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Biomedical subjects

U Moebius

Publications and source records attributed to U Moebius.

At least 19 recordsLinked to original sources
PubMed2005

Electromechanical delay of the knee extensor muscles is not altered after harvesting the patellar tendon as a graft for ACL reconstruction: implications for sports performance.

Although the scar tissue, which heals the donor site defect, has different elasticity from the neighbouring patellar tissue, it remains unclear if this scar tissue can lead to the changes of the electromechanical delay (EMD) of the knee extensor muscles. If such changes do exist, they can possibly affect both the utilization of the stored energy in the series elastic component, as well as the optimal performance of the knee joint movement. The purpose of this study was to investigate the influence of harvesting the patellar tendon during anterior cruciate ligament (ACL) reconstruction and the associated patellar tendon scar tissue development on the EMD of the rectus femoris (RF) and vastus medialis (VM) muscles. Seventeen patients who underwent an ACL reconstruction using the medial third of the patellar tendon were divided in two groups based upon their post-operative time interval. Maximal voluntary contraction from the knee extensors, surface EMG activity, and ultrasonographic measurements of the patellar tendon cross-section area were obtained from both knees. Our results revealed that no significant changes for the maximal voluntary contraction of the knee extensors and for the EMD of the RF and the VM muscles due to patellar scar tissue development after harvesting the tendon for ACL reconstruction. The EMD, as a component of the stretch reflex, is important for the utilization of the stored energy in the series elastic component and thus, optimal sports performance. However, from our results, it can be implied that the ACL reconstruction using a patellar tendon graft would not impair sports performance as far as EMD is concerned.

Adult↗
PubMed2001

The presence of proprioceptive mechanoreceptors in the remnants of the ruptured ACL as a possible source of re-innervation of the ACL autograft.

This study investigated the presence of neural mechanoreceptors in the remnants of the ruptured ACL as a possible source of reinnervation of the ACL autologous graft. The remainder of the torn ACL was selected for further histological investigation from 17 patients during ACL reconstruction 3 months to 3.5 years after injury. Perioperatively two types of ACL remnant were identified. Fifteen patients had portions of ACL adapted at the PCL. In all of these patients we found mechanoreceptors (I and II). In five patients we found mushroomlike remnants which included either none or small numbers of mechanoreceptors. Free neural ends were found in both patient groups. There was a significant difference between the groups in regard to the mean number of mechanoreceptors I and II per slice. In conclusion, in patients with an ACL remnant adapted to the PCL, mechanoreceptors exist even 3 years after injury. If we accept that restoration of proprioception is the result of reinnervation of the ACL, leaving the ACL remnants as a source, if this is surgically possible without risk of Cyclop's lesion, may be of potential benefit to the patient.

Adolescent↗
PubMed2001

Simultaneous ligation of CD5 and CD28 with monoclonal antibodies restores impaired immunostimulatory function in human renal cell carcinoma.

Tumor cells, including renal cell carcinoma (RCC) cells, do not effectively stimulate T lymphocyte responses against specific antigens presented on their surface. Reasons for this low immunogenicity may include low or absent expression of MHC class I and/or class II molecules, as well as accessory and costimulatory molecules. We used tumor cell pretreatment with cytokines, together with monoclonal antibodies (mAbs) directed at receptors for costimulatory molecules, to render RCC cells immunostimulatory. Interferon-gamma or tumor necrosis factor-alpha pretreatment enhanced expression of MHC class I and class II molecules, as well as CD54, but had only minimal effects on T cell activation. A CD28 mAb, or an even more effective combination of CD28 and CD5 mAb, induced strong primary proliferative responses of allogeneic resting T lymphocytes. Cytokine pretreatment further augmented this T cell response in vitro and allowed T cell expansion and establishment of T cell lines. Stimulation of T cells with autologous RCC cells resulted in a similar T cell activation but with the expansion of cytolytic T cells directed at autologous MHC class II molecules. These experiments demonstrate that cytokines combined with costimulatory mAbs are useful for increasing the immunogenicity of tumor cells. They also indicate. however, that autologous MHC class II expression on tumor cells, together with strong costimulation, may lead to the activation of autoreactive T cells.

