Biomedical subjects

M Mincheff

Publications and source records attributed to M Mincheff.

9 recordsLinked to original sources

PubMed2006

Immune responses against PSMA after gene-based vaccination for immunotherapy-A: results from immunizations in animals.

Two plasmid DNA vaccines, encoding either products that are retained in the cytosol and degraded in the proteasome (tVacs; hPSMAt), or secreted proteins (sVacs; hPSMAs) were evaluated for stimulation of cytotoxic cell or antibody responses. Immunization with both vectors led to generation of cell cytotoxicity providing granulocyte-macrophage colony-stimulating factor was administered with the vaccine. Spleen cells from animals immunized with hPSMAt demonstrated stronger cytotoxicity to the target cells. Priming with a vector that encoded a xenogeneic protein (hPSMAt; 'xenogeneic' construct) and boosting with a vector that encoded an autologous protein (rPSMAt; 'autologous' construct) gave the best protection against tumor challenge. Immunization with tVacs did not lead to formation of antibodies to the target protein as detected by Western blot or ELISA, while immunization with sVacs or with the protein did. Antibodies were of mixed Th1-Th2 isotype. Priming with tVacs and boosting with protein also resulted in antibody formation, but in this case the antibodies were from the cytotoxic, Th1 isotype. The best strategy to obtain a strong cellular cytotoxic response, therefore, seems to be gene-based vaccinations with tVacs, priming with the 'xenogeneic' and boosting with the 'autologous' constructs. When cytotoxic antibody production is the goal, priming should be performed with the tVacs while boosting with the protein.

Animals↗

PubMed2000

Naked DNA and adenoviral immunizations for immunotherapy of prostate cancer: a phase I/II clinical trial.

INTRODUCTION AND OBJECTIVES: Animal studies have indicated that the use of syngeneic dendritic cells that have been transfected ex vivo with DNA for tumor-specific antigen results in tumor regression and decreased number of metastases. Additional studies have also suggested the possibility to modulate the dendritic cells in vivo either by 'naked' DNA immunization or by injecting replication-deficient viral vectors that carry the tumor-specific DNA. Using the prostate- specific membrane antigen (PSMA) as a target molecule, we have initiated a clinical trial for immunotherapy of prostate cancer. The primary objective of the study was to determine the safety of the PSMA vaccine after repeated intradermal injections. METHODS: We have included the extracellular human PSMA DNA as well as the human CD86 DNA into separate expression vectors (PSMA and CD86 plasmids), and into a combined PSMA/CD86 plasmid. In addition, the expression cassette from the PSMA plasmid was inserted into a replication deficient adenoviral expression vector. Twenty-six patients with prostate cancer were entered into a phase I/II toxicity-dose escalation study, which was initiated in spring 1998. Immunizations were performed intradermally at weekly intervals. Doses of DNA between 100 and 800 microg and of recombinant virus at 5x10(8) PFUs per application were used. RESULTS AND CONCLUSION: No immediate or long-term side effects following immunizations have been recorded. All patients who received initial inoculation with the viral vector followed by PSMA plasmid boosts showed signs of immunization as evidenced by the development of a delayed-type hypersensitivity reaction after the PSMA plasmid injection. In contrast, of the patients who received a PSMA plasmid and CD86 plasmid, only 50% showed signs of successful immunization. Of the patients who received PSMA plasmid and soluble GM-CSF, 67% were immunized. However, all patients who received the PSMA/CD86 plasmid and sGM-CSF became immunized. The patients who did not immunize during the first round were later successfully immunized after a boost with the viral vector. The heterogeneity of the medical status and the presence in many patients of concomitant hormone therapy does not permit unequivocal interpretation of the data with respect to the effectiveness of the therapy. However, several responders, as evidenced by a change in the local disease, distant metastases, and PSA levels, can be identified. A phase II clinical study to evaluate the effectiveness of the therapy is currently underway.

Adenoviridae↗

PubMed1998

Changes in donor leukocytes during blood storage. Implications on post-transfusion immunomodulation and transfusion-associated GVHD.

Freshly drawn peripheral blood granulocytes, monocytes and NK cells express Fas-L following activation. Fully functional soluble Fas-L (sFas-L) is released and found in fresh plasma samples from healthy volunteers coupled to a Fas-L binding factor (PFBF). In the absence of plasma leukocytes undergo rapid apoptosis with 15% of the granulocytes and 25% of the monocytes showing signs of apoptosis immediately following separation. Apoptosis progresses during refrigerated storage even in the presence of plasma and all granulocytes disintegrate by day 15 of storage. The absence of plasma increases the rate of apoptosis. The resulting increased expression of PS on apoptosing leukocytes enhances blood procoagulant activity. Whereas transfusions of fresh blood lead to alloimmunization, the functional impairment and death of antigen-presenting cells during storage eliminates their ability to stimulate cytotoxic immune responses. Additionally, loss of function and death of donor T cells during storage preclude the possibility for development of post-transfusion GVHD. Continued storage leads to the secondary necrosis of apoptosed leukocytes and the release of soluble antigens. Depending on dose, transfusion of such products may lead to immunosuppression due to a T2 bias of the immune response.

Apoptosis↗

PubMed1998

Fas and Fas ligand expression on human peripheral blood leukocytes.

