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M Heberer

Publications and source records attributed to M Heberer.

At least 73 records · Page 4Linked to original sources
PubMed1995

Peptide-specific CTL in tumor infiltrating lymphocytes from metastatic melanomas expressing MART-1/Melan-A, gp100 and Tyrosinase genes: a study in an unselected group of HLA-A2.1-positive patients.

Peptide specificity of cultured tumor-infiltrating lymphocytes (TIL) was systematically investigated in a group of HLA-A2.1+ metastatic melanoma patients consecutively referred to our department for surgical treatment. Seven samples from 6 patients were studied. All surgical specimens showed evidence of gp 100, MART-1/Melan-A and Tyrosinase gene expression as detectable by reverse PCR (rPCR). Cultured TIL from 2 patients displayed cytotoxic activity against autologous or HLA-matched EBV-transformed cells previously pulsed with MART-1/Melan-A27-35 peptide. In contrast, no CTL activity against gp100(280-288) or tyrosinase1-9 peptides could be observed. TIL were then repeatedly stimulated in vitro with the same peptides. After 6 restimulation courses at weekly intervals, specific recognition of gp100(280-288) and MART-1/Melan-A peptides was detectable in 3 and 5 TIL populations, respectively. In one case Tyrosinase1-9-specific CTL could be demonstrated. Two TIL populations from metastases resected from a melanoma patient at 6 months' distance showed a different peptide specificity pattern, and no specific CTL could be generated from simultaneously sampled peripheral blood mononuclear cells (PBMC). All peptide-specific CTL populations also displayed significant cytotoxic activity against HLA-A2.1 matched melanoma cell lines expressing the antigens under investigation. Our data indicate that CTL specific for MART-Melan-A27-35, gp100(280-288) or Tyrosinase1-9 peptides could be expanded with varying frequency from TIL derived from 4 out of 6 HLA-A2.1+ patients whose tumors expressed the genes encoding these tumor-associated antigens (TAA).

Adult↗
PubMed1995

Identification and intracellular location of MAGE-3 gene product.

The human MAGE-3 gene encodes a melanoma antigenic epitope recognized by specific cytotoxic T lymphocytes, but its gene product has not been identified thus far. We produced a recombinant MAGE-3 gene product by expression cloning of the entire reading frame in the context of a fusion protein characterized by a 10-histidine tail, allowing purification by metal chelation on a nickel Sepharose column. The semipurified product was used to generate MAGE-3-specific monoclonal antibodies. One reagent could identify by immunoblotting the native MAGE-3 gene product as a M(r) 48,000 protein in lysates of cell lines showing evidence of MAGE-3 gene expression. No apparent cross-reactivity with recombinant or native MAGE-1 gene product was observed. Immunohistochemistry shows that, closely resembling the MAGE-1 gene product, MAGE-3 is a cytoplasmic protein.

Animals↗
PubMed1995

Expression of HLA-DR in granulocytes of polytraumatized patients treated with recombinant human granulocyte macrophage-colony-stimulating factor.

MHC class II determinants are the restriction elements involved in antigen-specific activation of helper T lymphocytes and interaction with CD4 molecules. They are typically expressed on a limited number of cell types, mostly endowed with antigen-presenting capacity. Recently, expression of HLA-DR has been detected on granulocytes stimulated "in vitro" with GM-CSF. However, no evidence of "in vivo" expression in humans has been presented so far. We report here that class II determinant expression is detectable in vivo on peripheral blood granulocytes of polytraumatized patients upon intravenous administration of rhGM-CSF. Expression of these molecules appears to be an early effect of rhGM-CSF treatment, independent from endotoxemia or endogenous production of IL-6 or TNF-alpha, and rapidly declining upon discontinuation of therapy. Thus, this treatment might increase the number of cells potentially capable of presenting class-II-restricted antigens in these patients.

