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Biomedical subjects

J Lebkowski

Publications and source records attributed to J Lebkowski.

At least 19 recordsLinked to original sources
PubMed2000

Ligand-mediated cytolysis of tumor cells: use of heregulin-zeta chimeras to redirect cytotoxic T lymphocytes.

New specificities may be engrafted onto lymphocytes by the transfer of genes for chimeric receptors that combine antigen recognition and signal-transducing elements. We have engineered and evaluated a new class of chimeric receptors that use the natural ligands of receptors found to be frequently overexpressed by cancer cells. The heregulin molecule, a ligand for Her3 and Her4 receptors when fused with the CD3 zeta-chain, was capable of redirecting T lymphocytes to recognize and respond to cancer cell lines that overexpress these receptors. Thus, CD8+ T lymphocytes were isolated from a healthy individual and transduced to express the chimeric heregulin-zeta receptor. These modified effector cells acquired the ability to specifically lyse a breast cancer cell line that overexpresses Her3 and Her4. A new class of chimeric receptors, such as heregulin-zeta, endowing anti-cancer effector cells with the potential to recognize and eliminate tumor targets, are likely to increase the effectiveness of adoptive immunotherapy for the treatment of cancer.

Adoptive Transfer↗
PubMed2000

Dendritic cells loaded with MART-1 peptide or infected with adenoviral construct are functionally equivalent in the induction of tumor-specific cytotoxic T lymphocyte responses in patients with melanoma.

Immunization with tumor-specific-associated antigen--pulsed dendritic cells has proved to be efficacious in various animal models and is being evaluated for the treatment of cancer in humans. Use of dendritic cells pulsed with specific peptides or transfected with tumor-associated antigen genes has been a focused area of investigation for inducing potent tumor and viral immune responses. In this study, the authors demonstrate transgene expression, including the lacZ and MART-1 genes, in dendritic cells infected with adenoviral constructs. These transiently transduced dendritic cells, derived from melanoma patients' monocytes cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4, express the transgene and can stimulate patients' CD8+ T cells to elicit an antitumor immune response comparable to dendritic cells loaded with a defined peptide. These cytotoxic T lymphocytes were able to recognize both known and unknown tumor-associated antigen epitopes and exhibited cytolytic activity against HLA-matched tumor cells expressing the antigen. The ability to induce tumor-specific cytotoxic T lymphocytes in vitro using gene-modified dendritic cells that transiently express tumor-associated antigens demonstrates the potential use of these antigen-presenting cells for developing in vivo cancer vaccines.

Adenoviruses, Human↗
PubMed1999

IL-13 can substitute for IL-4 in the generation of dendritic cells for the induction of cytotoxic T lymphocytes and gene therapy.

Immunization with tumor-associated antigen pulsed dendritic cells (DC) has been shown to elicit both protective and therapeutic antitumor immunity in a variety of animal models and is currently being investigated for the treatment of cancer patients in clinical trials. In this study we show that DC can be generated from peripheral blood mononuclear cells of healthy donors as well as breast and melanoma cancer patients using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-13 (IL-13) and that these DC have many of the same characteristics as DC differentiated using GM-CSF and IL-4. The DC generated in GM-CSF and IL-13 are CD14- and express high levels of the cell surface markers CD86, HLA-DR, and CD58, as do DC generated in GM-CSF and IL-4. The purity and yield of both DC populations are not significantly different. Furthermore, both populations of DC are effective at presentation of alloantigen as determined in a mixed lymphocyte response, and both are able to process and present soluble tetanus toxoid antigen to CD4+ T cells. Because we are interested in the generation of DC for antigen-specific cytotoxic T lymphocyte (CTL) generation, we compared the ability of peptide-pulsed DC differentiated in GM-CSF and IL-4 versus GM-CSF and IL-13 for the generation of influenza and MART-1 specific CTL. Both populations of DC induced CD3+ CD8+ CD4- and CD56- CTL, which could lyse the appropriate targets in an antigen-specific manner. Finally, both GM-CSF and IL-4 DC and GM-CSF and IL-13 DC yielded similar beta galactosidase expression levels after transduction with recombinant adenovirus containing the LacZ gene. These results suggest that DC generated in GM-CSF and IL-13 may be useful for immunotherapy and gene therapy protocols.