OBJECTIVES: Study of Fas and Fas ligand (Fas-L) expression, as well as sFas-L release, by fresh human peripheral blood leukocytes. METHODS: Flow cytometry, cytotoxicity, immunofluorescence staining of fresh smears. Western blotting. RESULTS: Granulocytes and monocytes express a low level of Fas receptor, but no Fas-L. These cells, as well as NK cells, contain presynthesized depots of Fas-L which they express following activation by brief storage (60 min) at room temperature or during separation from whole blood. Such activation also leads to Fas receptor upregulation. NK cells do not express Fas receptor. Once expressed on blood leukocytes, fully functional Fas-L can be released from the membrane and can be detected in plasma-free cell supernatants. CONCLUSION: Human peripheral blood granulocytes, monocytes and NK cells contain intracellular presynthesized Fas-L which they readily express following blood anticoagulation, blood storage or cell separation. Soluble Fas-L is released from those cells and can be detected in protein-free supernatants by immunoblotting.

Cell Separation↗

PubMed1986

Isolation and cryopreservation of human peripheral blood monocytes.

A modification of the Freundlich and Avdalovic method (J. Immunol. Methods 62, 31 (1983] is reported. Buffy coats, separated and pooled together, are used for isolation of monocytes (70% yield, 100% purity). Cell density of working suspension is increased up to 0.65 X 10(9) cells/75 cm2 surface by multiplication of the active fibronectin sites. For the purpose, cryoprecipitate is used instead of plasma for coating the glass-gelatin surface. Monocytes, isolated by that procedure, could be successfully cryopreserved with dimethyl sulfoxide cryoprotective solution.

Blood Preservation↗

PubMed1985

Aggregate formation in cryopreserved leukocyte suspensions.

During cryopreservation of leukocytes, a great part of the granulocytes is injured. The latter is responsible for aggregate formation in the suspension and a loss in the number of preserved cells. The formation of aggregates in the suspension is potentiated in the presence of serum in the cryoprotective medium. On the other hand, the presence of sugar (sucrose, lactose, dextrose) in the washing solution diminishes the aggregate formation, the latter effect being directly proportional to the final concentration of the sugar in the suspension. The aggregates contain mainly cells and very rarely amorphous or fibrous zones. They appear only in the presence of serum. Their formation is not connected with presence of traces of fibrinogen in the serum.

Blood Preservation↗

PubMed1984

A method for preparation of dry thrombin for topical application.

Thrombin for topical hemostasis can be prepared from bovine or human blood plasma. The prothrombin is isolated by means of adsorption on DEAE-Sephadex A-50 and consecutively activated by CaCl2 and thromboplastin. Thrombin is precipitated and purified by acetone. The specific activity of the thrombin preparation is 122 + 23 IU/mg protein while the yield is 36,360 +/- 6623 IU/liter plasma.

Administration, Topical↗

PubMedDate not supplied

In vivo transfection and/or cross-priming of dendritic cells following DNA and adenoviral immunizations for immunotherapy of cancer--changes in peripheral mononuclear subsets and intracellular IL-4 and IFN-gamma lymphokine profile.

In order to provoke an immune response, a tumor vaccine should not only maximize antigen-specific signals, but should also provide the necessary "co-stimulatory" environment. One approach is to genetically manipulate tumor cells to either secrete lymphokines (GM-CSF, IL-12, IL-15) or express membrane bound molecules (CD80, CD86). Furthermore, patient dendritic cells can be loaded with tumor-associated antigens or peptides derived from them and used for immunotherapy. Genetic modification of dendritic cells can also lead to presentation of tumor-associated antigens. Transfection of dendritic cells with DNA encoding for such antigens can be done in vitro, but transfection efficiency has been uniformly low. Alternatively, dendritic cells can also be modulated directly in vivo either by "naked" DNA immunization or by injecting replication-deficient viral vectors that carry the tumor specific DNA. Naked DNA immunization offers several potential advantages over viral mediated transduction. Among these are the inexpensive production and the inherent safety of plasmid vectors, as well as the lack of immune responses against the carrier. The use of viral vectors enhances the immunogenicity of the vaccine due to the adjuvant properties of some of the viral products. Recent studies have suggested that the best strategy for achieving an intense immune response may be priming with naked DNA followed by boosting with a viral vector. We have successfully completed a phase I and phase II clinical trials on immunotherapy of prostate cancer using naked DNA and adenoviral immunizations against the prostate-specific membrane antigen (PSMA) and phase I clinical trial on colorectal cancer using naked DNA immunization against the carcinoembryonic antigen (CEA). The vaccination was tolerated well and no side effects have been observed so far. The therapy has proven to be effective in a number of patients treated solely by immunizations. The success of the treatment clearly depends on the stage of the disease proving to be most efficient in patients with minimal disease or no metastases. A panel of changes in the phenotype of peripheral blood lymphocytes and the expression of intra-T-cell lymphokines seems to correlate with clinical improvement.

Adenoviridae↗

PubMedDate not supplied

Biochemical nature and mapping of PSMA epitopes recognized by human antibodies induced after immunization with gene-based vaccines.

BACKGROUND: A possible new target for immunotherapy is the prostate-specific membrane antigen (PSMA). The aim of the present study was to define potential PSMA epitopes for antibody binding using sera from patients immunized with gene-based anti-PSMA vaccines. MATERIALS AND METHODS: Sera from prostate cancer patients, immunized repeatedly with plasmid and adenoviral vectors, each encoding for the extracellular portion of human PSMA, were tested for anti-PSMA antibodies by Western blot. PSMA-producing LNCaP cells were used as a control. Recombinant PSMA protein cleaved with different proteinases was used for epitope mapping. Different enzymes were used to cleave the PSMA molecule. RESULTS: Specific anti-PSMA antibodies were detected in the studied patients' sera, mainly against the PSMA protein core. An alignment of the predicted enzyme-cleavage fragments was compared with Western blot results and several antibody epitopes were determined. CONCLUSION: These data demonstrate that multiple gene-based vaccinations induce an anti-PSMA humoral immune response. The antibodies are predominantly specific for the PSMA protein core.

Aged↗