Brorson et al., 1994). Human kidney tissue with amyloid A depositions, human fibrin, and human pituitary tissue were embedded, sections were deplasticized on grids, treated with anti-Aa, anti-fibrinogen or anti-ACTH (ACTH=adrenocorticotropic hormone), and reembedded on grids. Indications of significant antibody penetration were found only at the periphery of structures (ACTH-vesicles). This penetration was about 30 nm. The ratios of immunogold labeling of deplasticized and non-deplasticized sections were approximately 2, 5 and 1 for amyloid, fibrin and ACTH, respectively, and were independent of the gold particle size. No significant differences of gold labeling were found between thicker and thinner deplasticized epoxy sections regardless the gold particle size. No significant differences of gold labeling between deplasticized epoxy sections and LR-White sections were found on interior areas of ACTH-vesicles or amyloid A plaques. The increased labeling of deplasticized epoxy sections compared to normal epoxy sections seemed to be mainly a surface phenomenon. The practical significance of this observation is that deplasticizing of epoxy sections may be a better method for localizing antigens at the periphery of structures than the use of other resin embedding media. Deplasticizing of epoxy sections may be a method of choice in a pathological laboratory to detect antigens in routinely embedded tissues.

The purpose of this investigation is to study the ability of antibodies to penetrate sections of LR-WHITE resin. The methods used in this study were the following: (1) Reembedding of sections labeled with immunogold (1 nm) and peroxidase/DAB/gold chloride, (2) tilting of ultrathin sections treated with immunogold (1 nm), (3) immunolabeling of cylindrical structures embedded in LR-WHITE, (4) application of primary and secondary antibodies on opposite sides of ultrathin sections. Fibrin and human pituitary tissue was embedded in LR-WHITE and treated with anti-fibrinogen or anti-ACTH respectively (ACTH = Adrenocorticotropic hormone). No indication of antibody penetration into the section were found with either of the methods, contrary to findings in earlier publications. The significance of this result is that antigens cannot be demonstrated in the interior of LR-WHITE sections with post-embedding techniques. Furthermore, LR-WHITE resin may be used for quantitative immunoelectron microscopy, and the resin may be used for double immunogold labeling since the application of immunoreagents on opposite sides of the sections is completely safe.

Fifty renal biopsies were studied by immunoelectron microscopy after embedding in a partly hydrophilic polyacrylic resin (LR White). Immunofluorescence studies were carried out on frozen sections of parallel tissue samples. Polyacrylic embedding gave good preservation of the renal ultrastructure and precise localization of immunoglobulin and C3c antibodies within glomerular electron-dense deposits. Non-specific staining of plasma proteins within vascular lumina could easily be detected. There was good correlation between immunoelectron and immunofluorescence microscopy. Immunoelectron microscopy is a very sensitive method, which can detect small amounts of antigen. More cases were, however, positive by immunofluorescence than by immunoelectron microscopy. This discrepancy may be explained by difference in sample size, and by difference in resolution of morphological details (electron microscopy versus fluorescence microscopy).

Persistence of tissue spirochetes of Borrelia burgdorferi as helices and round bodies (RBs) explains many erythema-Lyme disease symptoms. Spirochete RBs (reproductive propagules also called coccoid bodies, globular bodies, spherical bodies, granules, cysts, L-forms, sphaeroplasts, or vesicles) are induced by environmental conditions unfavorable for growth. Viable, they grow, move and reversibly convert into motile helices. Reversible pleiomorphy was recorded in at least six spirochete genera (>12 species). Penicillin solution is one unfavorable condition that induces RBs. This antibiotic that inhibits bacterial cell wall synthesis cures neither the second “Great Imitator” (Lyme borreliosis) nor the first: syphilis. Molecular-microscopic techniques, in principle, can detect in animals (insects, ticks, and mammals, including patients) helices and RBs of live spirochetes. Genome sequences of B. burgdorferi and Treponema pallidum spirochetes show absence of >75% of genes in comparison with their free-living relatives. Irreversible integration of spirochetes at behavioral, metabolic, gene product and genetic levels into animal tissue has been documented. Irreversible integration of spirochetes may severely impair immunological response such that they persist undetected in tissue. We report in vitro inhibition and destruction of B. burgdorferi (helices, RBs = “cysts”) by the antibiotic Tigecycline (TG; Wyeth), a glycylcycline protein-synthesis inhibitor (of both 30S and 70S ribosome subunits). Studies of the pleiomorphic life history stages in response to TG of both B. burgdorferi and Treponema pallidum in vivo and in vitro are strongly encouraged.

