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Biomedical subjects

M Heberer

Publications and source records attributed to M Heberer.

At least 19 recordsLinked to original sources
PubMed2003

Development and validation of a bioreactor for physical stimulation of engineered cartilage.

A bioreactor has been developed to apply different regimes of physical stimulation to tissue specimens under highly controlled conditions. The computer-controlled device exposes specimens to compressive deformation at various strains and frequencies, measures the load applied to each sample and allows simultaneous medium stirring at different velocities. Validation tests confirmed the accuracy of the system in (i) its displacement (errors averaged 0.072+/-0.051 microm), and in (ii) setting the contact with the samples utilizing micrometer screws coupled to plungers (errors averaged 1.74+/-0.36% for samples of 1.60-3.18 mm thickness), thus ensuring accurate compressive deformation. The developed bioreactor, which represents an advance in the technology for physical stimulation of tissue specimens, is currently used to apply compressive deformation and hydrodynamic forces to human chondrocytes cultured in biodegradable polymer scaffolds, with the goals of (i) engineering functional grafts for the repair of cartilage defects (ii).

Bioreactors↗
PubMed2002

[Internet communication between family physicians and the university hospital].

The potential of electronic communication in medicine is assessed based on an analysis of a pilot project pertaining to internet based communication among referring and hospital physicians. Advantages of electronic data exchange in medicine pertain to speed and capacity for data transfer, availability of data and data integration, ultimately enabling consistent medical case management. Quality requirements of electronic communication of medical data are related to safety, availability, data integration, potential for case management and system qualities. Medical efficiency can be increased by use of electronic communication only if complex functions beyond the substitution of conventional mail by e-mail are implemented and an exhaustive use of the technology can be achieved.

Case Management↗
PubMed2001

Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro.

Adult human articular chondrocytes were expanded in a medium with 10% serum (CTR) or further supplemented with different mitogens (i.e., EGF, PDGFbb, FGF-2, TGF beta 1, or FGF-2/TGF beta 1). Cells were then induced to redifferentiate in 3D pellets using serum-supplemented medium (SSM), serum-free medium (SFM), or SFM supplemented with factors inducing differentiation of chondroprogenitor cells (i.e., TGF beta 1 and/or dexamethasone). All factors tested during expansion enhanced chondrocyte proliferation and dedifferentiation, as assessed by the mRNA ratios of collagen type II to type I (CII/CI) and aggrecan to versican (Agg/Ver), using real-time PCR. FGF-2/TGF beta 1-expanded chondrocytes displayed the lowest doubling times, CII/CI and Agg/Ver ratios, averaging, respectively, 50, 0.2 and 15% of CTR-expanded cells. Redifferentiation in pellets was more efficient in SFM than SSM only for EGF-, PDGFbb- or FGF-2-expanded chondrocytes. Upon supplementation of SFM with TGF beta and dexamethasone (SFM TD), CII/CI ratios decreased 4.4-fold for EGF- and PDGFbb-expanded chondrocytes, but increased 96-fold for FGF-2/TGF beta 1-expanded cells. Chondrocytes expanded with FGF-2/TGF beta 1 and redifferentiated in SFM TD expressed the largest mRNA amounts of CII and aggrecan and generated cartilaginous tissues with the highest accumulation of glycosaminoglycans and collagen type II. Our results provide evidence that growth factors during chondrocyte expansion not only influence cell proliferation and differentiation, but also the cell potential to redifferentiate and respond to regulatory molecules upon transfer into a 3D environment.

Adult↗
PubMed2001

Expression of MAGE tumour-associated antigens is inversely correlated with tumour differentiation in invasive ductal breast cancers: an immunohistochemical study.

