Improved diagnosis of idiopathic giant cell myocarditis and cardiac sarcoidosis by myocardial gene expression profiling.

AIMS: Improvement of clinical diagnostics of idiopathic giant cell myocarditis (IGCM) and cardiac sarcoidosis (CS), two frequently fatal human myocardial diseases. Currently, IGCM and CS are diagnosed based on differential patterns of inflammatory cell infiltration and non-caseating granulomas in histological sections of endomyocardial biopsies (EMBs), after heart explantation or postmortem. We report on a method for improved differential diagnosis by myocardial gene expression profiling in EMBs. METHODS AND RESULTS: We examined gene expression profiles in EMBs from 10 patients with histopathologically proven IGCM, 10 with CS, 18 with active myocarditis (MCA), and 80 inflammation-free control subjects by quantitative RT-QPCR. We identified distinct differential profiles that allowed a clear discrimination of tissues harbouring giant cells (IGCS, CS) from those with MCA or inflammation-free controls. The expression levels of genes coding for cytokines or chemokines (CCL20, IFNB1, IL6, IL17D; P < 0.05), cellular receptors (ADIPOR2, CCR5, CCR6, TLR4, TLR8; P < 0.05), and proteins involved in the mitochondrial energy metabolism (CPT1, CYB, DHODH; P < 0.05) were deregulated in 2- to 300-fold, respectively. Bioinformatic analyses and correlation of the gene expression data with immunohistochemical findings provided novel information regarding the differential cellular and molecular pathomechanisms in IGCM, CS, and MCA. CONCLUSION: Myocardial gene expression profiling is a reliable method to predict the presence of multinuclear giant cells in the myocardium, even without a direct histological proof, in single small EMB sections, and thus to reduce the risk of sampling errors. This profiling also facilitates the discrimination between IGCM and CS, as two different clinical entities that require immediate and tailored differential therapy.

A distinct subgroup of cardiomyopathy patients characterized by transcriptionally active cardiotropic erythrovirus and altered cardiac gene expression.

Recent studies have detected erythrovirus genomes in the hearts of cardiomyopathy and cardiac transplant patients. Assessment of the functional status of viruses may provide clinically important information beyond detection of the viral genomes. Here, we report transcriptional activation of cardiotropic erythrovirus to be associated with strongly altered myocardial gene expression in a distinct subgroup of cardiomyopathy patients. Endomyocardial biopsies (EMBs) from 415 consecutive cardiac erythrovirus (B19V)-positive patients with clinically suspected cardiomyopathy were screened for virus-encoded VP1/VP2 mRNA indicating transcriptional activation of the virus, and correlated with cardiac host gene expression patterns in transcriptionally active versus latent infections, and in virus-free control hearts. Transcriptional activity was detected in baseline biopsies of only 66/415 patients (15.9 %) harbouring erythrovirus. At the molecular level, significant differences between cardiac B19V-positive patients with transcriptionally active versus latent virus were revealed by expression profiling of EMBs. Importantly, latent B19V infection was indistinguishable from controls. Genes involved encode proteins of antiviral immune response, B19V receptor complex, and mitochondrial energy metabolism. Thus, functional mapping of erythrovirus allows definition of a subgroup of B19V-infected cardiomyopathy patients characterized by virus-encoded VP1/VP2 transcripts and anomalous host myocardial transcriptomes. Cardiac B19V reactivation from latency, as reported here for the first time, is a key factor required for erythrovirus to induce altered cardiac gene expression in a subgroup of cardiomyopathy patients. Virus genome detection is insufficient to assess pathogenic potential, but additional transcriptional mapping should be incorporated into future pathogenetic and therapeutic studies both in cardiology and transplantation medicine.

miRNA as activity markers in Parvo B19 associated heart disease.

Parvovirus B19 is a frequent virus detected in endomyocardial biopsies of patients with clinically suspected myocarditis or dilated cardiomyopathy (DCM). Viruses often cause a more symptomatic disease with increased tissue injury if they become reactivated. A disease-specific differential expression of microRNAs (miRNAs) has been described in the regulation of replicating viruses. Analyzing patients with latent and reactivated B19V infection, we found 29 differentially regulated miRNAs and, in order to test whether predicted genes are differentially expressed, selected mRNAs were tested by TaqMan-QPCR.

Prevalence of erythrovirus genotypes in the myocardium of patients with dilated cardiomyopathy.