The Ca(2+)/Calmodulin-dependent protein kinase (CaMK) family is activated in response to elevation of intracellular Ca(2+), and includes CaMK1 (as well as CaMK2 and CaMK4), which exists as different isoforms (alpha, beta, gamma and delta). CaMK1 is present in several cell types and may be involved in various cellular processes, but its role in bone is unknown. In situ hybridization was used to determine the spatial and temporal expression of CaMK1beta during endochondral bone development in mouse embryos and newborn pups. The cellular and subcellular distribution of CaMK1 was assessed by quantitative immunogold electron microscopy (EM). The role of CaMK1beta in mouse calvarial osteoblasts was investigated by using small interfering RNA (siRNA) to silence its expression, while in parallel monitoring cell proliferation and levels of skeletogenic transcripts. cRNA in situ hybridization and EM studies show that CaMK1beta is mainly located in developing long bones and vertebrae (from ED14.5 until day 10 after birth), with highest expression in epiphyseal growth plate hypertrophic chondrocytes. By RT-PCR, we show that CaMK1beta2 (but not beta1) is expressed in mouse hind limbs (in vivo) and mouse calvarial osteoblasts (in vitro), and also in primary human articular chondrocyte cultures. Silencing of CaMK1beta in mouse calvarial osteoblasts by siRNA significantly decreases osteoblast proliferation and c-Fos gene expression (approx. 50%), without affecting skeletogenic markers for more differentiated osteoblasts (i.e. Cbfa1/Runx2, Osterix (Osx), Osteocalcin (Oc), Alkaline phosphatase (Alp) and Osteopontin (Opn)). These results identify CaMK1beta as a novel regulator of osteoblast proliferation, via mechanisms that may at least in part involve c-Fos, thus implicating CaMK1beta in the regulation of bone and cartilage development.

The purpose of this study was to compare the level of immunogold labelling of both osmicated and non-osmicated epoxy sections when subjected to different antigen retrieval, etching and incubation temperature for the antibodies. Pure IgG protein gels were produced by glutaraldehyde fixation, eventually postfixed with 1% osmium tetroxide, and embedded in epoxy resin. Ultrathin sections were antigen retrieved in citrate solution at 95 or 144 degrees C and eventually etched with NaIO4. Immunogold labelling with anti-IgG was performed at 4 degrees C overnight or at 60 degrees C for 1 h. The level of labelling for osmicated gels was 140% higher when heated at 144 degrees C and incubated with primary antibodies at 60 degrees C than when heated at 95 degrees C, etched with NaIO4 and incubated with primary antibodies at 4 degrees C. Osmium-fixed IgG-gels antigen retrieved at 144 degrees C and incubated with anti-IgG at 60 degrees C showed more labelling than sections of non-osmicated gels heated at 95 degrees C. Non-osmicated gels gained significant intensity of immunolabelling when the antibody incubation occurred at 60 degrees C for 1 h than at 4 degrees C overnight. Resin embedding of pure protein gels was a useful tool for comparing different protocols for immunoelectron microscopy.