Antibodies, Monoclonal↗
PubMed1999

T cell activation by monoclonal antibodies bound to tumor cells by a cell surface displayed single-chain antibody.

Tumor cells often lack the costimulatory molecules necessary for T cell activation. However, the transformation of cells with more than one stimulatory molecule is a difficult procedure. We therefore developed a retroviral vector for the expression of a cell membrane anchored single-chain antibody fragment (scFv) directed against the hapten 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one (phOx). Proteins and peptides can be readily modified with this hapten, thus, enabling them to be bound to cells with the cell surface displayed anti-phOx scFv. To test combinations of surface-bound stimulatory molecules on T cell activation, SK-Mel63 human melanoma cells expressing the membrane anchored anti-phOx scFv were incubated with phOx-labeled mAbs against CD3, CD28 and CD5. Cells presenting a given mixture of modified anti-CD3 and anti-CD28 molecules stimulated T cell activation better than any single antibody and a given mixture of anti-CD3, anti-CD28 and anti-CD5 provided a stimulatory response higher than the best double combination. However, the relative concentrations are very important and must be carefully chosen. Concentrations of antibodies giving good T cell responses when used alone can block synergistic effects.

Antibodies, Monoclonal↗
PubMed1998

Strong immunogenic potential of a B7 retroviral expression vector: generation of HLA-B7-restricted CTL response against selectable marker genes.

The stimulation of a specific immune response is an attractive goal in cancer therapy. Gene transfer of co-stimulatory molecules and/or cytokine genes into tumor cells and the injection of these genetically modified cells leads to tumor rejection by syngeneic hosts and the induction of tumor immunity. However, the development of host immune response could be either due to the introduced immunomodulatory genes or due to vector components. In this study, human renal cell carcinoma cell lines were modified by a retrovirus to express the co-stimulatory molecule B7-1 together with the hygromycin/thymidine kinase fusion protein (HygTk) as positive and negative selection markers. These B7-1-transduced renal cell carcinoma cell lines were able significantly to activate allogeneic T cell proliferation. The cytolytic activity of these T cells was determined by employing several transduced and nontransduced renal cell carcinoma cell lines as targets. Evidence for a strong vector-specific T cell reactivity induced by the Hyg/Tk protein was obtained in autologous renal cell carcinoma systems. Antibody blocking experiments as well as peptide binding assays demonstrated an HLA-B7-restricted T cell response directed against both the Hyg and the Tk genes. Thus, the vector itself may mask the generation of immune reactivity against tumor antigens and may even detract from it. Vectors with immunogenic potential may be useful for tumor vaccination via cross priming in vivo, whereas antivector reactivities would be detrimental in situations where gene defects are being corrected and where long term expression of a therapeutic protein is required.

Antigens, Neoplasm↗
PubMed1998

Gene transfer of costimulatory molecules into a human colorectal cancer cell line: requirement of CD54, CD80 and class II MHC expression for enhanced immunogenicity.

Colorectal cancer is considered a non-immunogenic malignany. One strategy to augment the immunogenicity of such tumours is represented by the expression of costimulatory molecules by gene transfer. Using transfected variants of the human colorectal cancer cell line SW480 we tested various costimulatory molecules (CD80, CD86, CD54) and a class II major histocompatibility complex (MHC) allele (HLA-DR3) alone or in combination on their ability to support primary T-lymphocyte activation in vitro. Expression of CD80 or CD86 similarly as the combination of both was not sufficient to induce proliferation of human allogeneic T cells. Expression of CD54 together with CD80 strongly augmented the costimulatory function of CD80, as observed in the presence of a CD3 monoclonal antibody (mAb), but did not lead directly to a T-cell response against modified tumour cells. Importantly, SW480 cells coexpressing CD54, CD80 and the HLA-DR3 allele effectively promoted T-lymphocyte proliferation. Moreover, the use of such CD54+/CD80+/HLA-DR3+ SW480 variants for repetitive stimulations resulted in the generation of T-cell lines predominantly composed of CD8+ T cells exhibiting class I MHC restricted cytolytic activity towards untransfected SW480 tumour cells. This demonstrates that the generation of immunogenic tumour cell variants, i.e. for the use as cellular vaccines, requires multiple genetic alterations in the case of non-immunogenic human tumours cells, such as colorectal cancer cells.