Parvovirus B19 (PVB19) is a member of the human erythrovirus family detected frequently in endomyocardial biopsies from patients with dilated cardiomyopathy. Human erythroviruses cluster into three genotypes 1-3 which share a high degree of homology between major structural proteins and may cause indistinguishable infections clinically and serologically. In human cardiac tissue erythrovirus genotypes other than PVB19 have not yet been reported. Three hundred seventeen consecutive patients with symptomatic dilated cardiomyopathy (median left ventricular ejection fraction: 28.6%, range 5-45%) who underwent endomyocardial biopsy for the elucidation of the etiology, were analyzed using a new consensus PCR assay designed for the detection of the three erythrovirus genotype sequences. Endomyocardial biopsies of 151 (47.6%) patients were erythrovirus-positive. Genotype 1 specific sequences were detected in 43/151 (28.5%) of positive biopsy samples, whereas genotype 2-specific sequences so far considered rare in human disease and not yet been described in human heart tissue was identified in 108/151 (71.5%) of virus-positive endomyocardial biopsies with a preference in patients above 50 years of age. In spite of younger age, systolic left ventricular dysfunction of genotype 1-positive patients was significantly reduced as compared to genotype 2-positive patients (24.4+/-10.4% vs. 31.0+/-9.5%, P=0.0001) at the initial presentation. The data show that two genetically distinct erythrovirus variants with a different age distribution are detectable in endomyocardial biopsies of patients with dilated cardiomyopathy. The erythrovirus genotype 2, not described previously in human heart tissue, is highly prevalent in the heart but the less prevalent genotype 1 is associated with more severe disturbed cardiac function.

Culture of haematopoietic cells in a 3-D bioreactor made of Al2O3 or apatite foam.

Foams of Al2O3 and apatite ceramics with interconnecting pores were produced using a new technique. The surfaces of the ceramics served as substrates for the culture of human peripheral and bone marrow derived stem cells. Up to 27 days the cells were kept in culture where they proliferated and developed into different morphologies consistent with bone marrow cell lines.

In vivo comparison of bioactive glass particles in rabbits.

Bioglass particles of the compositions 45s5, 52s and 55s were implanted in the distal femoral epiphysis of rabbits. Animals were sacrificed at 7, 28, and 84 d postoperatively and specimens investigated using light microscopy and histomorphometry. Bone bonding occurred in a zentripetal fashion and fastest for 45s5. Bone formation was hampered at the core of the implantation bed where bone bonding showed a peak at 28d and diminished at 84d (except for 55s). This went along with a significant increase in numerous multinuclear giant cells (MNGC). Implantation model, particle size and surface-area-to-volume ratio are discussed as possible parameters determining bone regeneration.

Primary mineralization at the surfaces of implants.

Osteogenesis around implants is affected by the physical and chemical characteristics of the biomaterials used. The osteoprogenitor cells must migrate to the implant site and synthesize and secrete a mineralizable extracellular matrix. Because this is neo-bone formation, the mechanism by which the cells calcify their matrix involves extracellular organelles called matrix vesicles in a process termed "primary mineralization". Two different methods for assessing the effects of implant materials on primary mineralization are presented in this report. In the first approach, different implant materials used in dentistry and orthopedic surgery were placed in rat tibial bones after marrow ablation. Two groups of implants were used, bone-bonding and non-bonding materials. We examined the effects of the materials on calcification morphometrically by quantitating changes in matrix vesicle morphology and distribution in endosteal tissue around implants as compared with normal endosteal bone healing. In addition, matrix vesicles were isolated from the endosteal tissue around the implant as well as from the contralateral limb and were examined biochemically. The results demonstrated that bone-bonding materials induced a greater increase in matrix vesicle enzyme activity than did non-bonding materials. However, all materials caused changes in matrix vesicles that were different from those seen in normal endosteal bone formation following injury. The effects of implant materials on biochemical markers of mineralization, including specific activities of matrix vesicle alkaline phosphatase and phospholipase A2 and phosphatidylserine content, demonstrated a high correlation with the morphometric observations with regard to enhancement and/or delay of primary mineralization. In the other approach, we used a radioisotopic method to evaluate the effects of implant materials on primary mineralization. This analysis revealed that implants alter bone healing, as shown by the differential uptake of 99mTc and 32P in different bone compartments. Decreased 32P uptake by the organic phase in the presence of bone-bonding implants suggests that cleavage of 99mTcMD32P into its technetium and methylene diphosphonate moieties was inhibited by the presence of the implants. In summary, these approaches to evaluating the effects of materials on primary mineralization demonstrate that the marrow ablation model can easily distinguish between bone-bonding and non-bonding materials. The use of this model can be valuable in the development of new materials.

Woven bone formation around implants and the effect of bacterial infection.