Antigens, Neoplasm↗
PubMed1998

High proliferative potential-quiescent cells: a working model to study primitive quiescent hematopoietic cells.

Human adult hematopoietic stem cells are mostly quiescent or slow cycling. We have previously demonstrated that blocking of transforming growth factor-beta1 (TGF-beta1) is able to activate, in the presence of cytokines, primitive quiescent hematopoietic multipotent progenitors which could not grow in a two week semi-solid culture assay (short term culture). We have also shown that anti-TGF-beta1 can up-modulate c-KIT, the receptor of the stem cell factor (steel factor). To elucidate whether TGF-beta1 plays a central role in controlling the quiescence of hematopoietic primitive cells, it was necessary to demonstrate, as detailed in this study, that: (1) whatever the cytokine combination tested, addition of anti-TGF-beta1 releases from quiescence multipotent progenitors with a significantly higher hematopoietic potential than those activated by cytokines alone. (2) Other important cytokine receptors controlling the most primitive hematopoietic cells such as FLT3 and the IL6 receptor (IL6-R) are down-modulated by TGF-beta1 but rapidly up-modulated by anti-TGF-beta1. (3) Anti-TGF-beta1-sensitive multipotent and high proliferative potential progenitors express these cytokine receptors at a low level (FLT3(low) and IL6-Rlow). According to these results, we propose the working model of 'High Proliferative Potential-Quiescent cells' to refer to these primitive hematopoietic multipotent progenitors that are highly sensitive to the growth inhibitory effect of TGF-beta1. This model could be valid not only to study the human hematopoietic quiescent progenitors but also for other somatic stem cell systems.

Antibodies↗
PubMed1997

Low dose subcutaneous interleukin-2 after autologous transplantation generates sustained in vivo natural killer cell activity.

Autologous transplantation can induce extended remission in some patients with advanced breast cancer and lymphoma yet nearly 80% and 50%, respectively, will ultimately relapse. In vitro studies suggest that activated natural killer cells (NK) mediate lytic activity against breast cancer and lymphoma cell lines. Therefore, immunotherapy with interleukin-2 (IL-2, Amgen) to activate NK may improve long-term disease-free survival when administered in a post-transplant minimal residual disease setting. To determine the feasibility of administering IL-2 and activation of NK post-transplant, twelve patients (6 breast cancer, 6 lymphoma) were enrolled on a phase I dose escalation study after autologous transplantation (median day + 94, range 50-166). IL-2 was self administered at 0.25 x 10(6) (n = 6) or 0.5 x 10(6) (n = 6) U/m2/day subcutaneously for 84 consecutive days. The best tolerated dose was 0.25 x 10(6) U/m2/day (75% of planned doses given vs. 48% at the higher dose). Dose limiting toxicity occurred in 6 patients (n = 2 at 0.25 x 10(6) U/m2/day, n = 4 at 0.5 x 10(6) U/m2/day) consisting of decreased performance status (n = 2), thrombocytopenia (n = 3, 1 at the lower dose), and mild neutropenia (n = 1 at the lower dose). However, all symptoms resolved within a week following discontinuation of IL-2 and no patient required hospitalization. Circulating soluble IL-2 receptor levels were significantly increased in all patients receiving IL-2. Patients receiving at least 28 days of IL-2 exhibited a greater than 10-fold increment in circulating CD56+bright/CD3- NK. Furthermore, lytic function was increased against NK resistant targets, MCF-7 (breast cancer), and Raji (lymphoma). In vivo IL-2 primed NK cells obtained by lymphapheresis were activated in large-scale ex vivo incubation in high dose IL-2 (1,000 U/mL) at high cell density (10 x 10(6)/mL), in gas permeable bags, and using serum-free media. NK lytic function against MCF-7 and Raji targets was further enhanced. We conclude that low dose subcutaneous IL-2 based immunotherapy is feasible, relatively safe, can be administered in an outpatient setting and hypothesize that additional ex vivo incubation in IL-2 may be used to generate NK cells with potent antitumor effects in vivo.