B7-1 Antigen↗
PubMed1997

Detection of cytosine deaminase in genetically modified tumor cells by specific antibodies.

Bacterial cytosine deaminase (CD) converts the non-toxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), which is toxic for mammalian cells. Therefore, the CD gene is used in cancer gene therapy to achieve high local concentration of a toxic metabolite without significant systemic toxicity. To allow the detection of CD expression at the protein level, we raised both polyclonal rabbit antisera and a monoclonal antibody (mAb) against a histidine-tagged CD fusion protein. The specificity of the polyclonal antisera and the mAb was confirmed by immunohistochemistry, immunoblot analysis, and immunoprecipitation using CD-expressing tumor cell lines. Furthermore, the antibodies can be used for ELISA assays and flow cytometry. Finally, the CD protein could be demonstrated in frozen tissue sections of CD-modified tumors in a rat tumor model using the anti-CD serum. With these antibodies, CD expression can now be monitored throughout in vitro and in vivo gene transfer studies, including clinical protocols relying on the CD suicide gene strategy.

Animals↗
PubMed1997

Differential function of CD80- and CD86-transfected human melanoma cells in the presence of IL-12 and IFN-gamma.

Introduction of co-stimulatory molecules like CD80 and CD86 represents a means to augment the immunogenicity of tumor cells and to induce immune responses directed at tumor antigens. Here we compared CD80- and CD86-transfected human melanoma cells to induce primary immune responses by their capacity to promote proliferation of human allogeneic resting T lymphocytes. CD80- and CD86-transfected SkMel63 melanoma cells induced T cell activation to a comparable degree, which was found to be independent of the cell surface density of these co-stimulatory molecules. Co-expression of CD80 and CD86 did not result in a synergistic increase in T cell proliferation. Both CD80 and CD86 transfectants induced the proliferation of isolated CD4+ or CD8+ T cells. Exogenous IL-2, IL-4 and tumor necrosis factor-alpha respectively enhanced primary T cell proliferation independent of CD80 or CD86 expression. Interestingly, differential activities of CD80 and CD86 were observed following stimulation of resting T cells in the presence of IL-12. Whereas IL-12 increased T cell proliferation in the presence of CD86-transfected melanoma cells, it exhibited an inhibitory function in the presence of CD80-expressing SkMel63 cells. Experimental evidence indicates that this inhibitory effect was mediated by IFN-gamma since (I) IFN-gamma secretion of stimulated T cells was augmented by IL-12, (II) exogenous IFN-gamma also inhibited T cell proliferation induced by CD80- but not CD86-transfected SkMel63 cells and (III) the inhibitory effect of IL-12 was blocked by an anti-IFN-gamma mAb.

Antigens, CD↗
PubMed1997

A sensitive ELISPOT assay for detection of CD8+ T lymphocytes specific for HLA class I-binding peptide epitopes derived from influenza proteins in the blood of healthy donors and melanoma patients.

An enzyme-linked immunospot (ELISPOT) assay was adapted to detect peptide-specific CD8+ T lymphocytes in peripheral blood mononuclear cells (PBMCs). In HLA-A1-, HLA-A2-, and/or HLA-A3-positive individuals, we determined the release of IFN-gamma on a single cell level in response to three different peptide epitopes derived from the influenza matrix protein and nuclear protein containing the HLA-A2.1- and HLA-A1- or HLA-A3-binding motif, respectively. Comparison of the ELISPOT assay with the standard chromium release assay revealed a close correlation between the number of peptide-specific IFN-gamma-releasing T cells in PBMCs and the level of specific cytotoxicity after 14 days of in vitro expansion. The ELISPOT assay detected T cells with specificity for the HLA-A2. 1-binding epitope derived from the matrix protein in 76% of HLA-A2-positive healthy individuals (n = 25); the median frequency was 41 in 10(6) PBMCs. We also detected peptide-specific T cells in 10 of 12 HLA-A2-positive patients with metastatic melanoma with a median frequency of 20.5 in 10(6) PBMCs. In 10 of 24 HLA-A3-positive individuals and in 2 of 14 HLA-A1-positive individuals, peptide-specific T cells for a HLA-A3- and a HLA-A1-binding epitope derived from the nucleoprotein, respectively, were present. In conclusion, the ELISPOT assay may be suitable to monitor a peptide-specific T-cell response in vaccination protocols using peptides derived from tumor or viral antigens.