MAGE (Melanoma antigen E) family gene products encompass tumour-associated antigens (TAAs) recognised by human leukocyte antigen (HLA)-restricted specific T-cells. Agents inducing DNA demethylation, an event typically detectable in cellular de-differentiation processes, were shown to induce the expression of MAGE genes. By using a monoclonal antibody specific for MAGE family gene products, we have studied the expression of these TAAs in a group of 144 patients with invasive ductal breast cancers. Immunohistochemical data were correlated with tumour differentiation, lymphatic vessel invasion, oestrogen receptor expression, intratumoural necrosis, lymphocytic infiltration, perineural invasion, tumour microcalcifications and axillary lymph node metastases. MAGE immunoreactivity was undetectable in non-neoplastic cells. In poorly differentiated cancers positive staining was observed in 30/63 cases (47.6%) as compared with 13/51 (25.4%) and 5/30 (16.6%) in moderately and well-differentiated tumours, respectively (P<0.05). In addition, MAGE immunoreactivity was significantly correlated with lymphatic vessel invasion and intratumoural necrosis. Moreover, a significant inverse relationship with oestrogen receptor expression was also observed. However, no significant correlation could be established between MAGE immunoreactivity and defined phenotypic characteristics of tumour infiltrating lymphocytes, including expression of CD3, CD4, CD8, CD20 or granzyme B. Thus, expression of MAGE family gene products in invasive ductal breast cancers appears to be associated with poorly differentiated histological phenotypes. These data support the concept of specific immunotherapy in highly aggressive forms of breast neoplasms. Furthermore, they suggest that MAGE immunoreactivity could represent a tumour marker of potential prognostic relevance.

Antibodies, Monoclonal↗
PubMed2001

Modulation of dendritic cell phenotype and mobility by tumor cells in vitro.

To gain new insights into the functional interaction between DC and neoplastic cells, we have analyzed the effects of melanoma and colorectal cancer lines on the chemotaxis and the phenotype of monocyte-derived DC in vitro. Both types of tumor cells displayed effective chemoattractive capacity towards immature, but not mature DC. Furthermore, conditioned medium of discrete melanoma lines induced upregulation of CD80, CD86, MHC class I, and MHC class II molecules on immature DC. However, de novo expression of E-cadherin and strong upregulation of CD15 could also be detected in the absence of CD83 expression. Melanoma-conditioned DC exhibited an increased adhesion capacity to a melanoma cell line in vitro and did not migrate in response to SLC chemokine. Tumor-infiltrating CD15(+) cells displaying DC morphology could also be detected by immunohistochemistry in the original tumor specimens from which discrete melanoma cell lines under investigation were derived. Colorectal cancer cell lines, although able to chemoattract immature DC, were apparently unable to modulate their phenotype. Altogether our results suggest that tumor cells can attract immature DC in vitro and, eventually, modulate their phenotype. As a result, DC mobility could be severely impaired.

Animals↗
PubMed2001

Immunogenicity of nonreplicating recombinant vaccinia expressing HLA-A201 targeted or complete MART-1/Melan-A antigen.

The effect on immunogenicity of different tumor T cell epitope formulations was evaluated in vitro using nonreplicating recombinant vaccinia vector expressing two forms of the melanoma-associated MART-1/Melan-A antigen. The first recombinant virus expressed a minigene encoding a fusion product between an endoplasmic reticulum (ER)-targeting signal and the HLA-A201 binding 27-35 peptide. The second viral construct encoded the complete MART-1/Melan-A protein. The capacity of HLA-A201 cells infected with either viral construct to generate and to stimulate MART-1/Melan-A 27-35 specific cytotoxic T-lymphocytes (CTL), was comparatively characterized. The results obtained here with a tumor antigen confirmed the capacity of vaccinia virus-encoded ER-minigene to generate a very strong antigenic signal. In cytotoxicity assays, recognition of target cells infected with high amounts of both recombinant viruses with activated specific CTL clones, resulted in similar lytic activity. With regard to calcium mobilization, TCR down-regulation, IFN-gamma release, and T cell proliferation assays, the targeted epitope elicited 10- to 1000-fold stronger responses. Remarkably, the immunogenic difference between the two formulations, in their respective capacity to generate CTL from naive HLA-A2 peripheral blood mononuclear cells in vitro as measured by tetramer detection, was lower (2- to 3-fold). Recombinant vectors expressing complete antigens have demonstrated their capacity to generate specific responses and such vaccines might take advantage of a broader potential of presentation. However, as demonstrated here for the HLA-A201-restricted MART-1/Melan-A immunodominant epitope, nonreplicative vaccinia virus expressing ER-targeted minigenes appear to represent a significantly more immunogenic epitope vaccine formulation.