In February, 2003, a 61-year-old woman presented at our hospital with abdominal pain. Her past medical history was unremarkable. A physical examination and CT scan showed generalised lymphadenopathy with a retroperitoneal bulky tumour without the involvement of any other organ. Laboratory tests showed moderate anaemia (haemoglobin concentration 11·2 g/L) and an IgM/ monoclonal gammopathy (7·33 mg/L). No signs of nephrotic syndrome, renal failure, arthritis, congestive heart-failure, or coagulopathy were detected. A supraclavicular lymph node and a bone-marrow trephine were taken, fixed in 10% buffered formalin for 24 h, and then paraffin-embedded. The patient was diagnosed with nodal marginal-zone lymphoma (NMZL) stage IVA and received eight cycles of CIOP chemotherapy (cyclophosphamide, idarubicin, vincristine, prednisolone). At the patient’s last follow-up (October, 2003), she was in a good condition with stable disease, although the lymph node was only slightly smaller. The IgM/ value had decreased (3·90 g/L) compared with the concentration measured at presentation. Histological analysis of the lymph-node biopsy sample showed almost complete destruction of the normal structure because of the deposition of hyaline material, which stained positively with periodic acid Schiff (with and without diastase digestion), Van Gieson, and toluidine blue, but not with Congo red. Furthermore, the hyaline material was more abundant around the small vessels (figure 1A, 1B). Atypical lymphoid elements with a monocytoid profile were mixed with the hyaline deposition, either singly or in small aggregates, and contained some blast cells and numerous plasma cells (figure 1C). By use of the paraffin-embedded sections of biopsied lymph-node tissue, immunohistochemical analysis, and the alkaline phosphatase antialkaline phosphatase complex (APAAP) technique, the atypical elements were found to express the IRTA1 (immunoglobulin superfamily receptor translocationassociated 1) molecule, CD20, CD79a, CD45RA, CD27, IgM/ , and BCL2. The tissue was negative for IgG, IgD, Ig light-chain, BCL6, CD10, DBA44, CD5, and CD3 (figure 1D–G and figure 2A–C). About 15% of the cells expressed Nodal marginal-zone lymphoma associated with monoclonal light-chain and heavy-chain deposition disease

Calcium-induced calcium release is in principle an all-or-nothing mechanism and self-propagation of Ca2+ waves is a pathological manifestation of this positive feedback loop. However, since local release of Ca2+ only elicits elementary Ca2+ release events (Ca2+ sparks) that do not develop into Ca2+ waves, Ca2+ release can be graded. This local control is possible because of the spatial arrangement of L-type Ca2+ channels and clusters of ryanodine receptors at the sites of Ca2+ release (dyads) (Stern 1992). We propose that the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) also contributes to local control by limiting diffusion of Ca2+. Six days following conditional SERCA knockout, Ca2+ sparks exhibited broadened geometry and slowed kinetics (increase in spark width, time to peak, and duration by 33%, 33%, and 51%, respectively). At this time point, SERCA protein levels are reduced by 53% and SR Ca2+ content is decreased by 25% (Stokke et al. 2010). To determine the precise localization of SERCA we employed cryo immuno-gold electron microscopy on sections from the mouse papillary muscle. Normal cardiac myocytes showed preferential SERCA expression near the Z-lines (nearly twofold higher labeling density at the Z-line than the A-band) with SERCA molecules clearly located in the junctional SR in close proximity to the dyads. The conditional SERCA knockout resulted in preferential loss of labeling at these locations. From resin-embedded cross-sections of papillary muscles, we observed more abundant SR in the I-band compared to the A-band (by factor of 1.7 and 1.6 for control and KO respectively). These results suggest that SERCA contributes to local control of Ca2+ release by limiting diffusion of Ca2+ from the dyad. Thus, the processes of Ca2+ release and re-uptake are closely linked by a population of SERCA molecules in the junctional SR.

