Scaffold-Free Engineering of Human Cartilage Implants.

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.

Optic Atrophy 1 Controls Human Neuronal Development by Preventing Aberrant Nuclear DNA Methylation.

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.

Outer membrane vesicles extracted from Neisseria meningitidis serogroup X for prevention of meningococcal disease in Africa.

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.

NEIL3-Dependent Regulation of Cardiac Fibroblast Proliferation Prevents Myocardial Rupture.

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.

Syndecan-4 is a key determinant of collagen cross-linking and passive myocardial stiffness in the pressure-overloaded heart.

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.

Increased tartrate-resistant Acid phosphatase expression in osteoblasts and osteocytes in experimental osteoporosis in rats.

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 < 0.001) and ELF97⁺ osteocytes (p < 0.05) in cancellous bone, but no corresponding increase was observed in the osteocyte lacunar area. The level of TRAP⁺ vesicles in cortical osteoblasts (p < 0.01) in Ovx-D rats was also increased. Enhanced osteoclast activity was noted in healing rickets after 72 h (p < 0.05), but no differences in TRAP expression were detected in osteoblasts or osteocytes. Thus, increased osteoclast activity does not affect TRAP expression in osteoblasts and osteocytes, favoring the notion that increased TRAP in these cells is rather due to increased synthesis. Although the role of TRAP in osteoblasts and osteocytes remains elusive, we speculate that the function is related to the capability of the enzyme to regulate the phosphorylation of proteins known to be expressed by these cells.

The skeletal phenotype of chondroadherin deficient mice.

Chondroadherin, a leucine rich repeat extracellular matrix protein with functions in cell to matrix interactions, binds cells via their α2ß1 integrin as well as via cell surface proteoglycans, providing for different sets of signals to the cell. Additionally, the protein acts as an anchor to the matrix by binding tightly to collagens type I and II as well as type VI. We generated mice with inactivated chondroadherin gene to provide integrated studies of the role of the protein. The null mice presented distinct phenotypes with affected cartilage as well as bone. At 3-6 weeks of age the epiphyseal growth plate was widened most pronounced in the proliferative zone. The proteome of the femoral head articular cartilage at 4 months of age showed some distinct differences, with increased deposition of cartilage intermediate layer protein 1 and fibronectin in the chondroadherin deficient mice, more pronounced in the female. Other proteins show decreased levels in the deficient mice, particularly pronounced for matrilin-1, thrombospondin-1 and notably the members of the α1-antitrypsin family of proteinase inhibitors as well as for a member of the bone morphogenetic protein growth factor family. Thus, cartilage homeostasis is distinctly altered. The bone phenotype was expressed in several ways. The number of bone sialoprotein mRNA expressing cells in the proximal tibial metaphysic was decreased and the osteoid surface was increased possibly indicating a change in mineral metabolism. Micro-CT revealed lower cortical thickness and increased structure model index, i.e. the amount of plates and rods composing the bone trabeculas. The structural changes were paralleled by loss of function, where the null mice showed lower femoral neck failure load and tibial strength during mechanical testing at 4 months of age. The skeletal phenotype points at a role for chondroadherin in both bone and cartilage homeostasis, however, without leading to altered longitudinal growth.

Inflammatory markers CD11b, CD16, CD66b, CD68, myeloperoxidase and neutrophil elastase in eccentric exercised human skeletal muscles.

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.

The apolipoprotein E polymorphism and cardiovascular diseases--an autopsy study.

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSION: 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.

Destruction of spirochete Borrelia burgdorferi round-body propagules (RBs) by the antibiotic tigecycline.

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.

Calmodulin-dependent kinase 1beta is expressed in the epiphyseal growth plate and regulates proliferation of mouse calvarial osteoblasts in vitro.

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 intensity of immunogold labeling of deplasticized acrylic sections compared to deplasticized epoxy sections-Theoretical deductions and experimental data.

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.

Heat-induced retrieval of immunogold labeling for nucleobindin and osteoadherin from Lowicryl sections of bone.

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.

An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to tinidazole.