Several implant materials used in dental and orthopedic surgery were placed in rat tibial bones to study their effects on mineralization. The implants consisted of bone bonding and non-bonding materials. Changes in mineralization were defined by morphometric analysis of matrix vesicle distribution at the implant interface and in normal bone healing following marrow injury. Bone-bonding materials induced an increase in matrix vesicle activity. This finding was supported by study of the biochemical changes in the same model that manifested high correlations to the morphometrical observations with regard to enhancement or delay of primary mineralization. In addition, the study of healing using nuclear methods indicated that implants alter bone healing as shown by the different uptakes of 99mTc and 32P in the different bone compartments. Decreased 32P uptake by the organic phase in the presence of bone-bonding implants suggested that cleavage of 99mTc-MD32P into its technetium and methylene diphosphonate moieties was inhibited by administration of implants. Further studies on the effect of bacterial infection on the peri-implant tissues revealed a decrease in woven bone formation due to infection.

Tenascin labelling in colorectal biopsies: a useful marker in the diagnosis of collagenous colitis.

AIMS: This study was undertaken to clarify whether immunohistological detection of tenascin (TN), an extracellular matrix glycoprotein that is expressed during stromal remodelling, may allow a more precise diagnosis of collagenous colitis. METHODS AND RESULTS: We studied multiple colorectal biopsies specimens from 15 patients with clinically suspected collagenous colitis for TN expression by using a monoclonal antibody. Biopsies from further 15 patients without symptoms and signs of collagenous colitis served as controls. In seven of the 15 cases with clinically suspected collagenous colitis a prominent and selective subepithelial tenascin expression was identified. The TN expression pattern closely correlated with the conventional detection of a subepithelial collagen band diagnostic of collagenous colitis. The immunohistological labelling for TN allowed a quicker and more precise measurement of the thickness of the diagnostic collagen deposits than conventional staining. By this approach one further case could be reclassified as collagenous colitis. CONCLUSIONS: Our data show that immunohistological detection of TN allows a more correct and easy diagnosis of collagenous colitis.

Osteoblast reaction at the interface between surface-active materials and bone in vivo: a study using in situ hybridization.

Osteoblast activation after implantation of two kinds of surface-active material in bone was investigated chronologically using in situ hybridization with digoxygenin-labeled procollagen alpha 1(I) complementary RNA probe. The bioactive materials used were hydroxyapatite (HA) and apatite- and wollastonite-containing glass-ceramic (A-W GC). A hole was drilled bilaterally in the distal epiphysis of rabbit femurs with subsequent implantation of HA or A-W GC cylinders in a press-fit manner. Specimens were collected at 3, 7, 14, and 28 days after operation and decalcified. Then the undecalcified implant cores were pushed out of the hole without causing damage to the bony side of the interface. In situ hybridization documented no qualitative differences in the expression of procollagen alpha 1(I) RNA between HA and A-W GC. Few osteoblasts at the bone-material interface showed a specific signal at day 3, whereas many osteoblasts were positive around the materials at days 7 and 14, indicative of active new bone formation. The positive osteoblasts seemed to originate from preexisting trabeculae and lined the trabeculae, newly formed bone, and material surface. At day 28, many osteoblasts lining material-surrounding bone were negative, whereas those in remodeling canals were positive, suggesting that the bone was in the remodeling stage after bone formation. These findings were comparable to those with beta-tricalcium phosphate in a previous study, thus suggesting osteoconductive bone formation on HA and A-W GC.

Temporal and spatial patterns of osteoblast activation following implantation of beta-TCP particles into bone.

Temporal and spatial patterns of osteoblast activation around beta-TCP particles implanted into bone were analyzed by in situ hybridization with digoxygenin-labeled procollagen alpha 1(I) RNA probes. beta-TCP particles (150-300 microns in diameter) were implanted into rat tibiae, and specimens were collected 3, 5, 7, 14, and 28 days after operation. Activated osteoblasts displayed intense procollagen alpha 1(I) RNA specific labeling. At day 3, osteoblasts lining pre-existing trabeculae in places showed a specific signal. Additionally, scattered activated cells compatible with preosteoblasts also were observed in the vicinity of the trabeculae among red blood cells that filled the space between beta-TCP particles. Osteoblast activation on the surface of beta-TCP rarely was observed. At days 5 and 7, osteoblast activation and bone formation advanced centripetally. At the forefront of bone formation positive cells were scattered in the blood cell clots, and some of the positive cells colonized forming new bone matrix. Formation of new bone did not always begin at the surface of beta-TCP. At day 14, most of the beta-TCP particles were tightly associated with newly formed bone, and the number of positive osteoblasts was reduced. At day 28, absorption of the newly formed bone and the beta-TCP by multinuclear cells was sporadically demonstrated. Such cells often were accompanied by active osteoblasts, suggesting early bone remodeling. In conclusion, in situ hybridization with procollagen alpha 1(I) was employed to demonstrate precisely the mode of recruitment of bone cell precursors. beta-TCP does not positively guide collagen I expressing bone cells along its surface. It has no apparent effects on bone regeneration.