Adult↗
PubMed1995

Tyrosine kinase-dependent and -independent events induced by interleukin-2 stimulation: interleukin-2-mediated NO production required for the induction of lymphokine-activated killer cell activity in rat splenocytes is tyrosine kinase independent.

Nitric oxide (NO) has recently been shown to be an indispensable co-factor in the generation of lymphokine-activated killer (LAK) cells induced by interleukin-2 (IL-2). Upon stimulation with IL-2, cells endowed with specific receptors undergo phosphorylation of substrates mediated by protein tyrosine kinases (PTK). In this work we utilized a well-characterized PTK inhibitor, genistein (GEN), to address the role of PTK on NO-dependent LAK cell generation. The effects of GEN were tested on the expression of the inducible NO synthase (iNOS) gene, proliferation, generation of cytotoxic activity and production of NO upon IL-2 stimulation of rat splenocytes. We report here that GEN displays profound inhibitory effects on recombinant (r)IL-2 induced proliferation and on LAK cell generation, while only marginally affecting NO production, measured as NO2-. In contrast, a specific inhibitor of the NO synthetic pathway (NG-monomethyl-L-arginine; NMMA) blocked generation of LAK cells and NO production without affecting cell proliferation. If added directly to the cytotoxicity tests, GEN exerted minor inhibitory effects, not exceeding 25% of control tests, while NMMA was completely ineffective. Sodium nitroprusside (SNP), a non-enzymatic NO-releasing substance, restored LAK cell generation in cultures performed in the presence of NMMA, but not in those performed in the presence of GEN. These results indicate that IL-2-induced NO production is a PTK-independent event. IL-2-stimulated LAK cell generation obligatorily requires the concurrent activation of PTK dependent and independent signal transduction pathways.

Animals↗
PubMed1995

[Ischemic preconditioning improves postischemic function, but not energy metabolism of skeletal muscles].

Ischemic preconditioning (IP) refers to a phenomenon whereby short periods of ischemia reduce tissue damage after a subsequent sustained ischemia. The effect of IP before tourniquet ischemia of the extremities has not yet been evaluated. We developed a rat model of skeletal muscle ischemia and measured the effect of IP on postischemic function and high-energy phosphate levels. IP consisted in three cycles of 10 min ischemia and 10 min reperfusion each. IP improved significantly skeletal muscle function after 3 hours of ischemia and 2 hours of reperfusion. High-energy phosphate levels, however, remained unchanged. This study is the first that shows a protective effect of IP in skeletal muscles. These results furthermore suggest that the protection of energy metabolism is not a mechanism of IP in this model. IP could be easily performed before surgery of the extremities under tourniquet ischemia. The protective effect on postischemic skeletal muscle has therefore to be further investigated.

Animals↗
PubMed1995

[Functional and morphological results of transplantation of vascularized knee joints in a dog model--a preliminary report].

Functional and morphological results of transplantation (n = 3) and replantation (n = 4) of vascularised total knee joints were compared in a canine model. Follow up time was 6 months. All vascular anastomoses remained patent and all osteotomies healed. Replanted animals displayed normal static weight bearing in contrast to the transplant recipients, which showed reduced weight bearing after 6 months compared to preoperative values. Mild to moderate signs of chronic allograft rejection were detected in all transplanted animals. Functional results were correlated with morphological outcome demonstrating the relevance of adequate control of rejection.

Animals↗
PubMed1994

MAGE-1 gene product is a cytoplasmic protein.