The susceptibility of mobile and cystic forms of Borrelia burgdorferi to tinidazole (TZ) was examined. The minimal bactericidal concentration (MBC) of TZ against the mobile spirochetes was >128 microg/ml at 37 degrees C in micro-oxic atmosphere when incubated for 14 days. TZ significantly reduced the conversion of mobile spirochetes to cystic forms during incubation. The MBC for older (10-months-old) cysts at 37 degrees C in a micro-oxic atmosphere was >0.5 microg/ml, but >0.125 microg/ml for young (1-day-old) cysts. Acridine orange staining, dark-field microscopy and transmission electron microscopy revealed that, when the concentration of TZ was > or = MBC, the contents of the cysts were partly degraded, core structures did not develop inside the young cysts, and the amount of RNA in these cysts decreased significantly. When cysts were exposed to TZ, both the spirochetal structures and core structures inside the cysts dissolved, and the production of blebs was significantly reduced. These observations may be valuable in the treatment of resistant infections caused by B. burgdorferi, and suggest that a combination of TZ and a macrolide antibiotic could eradicate both cystic and mobile forms of B. burgdorferi.

A method for measurements of the efficiency of immunogold labelling of epoxy-embedded proteins subjected to different retrieval techniques.

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.

An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to tinidazole / Estudio in vitro de la susceptibilidad de las formas móviles y císticas de Borrelia burgdorferi al tinidazol

The susceptibility of mobile and cystic forms of Borrelia burgdorferi to tinidazole (TZ) was examined. The minimal bactericidal concentration (MBC) of TZ against the mobile spirochetes was >128 microg/ml at 37 degrees C in micro-oxic atmosphere when incubated for 14 days. TZ significantly reduced the conversion of mobile spirochetes to cystic forms during incubation. The MBC for older (10-months-old) cysts at 37 degrees C in a micro-oxic atmosphere was >0.5 microg/ml, but >0.125 microg/ml for young (1-day-old) cysts. Acridine orange staining, dark-field microscopy and transmission electron microscopy revealed that, when the concentration of TZ was > or = MBC, the contents of the cysts were partly degraded, core structures did not develop inside the young cysts, and the amount of RNA in these cysts decreased significantly. When cysts were exposed to TZ, both the spirochetal structures and core structures inside the cysts dissolved, and the production of blebs was significantly reduced. These observations may be valuable in the treatment of resistant infections caused by B. burgdorferi, and suggest that a combination of TZ and a macrolide antibiotic could eradicate both cystic and mobile forms of B. burgdorferi (AU)

Este estudio examina la susceptibilidad al tinidazol (TZ) de las formas móviles y císticas de Borrelia burgdorferi. La concentración bactericida mínima (CBM) de TZ para las espiroquetas móviles era >128 mg/ml a 37°C en atmosfera microóxica e incubación durante 14 días. El TZ redujo significativamente la conversión de espiroquetas móviles a la forma cística durante la incubación. La CBM para los cistos viejos (de 10 meses) a 37°C y en atmosfera microóxica era >0.5 mg/ml, mientras para los cistos jóvenes (de un día) era >0.125 mg/ml. La tinción con naranja de acridina, la microscopia de campo oscuro, y la microscopia electrónica de transmisión mostraron que cuando la concentración de TZ era ≥MBC el contenido de los cistos se degradaba parcialmente, no se desarrollaban las estructuras nucleares en el interior de los cistos jóvenes, y la cantidad de RNA en dichos cistos disminuía significativamente. Cuando los cistos se exponían a TZ, las estructuras espiroquetales y nucleares de su interior se disolvían, y la producción de vesículas se reducía significativamente. Estas observaciones pueden ser importantes en el tratamiento de infecciones resistentes causadas por B. burgdorferi, y sugieren que la combinación de TZ con un antibiótico macrólido podría erradicar tanto las formas císticas de B. burgdorferi como las móviles (AU)

The fate of monocytes during 24 h of culture as revealed by flow cytometry and electron microscopy.

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.

Allele frequencies of apolipoprotein E gene polymorphisms in the protein coding region and promoter region (-491A/T) in a healthy Norwegian population.

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.

Susceptibility of motile and cystic forms of Borrelia burgdorferi to ranitidine bismuth citrate

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 (AU)

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