Analysis of osteoblast activity at biomaterial-bone interfaces by in situ hybridization.

To investigate the effects of bioactive materials on bone formation in vivo, a new experimental model using in situ hybridization has been developed. A hole was drilled bilaterally in the distal epiphysis of rabbit femurs with subsequent implantations of beta-tricalcium phosphate (beta-TCP) cylinders in a press-fit manner. Specimens were collected at 3, 7, 14, and 28 days after operation. Femurs with empty drilling holes, and normal distal femurs without operation were used as controls. All specimens were decalcified and hybridized with a procollagen alpha 1(I) complementary RNA probe labeled with digoxygenin. In normal-bone sections, procollagen alpha 1(I) RNA was clearly demonstrated in periosteal osteoblasts, in osteoblasts in the mineralizing zone adjacent to growth plates, and in osteoblasts lining remodeling canals. As for beta-TCP, labeled osteoblasts around the material were not found at day 3, whereas they were most intensively observed at day 7 and a little less at day 14, in accordance with new-bone formation around the material. Weaker signals were also detected in fibroblasts at day 7. At day 28, osteoblasts lining the surface of newly formed bone were mainly negative, whereas those adjacent to the resorption sites of the beta-TCP showed positive signals, demonstrating an active remodeling at the material surface. The temporal expression of procollagen alpha 1(I) RNA in the beta-TCP specimens was fundamentally the same as that in the empty-hole specimens, suggesting no remarkable acceleration or suppression of bone-forming activity of osteoblasts by beta-TCP, which is consistent with osteoconductive bone formation. This in situ hybridization method was suggested to be a powerful tool in analyzing the biological effects of bioactive materials.

Gingival metastasis of merkel cell carcinoma: a case report.

An 82-year-old Caucasian man developed an ulcerated mass on the anterior mandibular gingiva. Five years previously he had been treated for a Merkel cell carcinoma (MCC) on his right cheek. Histopathologic examination showed small tumor cells with scanty cytoplasm, suggestive of malignancy. Immunohistochemical studies were performed with the use of nine antibodies. S-100 protein and leukocyte common antigen were helpful in ruling out melanoma and lymphoma. Pronounced reaction was shown for cytokeratin 20, a new histodiagnostic marker whose expression is almost entirely confined to Merkel cells, the gastric epithelium, and urothelium. The tentative diagnosis of metastasis of MCC was confirmed. Immunohistochemical studies are useful diagnostic aids in the establishment of the diagnosis of Merkel cell carcinoma.

Production of stable rabbit-mouse heterohybridomas: characterization of a rabbit monoclonal antibody recognizing a 180 kDa human lymphocyte membrane antigen.

Polyclonal rabbit antihuman thymocyte globulin (RATG) remains a key component of immunosuppressive strategies in transplantation. The human thymus immunization regimen that produces highly immunosuppressive RATG induces unique antibody specificities in the rabbit. Rabbit monoclonal antibodies (RAb MAbs) to human T cell antigens would be of value in the effort to investigate and reproduce the multiple specificities of RATG. We have fused mouse Sp2/0 cells with splenocytes from rabbits immunized with human thymus and have identified 52 rabbit-mouse heterohybridomas which secrete RAb MAbs directed against human lymphocyte surface antigens. The technical aspects of hybridoma isolation, stabilization and characterization are presented. Analysis by flow cytometry, preabsorption and immunoprecipitation suggests that RAb MAb 1A8 IgG may recognize LFA-1, one of the principal lymphocyte surface antigens recognized by RATG. The 1A8 antigen is 180 kDa and is expressed by 80-90% human PBL and thymocytes. LFA-1 and the 1A8 antigen exhibit 100% co-expression in two-color FACS analysis using four different murine anti-LFA-1 MAbs. 1A8 markedly inhibits the mitogenic response of lymphocytes to PHA, as do murine anti-LFA-1 MAbs. A combination of rabbit antilymphocyte MAbs may potentially reproduce the multiple specificities found in polyclonal RATG and lead to the production of a superior immunosuppressive clinical agent.

The early host and material response of bone-bonding and non-bonding glass-ceramic implants as revealed by scanning electron microscopy and histochemistry.