Antigen Presentation↗
PubMed2001

Quantitative analysis of gene expression in human articular cartilage from normal and osteoarthritic joints.

OBJECTIVE: To quantify the expression of genes encoding extracellular matrix (ECM) proteins in human cartilage from normal and osteoarthritic (OA) joints. DESIGN: Human cartilage samples were classified as control (CTR) or OA according to clinical evaluation and assessed histologically and biochemically to confirm the diagnosis. mRNAs encoding collagen types I, II and X, aggrecan, versican, osteopontin and osteocalcin were quantified by real-time reverse transcription-PCR assays and normalized to a reference mRNA (GAPDH). RESULTS: RNA from native cartilage could be reproducibly and efficiently amplified by real-time PCR only if isolated using purification membranes. Primers and fluorescent probes for real-time PCR, endowed with comparable (<6% difference from GAPDH) and high (>91%) amplification efficiencies, were designed and validated for the selected ECM genes. The expression of most genes under investigation displayed large variations and was not significantly different in CTR and OA cartilage. Only osteopontin mRNA levels were significantly higher in OA than CTR specimens. mRNA ratios of collagen type II to I and of aggrecan to versican, defined as indexes of chondrocyte differentiation, were less variable within each population than the single genes and markedly higher (27.0 and 7.6-fold, respectively) in CTR than OA cartilage, with high statistical significance (P = 0.00013 and P = 0.00007, respectively). CONCLUSIONS: Our results provide evidence that gene patterns related to chondrocyte differentiation discriminate between CTR and OA human cartilage with higher sensitivity than single ECM genes. The method described here has the potential to improve understanding of the progression of OA and could become a valuable diagnostic tool.

Adult↗
PubMed2001

The effect of pretreatment with ischaemic preconditioning or cromakalim on perfusion in skeletal muscle during ischaemia-reperfusion injury.

BACKGROUND: Ischaemia-induced damage of skeletal muscle may lead to side effects in orthopaedic and reconstructive surgery where tourniquet ischaemia is applied to ensure a bloodless operative field. In this study we investigated the effect of ischaemia-reperfusion injury with and without preconditioning by studying the skeletal muscle microcirculation. A further aim was to establish whether ischaemic preconditioning or pretreatment with cromakalim, a potassium channel opener reduces ischaemia-reperfusion injury. METHODS: Twenty-eight Wistar rats were randomised into four groups (n=7 per group). Group 1, control with no treatment; Group 2, two and a half hours tourniquet ischaemia followed by two hours of reperfusion to the left hindlimb. Furthermore, we pre-treated two groups prior to the ischaemia-reperfusion period; Group 3 with three short cycles of ischaemia-reperfusion (5'/5') and Group 4 pre treated with cromakalim (100 microg/kg bw). We monitored the gastrocnemius muscle blood flow in vivo. RESULTS: There were no significant changes in the skeletal muscle microcirculation and temperature at the baseline in the four groups (p=0.110). In the ischaemic reperfusion, ischaemia preconditioning and cromakalim groups, the recorded skeletal muscle microcirculation during ischaemia decreased significantly (p<0.001) with respect to the baseline. In Group 2 the microcirculation recovered rapidly after release of the tourniquet, but was significantly lower (37 percent of baseline value, p<0.001) within two hours of reperfusion. In the ischaemia preconditioning group the microcirculation as in the ischaemia-reperfusion group recovered rapidly after release of the tourniquet, although failing to reach the baseline value within two hours of reperfusion. The mean microcirculation value of the left limb was slightly higher than Group 2 but significantly lower compared to the baseline after two hours of reperfusion (p<0.001). The change in the skeletal muscle microcirculation with cromakalim after two hours of reperfusion was not significant when compared to baseline values (p>0.05). The cromakalim group after two hours reperfusion had significantly higher microcirculation values when compared with Groups 2 and 3 (p<0.001). During ischaemia-reperfusion in Groups 2-4, there was no significant alteration in the systemic haemodynamic circulation. CONCLUSIONS: This study supports the hypothesis that cromakalim reduces postischaemic skeletal muscle damage and reperfusion injury.