Culturing elutriation-purified and cryopreserved human monocytes gives a cell loss of about 60% after a week. The main loss is during the first 24 h when one cell population dies by apoptosis and secondary necrosis, while another survives with minimal signs of apoptosis and necrosis. We have studied this initial cell loss using flow cytometry (FCM) and electron microscopy (EM) in parallel. Thawed cells were cultured in ultra low attachment wells and studied by FCM using Annexin V, Propidium iodide (PI), JC-1, APO2.7 and APO-BrDU. The EM studies comprised both transmission EM (TEM) and scanning EM (SEM), the latter employing cells labelled with antiCD14/gold and Annexin V/gold. Cells were counted by light microscopy to provide cell recoveries. DNA ladder patterns were investigated by electrophoresis. Camptothecin (CAM) was used as an apoptosis inducer. In the first 6 h of culture, there was an apoptotic phase with Annexin V(+)/PI(-) positive cells in FCM, chromatin condensation in TEM, a rapid and short phase with Annexin V/gold positively labelled cells in SEM and the cells disappeared by 6 h. All of these effects were enhanced by CAM. The necrotic phase (6-24 h) was associated with Annexin V(+)/PI(+) in FCM, and the data at 24 h was in agreement with the semiquantitatvive results from TEM. Discrepancies in the results for CD14 and Annexin V between FCM and SEM indicated phagocytosis. APO2.7 and APO-BrDU increases also indicated an accumulation of ingested material in vital cells. Centrifugation of supernatants, labelling pellets with Annexin V/FITC and examination by flow cytometry revealed no Annexin V positive cell fragments. We found evidence of rapid and efficient phagocytosis. CAM not only induced apoptosis, but also appeared to stabilise the cell membrane and increase both cell recovery and phagocytosis.

Gastrointestinal symptoms accompanying Lyme disease have not been considered in the treatment of Lyme patients yet. Here we examine the effect of ranitidine bismuth citrate (RBC) on motile and cystic forms of Borrelia burgdorferi in vitro, to determine whether it could cure this bacterial infection in the gastrointestinal tract. When motile forms of B. burgdorferi were exposed to RBC for 1 week at 37 degrees C, the minimal bactericidal concentration (MBC) was > 64 mg/ml. At 30 degrees C, the MBC was > 256 mg/ml. When the incubation lasted for 2 weeks at 37 degrees C, the MBC dropped to > 2 mg/ml. Bismuth aggregates were present on the surface of B. burgdorferi when RBC > or = MBC, as shown by transmission electron microscopy (TEM). Cystic forms of B. burgdorferi, exposed to RBC for 2 weeks at 37 degrees C, were examined by cultivation in BSK-H medium (Sigma B3528). They were stained with acridine orange (pH 6.4, pH 7.4) and studied by TEM. The MBC for RBC for young cystic forms (1 day old) and old cysts (8 months old) was estimated to be > 0.125 mg/ml and > 2 mg/ml, respectively. Bismuth aggregates were attached to the cysts and, in some, the pin-shaped aggregates penetrated the cyst wall. The bismuth aggregates also bound strongly to blebs and granules of B. burgdorferi when RBC > or = MBC. When B. burgdorferi is responsible for gastrointestinal symptoms, bismuth compounds may be candidates for eradication of the bacterium from the gastrointestinal tract.

This study examines the distribution of apolipoprotein E (APOE) alleles in a population of healthy male and female Norwegians (n = 798) below the age of 40. The -491A/T polymorphism of the promoter region of the APOE gene was also examined. A seminested polymerase chain reaction was applied in the genotyping. The results showed that the E3 allele had the highest frequency (0.744), followed by E4 (0.198) and E2 (0.058). The APOE frequencies found in this study differ significantly from those obtained in earlier Norwegian APOE phenotypings. The allele frequencies in the -491 site of the promoter region were 0.845 for the A allele and 0.155 for the T allele. The genotype frequency was highest for AA (0.707), followed by AT (0.277) and TT (0.016). Moreover, the A allele was in linkage disequilibrium to E4.

The aim of this study was to examine the susceptibility of mobile and cystic forms of Borrelia burgdorferi to metronidazole. Because B. burgdorferi is a microaerobic bacterium like Helicobacter pylori, metronidazole (MZ) was chosen in the susceptibility test. For both microaerobic and aerobic incubation the normal mobile spirochetes were resistant to this antibiotic with an MBCor = 512 microg/ml. Conversion of mobile spirochetes to cystic forms was not observed when they were incubated with MZ. When they were incubated under microaerobic conditions, the biologically active cystic forms had an MBCor = 4 microg/ml, but the MBC wasor = 32 microg/ml with aerobic incubation at 37 degrees C. Staining with acridine orange (AO), dark field microscopy (DFM), and transmission electron microscopy (TEM) revealed that the contents of the cysts were degraded when the concentration of MZ wasor = MBC. Some cysts were also ruptured. When incubated with a sufficient concentration of MZ, core structures did not develop inside the cysts, and AO revealed less RNA in the cysts. Our observations may help efforts to treat resistant infections caused by B. burgdorferi with a combination of MZ and other antibiotics in order to eradicate both cystic and mobile forms of B. burgdorferi.