The interface of bone-bonding and non-bonding glass-ceramics in the femur of of rats with the concomitant material and host response has been investigated by scanning electron microscopy (SEM) and histochemistry after transverse fractures in the interface level. During wound healing around these implants, four overlapping phases could be distinguished: (1) blood clot formation, (2) formation of organization tissue, (3) formation of primary bone and calcification, and (4) remodelling which lasts from months to years. This sequence of healing was disturbed around metal ions containing non-bonding implants, as shown by a longer settlement of acid phosphatase positive macrophages and a disturbed calcification at the surface of the material. Only bone-bonding implants developed considerable changes in surface morphology due to leaching and corrosion phenomena. A preferential leaching of the glass moiety, starting at the phase transition between glass and ceramic, contributes to the production of surface elevations which provide adhesion points for fibres and fibrils. Subsequent mineralization of inserting fibres contributes to a tensile strength at the interface. These findings are essential for further understanding of bone-bonding mechanisms and for further development of surface-reactive materials.

Histological and ultrastructural observations at the interface of expanded polytetrafluorethylene anterior cruciate ligament implants.

Anterior cruciate ligament (ACL) grafts made from expanded polytetrafluorethylene (ePTFE) were examined after failure (n = 7; 3 instabilities, 2 partial ruptures, and 2 complete ruptures) to provide information about secondary fixation mechanisms via ingrowth of the different tissues. The ultrastructural evaluation clearly evidenced the existence of two main interface areas of ePTFE ACL-replacement: First, in areas without a dense cellular infiltrate there was interdigitating collagen producing a dense ePTFE-collagen network. Additionally, in a few areas of the graft/bone-tunnel interface a fibrous cartilage or bone regenerate could be demonstrated to be in contact with the prosthesis. Second, there was a dense infiltration of macrophages and multinuclear giant cells, partially containing birefringent material of implant origin, with and without a neosynovia-like reaction product, indicating a disturbance of tissue integration of the prosthesis. In areas of inflammation there was no bone development and only few collagen interdigitation with the graft material. This study provides further knowledge about mechanisms of secondary graft fixation due to tissue ingrowth. The interdigitation of collagen fibers and ePTFE filaments provides interfaces which should be at least partially resistant against load.

The interface of calcium-phosphate and glass-ceramic in bone, a structural analysis.

Bone bonding (BB) glass-ceramics and hydroxyapatite (HA) materials displayed considerable changes in their surface morphology after implantation which seem to be due to leaching, corrosion and active resorption by osteoclasts. Macrophages seem to play a central role in 'cleaning' and conditioning of the implant surface. Projections of the surface of BB glass-ceramics and HA serve as adhesion points for fibrillar structures. Thereafter, mineralization of fibres and interfibrillar spaces mediates and biomechanically stabilizes the interdigitation between surface elevations of implant and organic fibres and provides a tensile strength in the interface. Non-bonding (NB) glass-ceramic lacks considerable changes of surface morphology and displays a prolongated settlement of macrophages on their surfaces.

Tissues at the surface of the new composite material titanium/glass-ceramic for replacement of bone and teeth.

A new composite implant material titanium/glass-ceramic was tested in rabbits using light microscopy, histomorphometry, and biomechanical testing methods. Two rabbit implant models were used. The first premolar tooth was replaced and cylinders inserted into the trabecular bone of the distal femur below the patella sliding plane. There was bone bonding to the glass-ceramic component and additional mechanical interlocking, due to bone ingrowth between the titanium matrix into secondary pores. This was proved by measuring the tensile strength at the interface of the new composite material which was in the same range as compared to pure glass-ceramic implants. In tooth replacement there was a tight attachment of gingival epithelium and stroma to composite titanium/glass-ceramic. These results are of particular clinical interest: physicochemical bone bonding and additional mechanical interlocking result in a resistance of the implant material against shear and tensile loads at the interface. Therefore this new composite material should be suitable for further load-bearing applications.

Biocompatibility--the interaction of biomaterials and host response.

Future developments for better biocompatibility of dental implants are possible and are discussed here. On each level of local host response, which is subdivided into four phases, improvements of knowledge in basic mechanisms concerning the interaction of biomaterial and host are necessary. For the development of new biomaterials, the limits for load with foreign ions and particles acting on cellular and extracellular matrix should be determined. An example is given for ZrO2-containing glass ceramics. Biochemistry of soft and hard tissue bonding to biomaterials should be investigated. Biomechanical properties of implants could be adapted to individual needs of patients. New composite materials are candidates for the design of long-term functioning implants.