MAGE-1 gene encodes a human melanoma antigen, recognized by syngeneic cytotoxic T lymphocytes (CTL). MAGE-1 transcripts are also detectable in breast cancers, in non-small-cell lung carcinomas and in central nervous system tumors. In order to identify, in cellular preparations, the protein encompassing the antigenic peptide, we generated a panel of monoclonal antibodies (MAbs) against the MAGE-1 gene product by using, as immunogen, a full-length recombinant preparation (rMAGE-1), obtained through expression cloning of the relevant gene in E. coli. Four reagents were obtained recognizing both rMAGE-1 and the 46-kDa native protein in cell lines expressing MAGE-1 mRNA. No positivity could be detected in MAGE-1-mRNA-negative melanoma lines. No surface labelling of MAGE-1-positive cell lines could be observed. In contrast, on permeabilization of MZ2 melanoma cells, all 4 MAbs induced efficient staining, as detected by cytofluorography. Fluorescence microscopy shows that MAGE-1 gene product is a cytoplasmic protein clustered in paranuclear organelle-like structures. Thus, MAGE-1 protein location closely resembles that of P91A and P198 murine-tumor antigens.

Animals↗
PubMed1994

[Tumor-immune system interaction in renal cell carcinoma and melanomas. Cytokine transcription in tumors at the time of surgical resection].

Tumor infiltrating lymphocytes (TIL) of many tumors express surface activation markers. An antigen driven stimulation of T-lymphocytes is expected to induce not only cell membrane activation molecules but also a unique pattern of cytokine gene transcripts. These cytokines are relevant modulators and potent effectors of immune responses, and therefore play a crucial role in tumor-immune system interaction. The gene transcription of interleukin(IL)-2, IL-4, IL-7, IL-10 and interferon(IFN)-gamma of lymphocyte infiltrated, freshly excised tumor specimens from 10 renal cell carcinomas and 6 melanomas were investigated by reverse polymerase chain reaction (PCR) technique. Autologous, peripheral mononuclear cells (PBMC) and healthy tissue of the affected organs served as controls. In all samples the transcription of the beta-actin gene as a methodological control turned out to be positive. In contrast, no cytokine gene transcription was detected in healthy tissue specimens and PBMC. IL-2 transcripts were detectable in no melanomas but in half of the renal tumor samples. IL-10 never transcribed in melanomas but was positive in 7 out of 10 renal cell carcinomas. In only 2 respectively 1 of the resected tissue probes was transcription of IL-4 and IFN-gamma detected. IL-7 was positive in 1 melanoma and in 6 urological neoplasias. The most impressive fact is the frequent transcription of the inhibiting factor IL-10 in renal cell carcinomas (7/10). This pattern of cytokine gene transcription may explain functional deficits of TIL.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

The pattern of cytokine gene expression in freshly excised human metastatic melanoma suggests a state of reversible anergy of tumor-infiltrating lymphocytes.

Expression of an extended panel of cytokine genes was investigated by reverse polymerase chain reaction (PCR) in 10 freshly excised melanoma metastases infiltrated by lymphocytes (TIL). cDNA encoding for CD3-delta and tyrosinase could be amplified in all samples, confirming the presence of T lymphocytes and melanoma cells. Cytokine genes possibly transcribed by both cell types, such as GM-CSF, IL-6 and IL-10 could be amplified from 5, 2 and 2 samples respectively. In contrast, IL-1 beta and TNF-alpha mRNA were never detectable, IL-1 alpha, IL-3 and IL-7 mRNA could be observed only in one case each. Transcripts encoding for TGF-beta 1 were observed in 8 samples, while TGF-beta 2 and 3 mRNA were detectable in only 2 specimens. mRNA encoding for cytokine genes typically transcribed by antigen-stimulated T lymphocytes, such as IL-2, IL-4 and IFN-gamma were rarely or never detectable (none, none and 1 of the samples respectively). In one case, where no cytokine gene transcription was detectable at the time of surgery, we addressed the question of the antigenicity of the tumor and of the functional competence of TIL. A primary tumor cell line was generated and cultured TIL were induced to transcribe IL-2 and IFN-gamma genes by incubation with the autologous irradiated tumor cell line, but not with autologous EBV-transformed cells. In these conditions, tumor-specific cytotoxic T lymphocytes (CTL) could be generated only after 3 weekly re-stimulations. In contrast, if autologous irradiated EBV-transformed cells were added to the cultures, specific CTL could be detected after one single tumor stimulation. Thus, signs of active responsiveness in terms of lymphokine gene mRNA are seldom detectable in melanoma metastases. Tumor-specific responses, however, including IL-2 and IFN-gamma gene expression and generation of CTL can be produced in vitro from specimens in which no cytokine gene mRNA is detectable ex vivo.