Animals↗
PubMed2000

Naturally processed and concealed HLA-A2.1-restricted epitopes from tumor-associated antigen tyrosinase-related protein-2.

In this study, a computer-assisted reverse immunology approach was utilized in order to identify potentially antigenic peptides derived from the differentiation antigen TRP-2, a melanosomal protein frequently expressed in melanoma. Among the seven peptides complying with HLA-A2.1-binding motifs, two induced specific CD8(+) cytotoxic T lymphocytes. HLA-A2.1(+) melanoma cells expressing TRP-2 were lysed by clones specific for TRP-2(360-368) (TLDSQVMSL) peptide, thus identifying it as a naturally processed epitope. Other T-cell clones directed against TRP-2(476-484) (VMGTLVALV) were unable to lyse HLA-matched TRP-2(+) cell lines. The role of intracellular proteolytic processing in the generation of this epitope was investigated by transfecting mini-genes encoding the TRP-2(476-484) peptide alone or carrying N- or C-terminal extensions. Specific T-cell clones recognized target cells expressing the cytotoxic T-lymphocyte (CTL)-defined epitope or its C-terminally extended precursor, but failed to recognize cells expressing the N-terminally extended TRP-2(476-484) peptide, suggesting the presence of a negative processing signal (NPS). Regarding C-terminus-flanking regions, mutational analysis indicates that the GLY485 residue plays a key role in the processing of the TRP-2(476-484) epitope. Interestingly, proteasome inhibitors preventing the generation of the MART-1/Melan-A(27-35) immunodominant melanoma tumor-associated antigen (TAA) promoted detectable presentation of TRP-2(476-484) epitope in HLA-A2.1(+) and TRP-2(+) tumor lines, as witnessed by cytokine release by specific T-cell clones.

Acetylcysteine↗
PubMed2000

Identification of genes differentially expressed in melanoma sublines derived from a single surgical specimen characterised by different sensitivity to cytotoxic T-lymphocyte activity.

In this study we used differential display technology in an attempt to obtain an insight into the mechanisms underlying escape of tumour cells to the specific cytotoxic T cell response. A primary tumour cell line and autologous tumour infiltrating lymphocytes were raised from a metastatic melanoma sample (ME15). Upon co-culture of tumour infiltrating lymphocytes with irradiated tumour cells, CTL specific for neoplastic cells were generated and cloned. Using a CTL clone, a cytotoxicity resistant tumour subline (ME15R) was immunoselected. We applied a PCR-based differential display technique to amplify DNA sequences differentially expressed in ME15 sublines sensitive (S) or resistant (R) to specific CTL killing. 10 different sequences whose expression was exclusively detectable in ME15S cells were identified. Five of them matched with known expressed sequence tags encoding products of unidentified function. 2 showed high homology with a mitochondrial mRNA and with the gene encoding the S24 ribosomal protein. Most interestingly, genes coding for glutamine synthetase, TGF-beta-3 and PAX3, a well-characterised transcription factor, were only expressed in ME15S cells. The latter gene was found to be transcribed in all healthy tissues tested, but only in a subgroup of established melanoma cell lines. Taken together, our data underline the relevant potential of differential display technology in the molecular analysis of paired tumour lines endowed with different phenotypic characteristics. Cloning of entire open reading frames and transfection studies are warranted to clarify the role of individual differentially displayed genes in the escape of tumour cells from cellular immune response.

Base Sequence↗
PubMed2000

FLT3 ligand gene expression and protein production in human colorectal cancer cell lines and clinical tumor specimens.

