RESUMO
We have used cationic liposomes to facilitate adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. AAV plasmid DNA complexed with liposomes showed levels of expression several fold higher than those of complexes with standard plasmids. In addition, long-term expression (> 30 days) of the gene, unlike the transient expression demonstrated by typical liposome-mediated transfection with standard plasmids, was observed. Southern analysis of chromosomal DNA further substantiated the hypothesis that the long-term expression was due to the presence of the transgene in the AAV plasmid-transfected group and not in the standard plasmid-transfected group. AAV plasmid-liposome complexes induced levels of transgene expression comparable to those obtained by recombinant AAV transduction. Primary breast, ovarian, and lung tumor cells were transfectable with the AAV plasmid DNA-liposome complexes. Transfected primary and cultured tumor cells were able to express transgene product even after lethal irradiation. High-level gene expression was also observed in freshly isolated CD3+, CD4+, and CD8+ T cells from normal human peripheral blood. Transfection efficiency ranged from 10 to 50% as assessed by intracellular interleukin-2 levels in interleukin-2-transfected cells. The ability to express transgenes in primary tumor and lymphoid cells may be applied toward tumor vaccine studies and protocols which may eventually permit highly specific modulation of the cellular immune response in cancer and AIDS.
Assuntos
Neoplasias da Mama/metabolismo , DNA/genética , Dependovirus/genética , Expressão Gênica , Interleucina-2/biossíntese , Neoplasias Pulmonares/metabolismo , Neoplasias Ovarianas/metabolismo , Plasmídeos/administração & dosagem , Linfócitos T/metabolismo , Transfecção/métodos , Células Tumorais Cultivadas/metabolismo , Síndrome da Imunodeficiência Adquirida/imunologia , Animais , Southern Blotting , Células Cultivadas , Cloranfenicol O-Acetiltransferase/análise , Cloranfenicol O-Acetiltransferase/biossíntese , DNA/administração & dosagem , Portadores de Fármacos , Feminino , Vetores Genéticos , Humanos , Interleucina-2/genética , Lipossomos , Masculino , Neoplasias da Próstata , Ratos , Subpopulações de Linfócitos T/metabolismo , Neoplasias da Bexiga UrináriaRESUMO
A rapid method has been developed which combines immunofluorescence and autoradiography and permits the double labeling of DNA. P388 murine leukemic cells were incubated with bromodeoxyuridine and tritiated thymidine simultaneously. After fixation, the sample was first processed with a monoclonal antibody to bromodeoxyuridine (RPMB I) so that any cell in S-phase was brightly fluorescent (RPMB technique). Next, tritiated thymidine grains were developed by autoradiography, and the result demonstrated fluorescence as well as black grains in each S-phase cell. P388 cells sensitive (P388S) and resistant (P388R) to 1-beta-D-arabinofuranosylcytosine (ara-C) were incubated with bromodeoxyuridine and [3H]ara-C simultaneously. Processing by autoradiography and RPMB techniques revealed that all S-phase cells in the P388S sample demonstrated vivid "double labeling," whereas P388R cells only revealed bright green fluorescence in S-phase cells, but no grains, confirming a lack of ara-C incorporation into the DNA by this line. Finally, a computerized digital analysis system attached to a microphotometer was used to quantitate fluorescence and grains per cell, and the data demonstrated that the number of [3H]ara-C grains in each P388S cell was inversely proportional to the degree of fluorescence in that cell, indicating that DNA synthesis was inhibited by ara-C. In conclusion, a simple, easy-to-use double-labeling method has been introduced which will be useful to a wide variety of researchers, because this technique together with the digital analysis system offers the possibility of measuring drug sensitivities in individual cells.
Assuntos
Bromodesoxiuridina , DNA/biossíntese , Animais , Anticorpos Monoclonais , Autorradiografia , Ciclo Celular , Citarabina/metabolismo , Leucemia P388/metabolismo , Camundongos , Timidina/metabolismo , TrítioRESUMO
The majority of human neuroblastomas express low to undetectable levels of major histocompatibility complex (MHC) class I and II antigens (MHC-I and -II). We studied the effects of gamma interferon (gamma-IFN) transduction on expression of these antigens in six human neuroblastoma cell lines with and without genomic amplification of the N-myc oncogene. All six were stably transduced with an MoMLV-based gamma-IFN retroviral vector (DAh gamma-IFN). G418-resistant cells were assayed for MHC-I, MHC-II, B7-1, and neuroblastoma-associated antigen expression, as well as for gamma-IFN levels in cell culture supernatants. Sustained gamma-IFN production, 2 to > 1000 units/10(6) cells/d, was attained for five of six transduced cell lines and persisted for up to 9 months. This resulted in marked upregulation of MHC-I and MHC-II expression in LA-N-1, LA-N-6, and CHLA-127 cells and moderate upregulation in SK-N-Fi and SK-N-AS cells. One cell line (LA-N-1) had marked induction of MHC-I and MHC-II despite marginal levels of gamma-IFN production. Expression of CD28 ligand B7-1 (as determined by BB1 antibody) remained unchanged in all gamma-IFN-transduced cell lines tested. Expression of several neuroblastoma-associated antigens (NKH1A, 126-4, HSAN 1.2, HNK, 459, and 390) was upregulated in some of the gamma-IFN-transduced cell lines. These results demonstrate that preparation of gamma-IFN expressing neuroblastoma cells for immunotherapeutic purposes is feasible and that gamma-IFN transduction results in phenotypic changes that may improve immunogenicity of human neuroblastoma cells.