Adult↗
PubMed1994

Generation of lymphokine-activated killer activity in rodents but not in humans is nitric oxide dependent.

The role of nitric oxide (NO) in the generation of lymphokine-activated killer (LAK) cells was investigated. Here we report that L-arginine analog NG-monomethyl-L-arginine (NMMA), a specific inhibitor of nitric oxide synthase, prevents LAK cell generation from cultured rat splenic cells. Accumulated NO endproduct nitrite (NO2-), as measured in the supernatants of rat splenic cells, correlated well with the generation of LAK cells. In contrast, cell proliferation induced by rIL-2 or by Con A was not affected by NMMA. Similarly, phenotypic expression of CD25 in rIL-2-stimulated cultures was unaffected. Furthermore, we could not observe differences in percentages of CD5-CD8+ cells (NK and LAK cell phenotype markers in rats) between rIL-2-stimulated cultures performed in the presence or absence of NMMA. LAK cell generation could no longer be blocked if NMMA was added to the rat cell cultures 24 hr after rIL-2 stimulation. To further confirm the role of NO in LAK cell generation, rat splenic cells were cultured in medium without L-arginine. Under such conditions rIL-2 could not induce LAK cell generation. Hemoglobin, which is a scavenger of NO, also inhibited LAK cell generation. Finally, addition of sodium nitroprusside (SNP) which releases NO in cultures was able to overcome blocking effects of NMMA. To attempt the identification of NO-producing cells, lysosomotropic agent, L-leucine methyl ester (LME), was used. Generation of LAK cell activity was virtually abolished in cell cultures treated with LME. Addition of SNP to cultures, however, sufficed to restore LAK cell generation. These results suggest that LAK cell precursors depend on a exogenous NO supply from other cell types in order to display their full cytotoxic potential. Similar results were also obtained by using mouse splenocytes as responder cells. In contrast, NMMA did not affect generation of LAK cells from human peripheral blood or spleen mononuclear cells.

Animals↗
PubMed1994

Glutamine requirements in the generation of lymphokine-activated killer cells.

The role of glutamine (GLN) in the generation of lymphokine-activated killer (LAK) cell activity was investigated. LAK cells were derived from healthy donors and peripheral blood mononuclear cells (PBMC) were obtained using either unseparated PMBC or DR(-) CD3(-) CD16(+) CD56(+) enriched cells. PBMC were cultured for 6 or 10 days in medium supplemented with recombinant interleukin-2 (rlL-2; 100 U/ml) in the presence of different concentrations of GLN. K562 (natural killer-NK-sensitive targets), 1301 and U-937 (NK-resistant targets) cells were used as targets in the cytotoxic assays. Furthermore, the limiting dilution (LD) culture system was applied as an alternative to the bulk cell culture system. It was found that GLN affects the lytic potential of cultured cells while the frequency of responding cells did not significantly differ between the compared cell cultures performed in the presence of different amounts of GLN. Data on cell proliferation with IL-2 stimulation showed significant differences in cultures performed in the presence or absence of GLN. The results of present investigation suggest a supportive role of GLN in the generation of LAK cells. GLN deficit affects LAK cell killing activity by limiting the number of generated effector cells while acquisition of broad-range killing capacity was not affected.

Journal Article↗
PubMed1994

[Surgical training in Switzerland].