CD8-Positive T-Lymphocytes↗
PubMed1996

Cytosine deaminase gene as a potential tool for the genetic therapy of colorectal cancer.

The bacterial enzyme cytosine deaminase (CD) catalyzes the conversion of 5-fluorocytosine (5-FC) to the lethal 5-fluorouracil (5-FU) and so provides a useful system for selective killing of gene-modified mammalian tumor cells. Cloning of the CD gene from Escherichia coli and expression in human tumor cell lines enabled these cells to convert 3H-labeled 5-FC into 3H-5-FU. Two CD-expressing human tumor cell lines (adenocarcinoma cell line KM12 and glioblastoma cell line T1115) became 200-fold more sensitive to 5-FC than the nonexpressing parental cell lines. At least 90% of the cells are killed within 7 days. CD-expressing cells are able to kill nonexpressing cells when grown in the same culture flask (bystander effect). The CD gene may be used as a suicide system for in situ chemotherapy or as a safety mechanism abrogating the expression of other genes.

Base Sequence↗
PubMed1996

Immune reactions induced by interleukin-2 transfected colorectal cancer cells in vitro: predominant induction of lymphokine-activated killer cells.

One aim of the genetic modification of tumor cells is the generation of immunogenic variants that can be used for the induction of immune responses against tumors. We engineered the human colorectal carcinoma cell line SW480 by means of plasmid transfection to secrete interleukin (IL)-2. Transfection of SW480 cells resulted in stable IL-2 secretion at 5-30 ng/ml per 10(5) cells in 24 h and, unexpectedly, in CD54 expression on the cell surface. SW480 variants expressing IL-2 and CD54 were tested for their capacity to induce T lymphocyte activation in vitro in comparison to untransfected and CD54 transfected cells. The cytolytic effector function of a class I MHC restricted CD8+, peptide antigen specific T cell clone was augmented following expression of CD54. IL-2 secreting SW480 variants did not further increase antigen-dependent cytolysis. Primary activation of resting T lymphocytes was assessed following allogeneic stimulation. When compared with unmodified SW480 cells, CD54 expressing variants did not initiate T cell proliferation. In contrast, IL-2 secreting SW480 cells strongly promoted primary T cell proliferation. Similarly, exogenous IL-2 and SW480 cells induced T cell proliferation which was not only due to IL-2 but was dependent on tumor cells. However, following the initial wave of cell growth in response to IL-2 secreting SW480 cells T lymphocytes could not be restimulated with SW480 or IL-2 secreting variants and could not be further expanded. T cells initially activated by IL-2 secreting SW480 cells exhibited cytolytic activity towards SW480 cells. This reactivity, however, was transient and completely blocked by K562 cells, suggesting MHC-unrestricted, nonspecific cytotoxicity. We conclude that endogenous IL-2 secretion by the colorectal carcinoma cell line SW480 does not result in the activation of MHC restricted specific T lymphocytes but predominantly induces lymphokine-activated killer cells. Considering that tumor cell vaccines are aimed at inducing tumor-specific immune responses, our in vitro observation would rather argue against the in vivo application of such a tumor cell modification in colorectal cancer.

Amino Acid Sequence↗
PubMed1995

Development of immunogenic colorectal cancer cell lines for vaccination: expression of CD80 (B7.1) is not sufficient to restore impaired primary T cell activation in vitro.