The purpose of this study was to examine the intensity of the immunogold labeling of kappa light chains as single molecules and as parts of whole immunoglobulin molecules in LR-White sections and epoxy sections both practically and theoretically. Human renal tissues including deposits of kappa light chains and immune complex deposits of IgA were embedded in both LR-White resin and epoxy resin. Immunogold labeling was performed on unetched thin sections of both resins with anti-kappa or anti-IgA. In all relevant cases the immunolabeling was intense on the LR-White sections. Single kappa light chains were intensely labeled also on the epoxy sections, although not as intensely as on LR-White sections. In contrast, the immunogold labeling of whole immunoglobulins with anti-kapp and anti-IgA was weak and hardly positive on the epoxy sections. Consequently, the quotient labelinglr-white/labelingepoxy for anti-kappa was significantly lower for labeling of single light chains (3.6) than for labeling of whole immunoglobulins (15.9). The corresponding quotient for labeling of whole immunoglobulins with anti-IgA was 17.0. Supported by theoretical considerations, it is believed that the copolymerization between the epoxy resin and the antigens allows the knife to cleave the large whole immunoglobulins more easily than the smaller single kappa chains. This prevents satisfactory immunolabeling of whole immunoglobulins on epoxy sections whether anti-kappa or anti-IgA is used as antibodies, while single kappa chains are easily immunolabeled.

Mobile Borrelia burgdorferi were transferred to distilled water (10(6) per ml). The cultures were observed by dark field microscopy (DFM), interference contrast microscopy (ICM) and transmission electron microscopy (TEM). 95% of the spirochetes were converted to cysts after 1 min, and after 4 h no normal mobile borreliae were observed. When transferred to growth medium (BSK-H), the cysts became smaller and more irregular, and were filled with organic substances. After 1 day, 1-5 thin structures sprouted from the cysts. They continued to grow in both length and thickness until they attained a normal spirochetal structure. Finally, these new-born spirochetes detached from the cysts, by which time their mobility had become normal. The present method for producing large amounts of cystic forms of B. burgdorferi is well suited for further studies of this unique microbe.

The purpose of this study was to examine how antigen retrieval affected the yield of immunogold labeling on epoxy sections based on embedding with different amounts of accelerator. The concentration of accelerator DMP-30 (tri(dimethyl amino methyl) phenol) was varied in the range of 0–8% in the processing of the tissue for epoxy embedding. Immunogold labeling was performed on epoxy sections and LR-White sections of fibrin clots and renal tissue with IgG-deposits, and the antibodies used were anti-fibrinogen anti-IgG and, respectively. For some of the sections antigen retrieval was performed by heating the sections in citrate buffer. In all cases, the yield of immunogold labeling increased following antigen retrieval. The increase (%) in the yield of immunogold labeling as a result of antigen retrieval was larger for epoxy sections than for LR-White sections. The immunolabeling on high-accelerator epoxy sections exposed to antigen retrieval was about 20% more intense than on untreated LR-White sections both for IgG and fibrinogen. In addition to breaking fixations bonds introduced by the chemical fixation, we believe that the antigen retrieval also breaks bonds between the epoxy resin and the embedded tissue. The combination of increased amount of accelerator during tissue processing for epoxy embedding and antigen retrieval by heating in citrate buffer is a potent method for increasing specific immunolabeling on epoxy sections.