Assuntos
Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe I/biossíntese , Interferon gama/genética , Neuroblastoma/imunologia , Transdução Genética , Antígeno B7-1/metabolismo , Antígenos CD28/metabolismo , Antígenos CD57/metabolismo , Vetores Genéticos , Humanos , Imunofenotipagem , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/metabolismo , Interferon gama/biossíntese , Neuroblastoma/patologia , Fenótipo , Tolerância a Radiação , Retroviridae/genética , Células Tumorais CultivadasRESUMO
This article has summarized known congenital and acquired alterations of hemostasis leading to thrombosis. Decreases in coagulation inhibitors, including antithrombin III, heparin cofactor II, and protein C and protein S, are of major importance in assessing patients with hypercoagulable states or patients with unexplained thrombosis. Newer assays for components of the fibrinolytic system, plasminogen, t-PA and t-PA inhibitor are also now readily available and are important for defining congenital or acquired fibrinolytic defects leading to hypercoagulability and thrombosis. By judicious use of these assays, combined with clinical evaluation, many patients with thrombosis will have an underlying etiologic blood protein defect defined. Delineating reasons for a thrombotic event is of obvious importance for planning long-term prophylactic therapy and for diagnosing and counseling afflicted family members. In this manner, newly found patients can be treated prophylactically before unalterable morbidity or mortality occurs.
Assuntos
Transtornos da Coagulação Sanguínea/etiologia , Trombose/etiologia , Antitrombina III/fisiologia , Deficiência de Antitrombina III , Fatores de Coagulação Sanguínea/antagonistas & inibidores , Fibrinólise , Cofator II da Heparina/deficiência , Cofator II da Heparina/fisiologia , Humanos , Proteína C/fisiologia , Deficiência de Proteína C , Proteína S/metabolismo , Proteína S/fisiologia , Deficiência de Proteína SAssuntos
Citarabina/uso terapêutico , Leucemia Mieloide/tratamento farmacológico , Leucemia/tratamento farmacológico , Adulto , Idoso , Medula Óssea/patologia , Bromodesoxiuridina/metabolismo , Citarabina/administração & dosagem , DNA/biossíntese , Resistência a Medicamentos , Feminino , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Interfase , Leucemia/metabolismo , Leucemia/patologia , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
B16-C3 murine melanoma, A375P human melanotic melanoma, and C32 human amelanotic melanoma cells were incubated in the presence of (0-4 mM) H2-antagonists, ranitidine and cimetidine. Cell proliferation, tyrosinase activity and melanin content were monitored. H2-antagonists stimulated tyrosinase activity and melanin accumulation in B16-C3 cells in a dose- and time-dependent manner. Stimulation of enzyme activity and pigment production was accompanied by inhibition of cellular proliferation in B16-C3 cells. The inhibitory concentration of cimetidine was approximately 2-fold higher than that of ranitidine. H2-antagonists failed to stimulate melanogenesis in A375P or C32 cells, but inhibited cellular proliferation in both cell lines. These results are the first demonstration of H2-antagonist induced phenotypic changes in malignant melanoma cells in vitro, and represent a novel mechanism for the previously described in vivo antitumor effects of these agents.