Dendritic cells (DC) are professional antigen presenting cells (APC) whose proliferation and functional differentiation can be induced by hematopoietic growth factors including GM-CSF and FLT3 ligand (FL). Colorectal cancers are known to be infiltrated by dendritic cells (DC) and neoplastic cells have been shown to produce GM-CSF. In this work we investigated FLT3 ligand (FL) gene expression and protein production in human colorectal cancer cell lines and clinical tumor specimens. Using reverse transcription polymerase chain reaction (RT-PCR), 6 out of 6 established tumor lines were found to express to variable extents FL gene. In 1 of them, SW480, FL immunoreactivity could be observed by taking advantage of specific antibodies. In contrast, soluble FL could not be detected in any culture supernatant. FLT3 receptor (FR) gene was not expressed and exogenous addition to the cultures of recombinant FL (rFL) did not affect the proliferation of the tumor lines. FL gene expression was investigated using a densitometry-assisted, semiquantitative RT-PCR in clinical tumor specimens. Specific FL gene transcripts were amplified from 12 of 12 surgical samples. In these cases, FL gene expression of significantly lower intensity was also detected in healthy mucosa sampled in the vicinity (2 cm) or at a distance (10 cm) from neoplastic outgrowth. Immunohistochemical studies identified FL-positive cancer cells in 5 of 5 cases tested. No positivity was detected in healthy mucosa epithelia at a distance from the tumor or in stromal cells. FL content in preoperative sera from colorectal cancer patients (n = 13) did not exceed the levels detected in healthy donors (</= 100 pg/ml).

Colorectal Neoplasms↗
PubMed2000

Mechanisms of ischemic preconditioning in skeletal muscle.

BACKGROUND: Ischemic preconditioning (IP) (one or more cycles each consisting of a short period of ischemia and a short period of reperfusion, before the sustained ischemia) reduces ischemia-related organ damage in heart and skeletal muscle but the underlying mechanisms are not clear. This study was intended to assess the possible involvement of K(ATP) channels and of adenosine receptors in IP of skeletal muscle in a rat model of skeletal muscle ischemia. MATERIALS AND METHODS: Groups of 8-15 rats were given the following in vivo treatments: ischemia-reperfusion (I-R: 2.5 h tourniquet-induced ischemia of the right hindlimb, then 2 h reperfusion); IP (three cycles of 5 min ischemia, then 5 min reperfusion) before I-R; cromakalim and I-R; glibenclamide, cromakalim, and I-R; glibenclamide, IP, and I-R; [R]-N(6)-[1-methyl-2-phenylethyl]adenosine (R-PIA) and I-R; adenosine and I-R; and glibenclamide, IP, and I-R. Parameters of muscle function (postischemic maximal force, performance, contraction index, and force after 1 min of stimulation) were then assessed in vitro in the extensor digitorum longus muscle. RESULTS: Pretreatment with either IP or the K(ATP) channel opener cromakalim significantly improved postischemic muscle function. The protective effect of cromakalim was not seen when the K(ATP) channel blocker glibenclamide was added. Glibenclamide, however, did not block IP-induced protection. Pretreatment with the adenosine A(1) receptor agonist 8-(p-sulfophenyl)-theophyllin (8-SPT) or with adenosine did not improve postischemic muscle function. The adenosine receptor agonist did not block IP-induced protection against ischemic damage. CONCLUSIONS: The results show significant improvements in postischemic skeletal muscle function after IP or cromakalim pretreatment but they do not support a role for K(ATP) channels or for adenosine receptors in IP of skeletal muscle.

Animals↗
PubMed2000

Functional outcome of transplanted knee joints in dogs.

Total knee joint transplantation has been performed in animal models and humans. This study investigates the impact of this operation on knee joint function in a dog model. Therefore, replantation was compared to transplantation during a 6-month follow-up period in four dogs in each group. The peak vertical ground reaction force normalized in all legs undergoing replantation and in two of four after transplantation. A third transplant recipient reduced loading from the 4th month due to a local complication, and the fourth succumbed to sepsis 3 months postoperatively. A weight-bearing index (WBI), defined as loading of the grafted divided by loading of both hind-limbs decreased from 0.48 +/- 0.08 preoperatively to 0.13 +/- 0.10 by 1 month after replantation and from 0.53 +/- 0.07 to 0 after transplantation. After 6 months, weight-bearing of all replant recipients was restored, but reduced in two transplant recipients with graft function. Full recovery after replantation, but impaired function after transplantation, was also reflected in the histological results: normal histological pictures of blood vessels, cartilage, bone and soft tissues were found in all replant recipients, but infiltrative vasculopathy indicating chronic rejection was found in the transplanted joints. The results of this animal study confirm that the procedure can lead to satisfactory functional results but also emphasize the need for perfect control of immunosuppression.

Animals↗
PubMed2000

NY-ESO-1 tumour associated antigen is a cytoplasmic protein detectable by specific monoclonal antibodies in cell lines and clinical specimens.