Adjuvants, Immunologic↗
PubMed1996

Transient protection of human T-cells from human immunodeficiency virus type 1 infection by transduction with adeno-associated viral vectors which express RNA decoys.

RNA decoys are oligonucleotides corresponding to the TAR and RRE sequences of HIV which inhibit the HIV-encoded regulatory proteins Tat and Rev, respectively. Adeno-associated viral vectors encoding RNA decoys stably transduced into the human T-cell line CEM-SS expressed transactivating region (TAR) and Rev-responsive element (RRE) RNA decoys from tRNA polIII promoters at high levels, without any apparent deleterious effects on cell growth or expression of CD4. DNA blot analysis indicated that RNA decoy-encoding vectors were not rearranged and were integrated into the genomic DNA of selected cell lines. Vector DNA with the appropriate TAR and RRE sequences was isolated from transduced cell lines after prolonged growth in culture, further confirming that the vector DNA was present in a stable form through multiple cell cycles. Cell lines expressing TAR and RRE decoys transiently inhibited HIV gene expression and replication by 70-99% as determined by measurement of intracellular and extracellular HIV p24 production. Adeno-associated vectors encoding RNA decoys may be useful for gene therapy of HIV infection.

Cell Line↗
PubMed1996

Gene modification of primary tumor cells for active immunotherapy of human breast and ovarian cancer.

We have previously shown that cationic liposomes facilitate adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. To test the clinical feasibility of using genetically modified tumor vaccines for the treatment of breast and ovarian cancers, we have constructed an expression plasmid pMP6IL2 and investigated the use of liposome-mediated gene delivery into primary, uncultured human breast and ovarian tumor cells to produce interleukin 2 (IL-2)-secreting tumor cells. We have demonstrated significant levels of IL-2 expression in tumor cell lines and primary breast and ovarian tumor cells using this AAV-based expression plasmid complexed to cationic liposomes. Transfections with the non-AAV plasmid containing the identical expression cassette as the AAV plasmid induced IL-2 expression in the tumor cell line but failed to produce IL-2 in primary tumor cells. Significant levels of IL-2 were induced with the AAV plasmid regardless of liposome compositions used for transfection. The transfected breast cell line and primary tumor cells were able to express the transgene product for up to 28 days after lethal radiation. The transfection efficiency was comparable for both the tumor cell line and primary tumor cells and ranged from 20 to 50% for both cell types as assessed by intracellular IL-2 staining. Although the primary tumor cell preparations consist of mixed population of cells, at least 40% of the tumor cells expressed the transgene as assessed by immunostaining for IL-2. The ability to efficiently express transgenes in freshly isolated, nondividing tumor cells may potentiate active immunotherapy strategies for gene-based cancer treatment.

Blotting, Southern↗
PubMed1995

Functional reconstitution of the NADPH-oxidase by adeno-associated virus gene transfer.

Chronic granulomatous disease (CGD) comprises a heterogeneous group of inherited conditions characterized biochemically by disordered function of a unique multicomponent enzyme system present in phagocytic cells, the NADPH-oxidase. Clinically, it is characterized by recurrent bacterial and fungal infections that are relatively resistant to treatment by conventional means. Curative bone marrow transplantation has been successfully achieved in a small number of cases, but the wider application of this procedure is limited by availability of suitable donor material. Somatic gene therapy would overcome this problem, and several groups have now shown correction of the biochemical defect in hematopoietic cells by retrovirus-mediated gene transfer. However, the failure of the current generation of retroviral vectors to efficiently transduce quiescent cells greatly restricts their potential for gene transfer to pluripotent hematopoietic stem cells. Given these limitations, we have constructed vectors based on adeno-associated virus and used these to transfer a functional copy of the p47phox gene to immortalized B cells derived from patients with p47phox-deficient autosomal recessive CGD. We show stable expression of protein and restoration of NADPH-oxidase function in these cells in the absence of selection. Adeno-associated virus vectors may overcome some of the limitations of retroviral gene delivery systems and may therefore be a useful vehicle for curative gene therapy of CGD and other primary immunodeficiencies.