Assuntos
Cimetidina/farmacologia , Melaninas/biossíntese , Melanoma Experimental/metabolismo , Melanoma/metabolismo , Ranitidina/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Cinética , Melaninas/análise , Melanoma/patologia , Melanoma Experimental/patologia , Camundongos , Monofenol Mono-Oxigenase/metabolismoRESUMO
A monoclonal antibody against bromodeoxyuridine (BrdUrd) was produced, and a rapid slide technique (RPMB technique) was developed for the estimation of S-phase cells in a population using this antibody. Bone marrow cells from patients with acute nonlymphocytic leukemia (ANLL) were studied by both the RPMB technique and tritiated thymidine (3HdThd) labeling index studies. The percentage of S-phase cells obtained by each method was compared in 50 samples, and the correlation coefficient was r = 0.89. A "double label" method is also described in which cells were simultaneously incubated with either BrdUrd and 3HdThd or BrdUrd and tritiated cytosine arabinoside (3HAra-C). The samples were first processed by the RPMB technique and then by autoradiography. Results showed only black grains overlying the nuclei of fluorescent cells in each group. An automated microphotometer was used to quantitate grains and fluorescence from each cell. This demonstrated an almost direct relationship between grains and fluorescence from BrdUrd + 3HdThd slides, whereas different patterns of relationship were noted from BrdU + 3HAra-C slides of leukemic patients. Their implications are discussed in the text. Finally, intravenous infusions of BrdUrd was given to five leukemic patients. S-phase cells were recognized distinctly within 5 min of starting the infusion. The percentage of S-phase cells was almost identical from in vivo and in vitro samples. Various possibilities of studying the biological behavior of acute leukemias and analyzing cell cycle characteristics are discussed.
Assuntos
Bromodesoxiuridina , Ciclo Celular , Leucemia/patologia , Automação , Autorradiografia , Células da Medula Óssea , Citarabina/farmacologia , DNA de Neoplasias/biossíntese , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , TimidinaRESUMO
OBJECTIVE: To describe the current indications and side effects of Interferons in the treatment of human disease together with sufficient background information to understand the rationale for their use. DATA SOURCES: Literature searches on interferons extended back to the time of their discovery. Citations were limited to English and human aspects. Reference lists from recent reviews were also consulted. STUDY SELECTION: Reviews of fundamental mechanisms were selected together with studies of individual clinical trials and anecdotal experiences with human use of interferons. CONCLUSIONS: Interferons are accepted therapy for a number of conditions. Their range of therapeutic uses will undoubtedly increase as further knowledge is obtained.
Assuntos
Interferons/uso terapêutico , Interações Medicamentosas , Humanos , Interferons/química , Interferons/farmacologiaRESUMO
A rapid and convenient method for estimating S-phase cells in a population was developed which detects bromodeoxyuridine (BrdU) incorporation into DNA by means of monoclonal anti-BrdU antibodies. This immunofluorescence technique (RPMB technique) was compared to autoradiographic (ARG) detection of tritiated thymidine (3HTdr) grains incorporated into the DNA. Using incubation periods for BrdU and 3HTdr ranging from one minute to one hour and detecting their incorporation by ARG and RPMB techniques, it became apparent that the RPMB technique was far more sensitive than ARG in addition to being extremely easy to perform. Some possible utilities of the RPMB technique are discussed.
Assuntos
Bromodesoxiuridina/imunologia , Imunofluorescência , Interfase , Anticorpos Monoclonais , Autorradiografia , Bromodesoxiuridina/metabolismo , DNA/biossíntese , Humanos , Leucemia/metabolismo , Leucemia/patologiaRESUMO
The reliability of DNA histogram analysis in accurately estimating S-phase cells from human tumors was tested by comparing the results to those of simultaneously obtained tritiated thymidine labeling index (LI) studies. Patients with chronic myelocytic leukemia (CML) during chronic phase were selected for study because the Philadelphia chromosome (Ph) was the only cytogenetic abnormality in each case and, since it is a balanced translocation, the frequently encountered problem of aneuploidy in human neoplastic cells was avoided. Unfortunately, when 30 CML patients were studied simultaneously by DNA histogram analysis and LI studies, the correlation coefficient between the two results was only r = 0.611. A comparison of three different mathematical programs for DNA histogram analysis showed that none was completely satisfactory. We conclude that DNA histogram analysis does not provide the same data as autoradiographically processed labeling index studies even in patients with Ph-positive CML during the chronic phase when the situation is not complicated by additional aneuploidy.
Assuntos
DNA/análise , Citometria de Fluxo/normas , Leucemia Mieloide/patologia , Medula Óssea/patologia , Células Cultivadas , DNA/biossíntese , Humanos , InterfaseRESUMO
The proliferative characteristics of myeloid leukemias were defined in vivo following intravenous bromodeoxyuridine (BrdU). Fifteen patients received a 2-hour infusion of BrdU. A monoclonal anti-BrdU antibody was used to detect the in vivo incorporation of BrdU by S-phase cells. The percentage of S-phase cells obtained from the biopsies (mean 17.3%) was significantly higher (p = 0.00001) than the percentage determined from the aspirates (7.8%). It is concluded that the true estimate of S-phase cells can only be obtained from biopsies following in vivo labeling of cells synthesizing DNA. The persistence of BrdU-labeled cells in follow-up studies can be used to recognize 'residual leukemia', and the subsequent fate of these cells can be defined in vivo.