NY-ESO-1 gene encodes a novel member of the cancer/testis (CT) family of human tumour-associated antigens (TAA). Specific monoclonal antibodies (mAb) have identified the corresponding gene product in lysates of tumour cell lines as a 22 kDa protein but no data are available concerning its intracellular location or distribution within neoplastic tissues. We have generated NY-ESO-1 specific mAbs recognizing the target molecule in cytospin preparations and in sections from clinical tumour specimens. These reagents identify NY-ESO-1 TAA in melanoma cell lines expressing the specific gene as a cytoplasmic protein, sharing the intracellular location of most MAGE TAA. In a series of 12 melanoma specimens, specific staining, limited to neoplastic cells, was detectable in the five cases where NY-ESO-1 gene expression was observed. In two of them over 90% of tumour cells showed evidence of positive staining. Lower percentages of positive neoplastic cells ranging between single cells and 50% were observed in the remaining tumours. These data suggest that active specific immunotherapies targeting NY-ESO-1, alone or in combination with other TAA could be of high clinical relevance in sizeable subgroups of melanoma patients.

Antibodies, Monoclonal↗
PubMed2000

Function of fast- and slow-twitch rat skeletal muscle following ischemia and reperfusion at different intramuscular temperatures.

Fast- (peroneal) and slow-twitch (soleus) skeletal muscles of anesthetized Wistar rats were subjected to 3 h of tourniquet ischemia. The intramuscular temperature of the leg was adjusted to 22, 30 or 35 degrees C (n = 12 per group) during ischemia. After 2 h of reperfusion, the muscles were electrically stimulated in vitro and muscular function was analyzed for maximal force, performance, contractility and fatigue. Contralateral nonischemic muscles served as controls. Three hours of ischemia at 30 degrees C did not reduce the function of the peroneal muscles compared to nonischemic controls. The same ischemic stress significantly reduced the function of the soleus muscles compared to nonischemic controls. The postischemic function of the soleus muscles declined with increasing temperature. The postischemic function of the 35 degrees C group of peroneal muscles was significantly reduced compared to the 22 and the 30 degrees C groups, which did not differ. These results provide evidence that fast-twitch muscles are more resistant to ischemia than slow-twitch muscles. They furthermore show a fiber type-specific dependency of postischemic muscle function on intramuscular temperature during ischemia. Hypothermia-sensitive fast-twitch fibers predominate in the skeletal muscles of the extremities. Mild hypothermia could, therefore, reduce tourniquet ischemia-induced injury after surgery of the extremities.

Animals↗
PubMed2000

Preconditioning with short cycles improves ischemic tolerance in rat fast- and slow-twitch skeletal muscle.

AIM: The aim of this study was to investigate whether the efficacy of ischemic preconditioning (IP) in rat skeletal muscle depends on the duration of the preconditioning cycles. METHODS: Rats were divided into four groups (n = 10 each). The right hindlimb of rats in group A were subjected to 2.5 h of tourniquet ischemia followed by 2 h of reperfusion (I-R). Thereafter, muscular function was analyzed in vitro and high-energy phosphates (HEP) were determined by HPLC. Before I-R, right hindlimbs of rats in groups B-D subjected to IP with three cycles each consisting of 2.5, 5 or 10 min of ischemia followed by reperfusion for the same duration. RESULTS: Postischemic function of the extensor muscle was significantly improved with all three preconditioning protocols. Postischemic function of the soleus muscle was only improved by IP with three cycles of 5 min of ischemia and 5 min of reperfusion. Postischemic HEP tissue levels were not influenced by IP. CONCLUSION: This study shows for the first time that IP increases ischemic tolerance not only of fast-twitch but also of slow-twitch skeletal muscle. The efficacy of IP seems to be less dependent on the duration of the single preconditioning cycle than on the number of cycles performed. Three cycles each of 2.5, 5 or 10 min ischemia and reperfusion significantly improved postischemic skeletal muscle function. Tissue levels of HEPs, however, were not influenced by IP indicating that preservation of HEPs does not play a major role in the effects of IP on rodent skeletal muscle.

Adaptation, Physiological↗