The training protocol for Swiss surgeons has become obsolete and too many new surgeons are being granted specialist diplomas. A revision of surgical training will have to take into account increasing patient demands, the multidisciplinary nature of treatments, and rapid progress in surgical techniques. It should allow the possibility of earlier specialization within the field of general surgery, but reserved to those surgeons who will remain in major hospital centres in the long term. It will have to integrate surgical research activities and recognize them as part of training. The introduction of examinations is essential to establish equivalencies with European countries and to ensure an identical level of training in all countries. This restoration of objective competitiveness will help to control the plethora of surgeons and the decreased number of active surgeons will help to group patients in a hospital system readjusted to needs and to current costs.

Curriculum↗
PubMed1994

[Surgical technique in vascularized auto- and allotransplantation of the knee joint in a dog model].

Experimental transplantation of vascularized canine knee joints has so far been associated with high complication rates in both auto- and allografts. We have now compared 4 autografts and 4 allografts that were successfully performed at our institution. This report details our surgical technique. Stable internal fixation enabling immediate postoperative weight bearing, microsurgical end-to-end anastomoses of popliteal artery and vein and postoperative control of immunosuppression in transplanted animals are crucial. Thus, an interdisciplinary approach of experts in microsurgery, orthopedic and trauma surgery and immunologists appears to be a prerequisite for successful joint transplantation.

Animals↗
PubMed1994

[Stable joint-bridging extension of malleolar dislocations and pilon fractures with the AO pinless external fixator].

The pinless external fixator, introduced into clinical practice for open tibial fractures, suggested itself for use as a traction substitute because of its pinless frame. The aim of this feasibility study was to replace the conventional calcaneal pin traction by a joint-bridging pinless fixator, inserted under local anesthesia. 10 patients with 6 malleolar dislocation fractures, 3 pilon tibial and 1 open distal tibial fracture were immobilised by a joint-bridging pinless fixator during 10.4 days (5-16 days) till swelling had subsided and definitive fracture treatment, consisting of plate fixation, took place. The implantation of the joint-bridging pinless fixator in local anaesthesia was well tolerated by all patients. This traction substitute offered good patient comfort and easy care. Although the provided stability was less than a conventional fixator, all patients were able to lift up their fractured extremity without pain.

Aged↗
PubMed1993

[The DHS (dynamic hip screw) buttress plate in the management of unstable proximal femoral fractures].

The DHS-implant system is a technically simple and widely used operative treatment modality for pertrochanteric fractures of the femur. In unstable 4-part fractures rotation of the head and neck fragment around the lag screw and significant impaction might lead to lateralisation of the greater trochanter and therefore to marked shortening. To prevent these effects we treated 17 patients with 4-part fractures with a prototype of a modular trochanteric DHS buttress plate. With this additional implant lateralisation of the greater trochanter could be prevented in all cases. This also leads to a limitation of telescoping, with less shortening even with immediate full weight bearing.

Adult↗
PubMed1993

Cytokine gene transcription in renal cell carcinoma.

Cytokines are powerful modulators of immune responses, the local production of which could be relevant to the interaction between tumour and immune system. This study investigated the transcription of genes encoding interleukin (IL) 2, IL-4, IL-10 and interferon (IFN) gamma in lymphocyte-infiltrated renal cell carcinoma biopsies from ten patients using the reverse polymerase chain reaction technique. Autologous peripheral blood mononuclear cells and healthy renal parenchyma tissue were tested in parallel. The beta-actin gene, used as a positive control, was transcribed in all samples. In contrast, transcription of cytokine genes was confined to tumour biopsies: IL-2 gene transcripts were detectable in five biopsies and IL-10 transcripts in seven. IL-4 and IFN-gamma gene transcripts were detectable in one biopsy each. In two patients no cytokine gene transcription could be identified. These data underline that heterogeneous patterns of cytokine gene transcription can be observed in renal cell carcinoma biopsies. Although transcription of an immunostimulatory lymphokine such as IL-2 was observed in 50 per cent of biopsies, the most frequently transcribed cytokine gene coded for an inhibitory factor, IL-10.

Aged↗