B-Lymphocytes↗
PubMed1995

Generation of recombinant adeno-associated virus (rAAV) from an adenoviral vector and functional reconstitution of the NADPH-oxidase.

The human parvovirus, adeno-associated virus-2 (AAV-2), has many attributes that recommend its use as a gene transfer vehicle, including a broad tissue tropism, the ability to integrate stably into the host genome, and efficient transduction of cells which proliferate slowly. However, application to human gene therapy is currently limited by existing methods for generation of recombinant AAV (rAAV), resulting in relatively low transducing titres. In an attempt to overcome some of these problems, we have developed a defective adenoviral vector which improves the efficiency of rAAV vector delivery to cells in which rAAV is propagated, and from which the rAAV genome can be efficiently rescued. A functional copy of the p47phox gene was successfully transferred to cell lines derived from patients with autosomal recessive chronic granulomatous disease (CGD) by rAAV recovered in this way, and function of the NADPH-oxidase was restored to levels which were stable for at least 8 weeks. This method for generation of rAAV, although still limited by the need for cotransfection of AAV Rep and Cap functions, may permit recovery of higher titre transducing stocks from cell lines in which these genes are stably incorporated, and significantly reduces the risk of contamination with wild-type adenovirus (wtAd).

Adenoviridae↗
PubMed1993

Direct isolation and expansion of human CD34+ hematopoietic stem cells.

The isolation and expansion of CD34+ cells has numerous applications as supportive therapy for cytopenias that occur postchemotherapy. In this report, we describe a simple system to directly isolate and culture human CD34+ cells. Using this system, CD34+ cells isolated from bone marrow were cultured in the presence of GM-CSF, G-CSF, erythropoietin, stem cell factor, IL-1, IL-3, and IL-6. Cell expansions up to 100-fold were noted during the first 2 weeks of culture with high maintenance of CD34+ cells during the first week of culture. Maximal expansions of progenitors were observed at one week of culture, with, on average, 20-fold increases in CFU-GM number. The results support the feasibility of CD34+ cell expansion for clinical application.

Antigens, CD↗
PubMed1988

Platelet aggregability in patients with common carotid artery ligation.

Increased platelet aggregability is regarded as being a sensitive indicator of the initiation of thrombotic processes. Platelet aggregation was analysed in blood taken from the common carotid artery before and 30 min after its ligation in 3 patients, as well as in the venous blood of 14 patients in the late postoperative period. No tendency towards increasing platelet aggregation was observed in either of the groups investigated.

Adult↗
PubMed1987

Late rheoencephalographic assessment of the cerebral circulation after ligation of the common carotid artery.

A series of 17 patients was investigated following common carotid ligation. The period between operation and examination ranged from 2-7 years. Cerebral circulation was estimated by use of the rheoencephalographic method. Statistical analysis of the reg plot was performed by comparing an age-matched normal population with patients. A comparison was also made of the ligated and nonligated sides for the patients. The following results were obtained: a significant decrease in the amplitude of the reg waves, an extension in the anacrotic part of the wave, and a decrease in the angle of its inclination. This was intensified more on the side of the common carotid ligation. These findings may suggest that after common carotid ligation the cerebral circulation is changed for a long period of time. They would also seem to indicate that postoperatively, the cerebral circulation does not return to normal--if it ever does.

Adult↗
PubMed1987

Some aspects of measuring intracranial pressure.

The problem of measurement of intracranial pressure in the epidural space is discussed in the light of the authors' own experience. Three forms of transducers have been used consecutively: (1) a capsular form which slides into the epidural space, (2) a screw-shaped form, fixed firmly in a burr hole and (3) a cylindrical tape, designed to be fixed in a burr hole with the aid of a protective socket. The zero point may be adjusted during measurement. The merits and disadvantages of these three types of transducer, as well as the practical problems connected with their use are discussed.

Epidural Space↗