The capacity of colorectal carcinoma and melanoma cell lines to induce primary versus effector T lymphocyte activation in vitro was investigated. Established epithelial tumour cell lines derived from colorectal carcinoma and melanoma did not activate a primary proliferative response of resting T lymphocytes in allogeneic mixed lymphocyte tumour cell cultures (MLTCs). In contrast, the same tumour cells were effectively lysed by preactivated cytolytic T cell clones. This demonstrates that tumour cells are impaired in inducing a primary immune response but are susceptible to effector immune responses. Attempts at improving primary T cell activation revealed that exogenous cytokines, including interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-2 (IL-2), were not effective. Expression of CD80 (B7.1), by transfecting a CD80 cDNA into the melanoma cell line SkMel63, improved T cell proliferation considerably. In contrast, CD80 expression in two colorectal carcinoma cell lines (SW480, SW707) did not result in T cell activation. This was not due to lack of class II MHC expression on SW480 since coexpression of a HLA-DR3 alloantigen and CD80 had no effect. Our data suggest that de novo CD80 expression is not, in general, sufficient to improve primary T cell activation by human tumour cells.

Antigens, Neoplasm↗
PubMed1995

CD80-transfected human breast and ovarian tumor cell lines: improved immunogenicity and induction of cytolytic CD8+ T lymphocytes.

Human tumor cell lines derived from breast and ovarian carcinomas have been found to be ineffective in stimulating the induction phase of an immune response such as T cell proliferation in allogeneic mixed tumor cell lymphocyte cultures. Since representative tumor cell lines are effectively lysed by activated T lymphocytes, the induction of an effector phase is not impaired. In order to reconstitute the potential to induce primary T cell activation, we transfected CD80 into a breast (KS) and an ovarian carcinoma (GG) cell line. CD80 expression in KS cells resulted in improved primary T cell activation, whereas it was ineffective in the case of GG cells. However, treatment of CD80-transfected GG cells with INF-gamma rendered them immunogenic, and resulted in T cell proliferation. Likewise, TNF-alpha and/or INF-gamma augmented T cell proliferation induced by CD80-transfected KS cells. Furthermore, T lymphocytes stimulated with cytokine-treated CD80+ KS cells gave rise to a long term proliferating CD8+ CTL line with class I MHC restricted cytolytic antitumor activity. These studies emphasize the requirement for costimulation in generating tumor-specific immunity, and demonstrate the efficacy of CD80 in generating CD8+ cytolytic T lymphocytes.

Antigens, CD↗
PubMed1994

Identification of a novel dimeric phosphoprotein (PP29/30) associated with signaling receptors in human T lymphocytes and natural killer cells.

Two-dimensional gel electrophoresis of in vitro phosphorylated proteins coprecipitated by CD2 monoclonal antibody (mAb) from Brij58 lysates of resting human T lymphocytes and natural killer (NK) cells resulted in the identification of a novel 29/30-kD disulfide-linked dimer (pp29/30). Comparative two-dimensional analysis of CD2, CD3, CD4, CD5, and CD8 immunoprecipitates revealed that pp29/30 associates with these signaling receptor complexes but not with CD18, CD27, and CD29 in human T lymphocytes. Analysis of CD2 immunoprecipitates prepared from T cell antigen receptor/CD3-modulated T lymphocytes indicated that pp29/30 preferentially associates and comodulates with the human T cell antigen receptor (TCR). Since tyrosine phosphorylated pp29/30 selectively interacts with the Src homology type 2 domains (SHZ) of the protein tyrosine kinases p56lck and p59fyn but not ZAP70 the present data suggest that pp29/30 represents a novel signaling receptor associated phosphoprotein likely involved in the activation of human T lymphocytes and NK cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1993

Delineation of an extended surface contact area on human CD4 involved in class II major histocompatibility complex binding.

We describe a detailed mapping of the class II major histocompatibility complex (MHC) binding site using site-directed mutagenesis in conjunction with high-resolution CD4 structural data. Residues on all lateral surfaces of domain 1 and the neighboring portions of domain 2 participate in contacting class II MHC. Thus, in addition to the C'C" ridge that forms the human immunodeficiency virus type 1 gp120 binding site, apparent MHC contacts extend over the BED face of domain 1 and across the interdomain groove onto the FG loop of domain 2. Several models of the CD4/class II MHC interaction accounting for the extent of the CD4 surface involved are discussed, including the possibility that CD4 may contact more than one class II MHC molecule using different surfaces.

Amino Acid Sequence↗