Twenty renal biopsies were studied by immunoelectron microscopy (IEM) after embedding in epoxy resin. Immunogold labeling for immunoglobulins and complement C3 was performed on the epoxy sections, which were not subjected to any kind of etching or deplasticizing prior to the immunolabeling. The concentration of accelerator, DMP-30 (Tri (Dimethyl Amino Methyl) Phenol), was increased in the infiltration and embedding steps far beyond the values normally used to make immunolabeling of these antigens possible on epoxy sections. The sections were stained with tannic acid accompanied by uranyl acetate and lead citrate. Immunofluorescence (IF) for light microscopy was carried out on frozen sections of parallel tissue samples. Some cases with IgA-nephritis demonstrated a higher sensitivity for IEM than IF, in the sense that smaller amounts of antigen were detectable with IEM. Ultrastructural preservation with this method was approximately the same as that usually seen on epoxy-embedded material. By combining excellent immunolabeling with nearly optimal ultrastructural morphology in one procedure, this method is useful particularly in situations where the material available is limited, such as in studies of renal biopsies. As far as we know, this is the first time that immunoglobulins have been satisfactorily immunolabeled on epoxy sections without etching or deplasticizing.

The purpose of this investigation was to develop a method that could be used to estimate how damaging sodium ethoxide is to different antigens with respect to immunolabeling when epoxy sections are deplasticized. If we obtain weak labeling for an antigen on deplasticized epoxy sections, this might be caused by the damaging effect of the ethoxide solution. It is therefore interesting to develop a method to check if this really is the reason. Fibrin clots and tissues of human kidney and thyroid were embedded in LR White resin. Some thin sections from these specimen blocks were exposed to sodium ethoxide in the same way as epoxy sections are when being deplasticized. Other sections from the same blocks were not exposed to sodium ethoxide. Both categories of sections were immunogold-labeled with anti-fibrinogen, anti-thyroglobulin, anti-IgA, anti-IgG, or anti-IgM. The intensity of immunolabeling of sections treated with ethoxide was compared with the immunolabeling of corresponding sections that were not treated with ethoxide. No significant differences were found in immunolabeling for fibrinogen, IgA, IgG, and IgM. For thyroglobulin, the intensity was approximately 30% less in tissues that were exposed to sodium ethoxide. The practical significance of this method is that we easily can examine the degree to which a given antigen is affected by sodium ethoxide, which is the agent used for deplasticizing epoxy sections.

The purpose of this study was to predict the ratio of immunogold labeling of deplasticized epoxy sections and LR-White sections on the basis of theoretical considerations. Tissues used in the experiments were pancreas, pituitary, kidney, thyroid, and fibrin. Antigens used as test proteins were glucagon, somatostatin, thyroglobulin, chromogranin A, ACTH (=Adrenocroticotropt hormone), amyloid A, and fibrinogen. These are proteins of different sizes. The quotient labeling deplasticized /labeling LR-white was deduced theoretically and compared to measurements based on immunogold experiments to obtain a theoretical model with acceptable correlation to the measurements. This study describes a theory—expressed mathematically—for what happens at the molecular level in immunoelectron microscopy at the surface of deplasticized epoxy sections and acrylic sections. The theory explains why we normally get about the same amount of immunogold labeling using LR-White sections (acrylic resin) and deplasticized epoxy sections. Taking the nuances into account, the theory indicates increased usefulness of deplasticized epoxy sections when the diameter of the protein carrying the epitopes decreases.

Tartrate-resistant acid phosphatase (TRAP) is known as an osteoclast marker, but osteoblasts and osteocytes in the vicinity of bone remodeling sites also express TRAP. Cell culture studies suggest that osteoblasts endocytose osteoclastic TRAP for inactivation. To evaluate whether changes in osteoclast activity could alter TRAP expression in osteoblasts and/or osteocytes in vivo, we studied the ovariectomized and vitamin D-deficient rat (Ovx-D) and rats healing from rickets. Bone sections were analyzed for TRAP gene expression by in situ hybridization, TRAP protein by immunogold labeling, and TRAP enzyme activity using the fluorescent substrate ELF97. Osteoblasts and osteocytes close to intracortical remodeling sites and bone surfaces demonstrated TRAP, most prominently in cancellous bone and osteocytes. Intracellular TRAP was located to electron-dense vesicles with similar morphology in both cell types. Ovx-D increased osteoclast activity (p