Purified hybrid cells from dendritic cell and tumor cell fusions are superior activators of antitumor immunity.

The use of fusions between dendritic cells (DCs) and tumor cells as vaccines has been proved very effective in stimulating antitumor immune responses, both in animal studies and in early human clinical trials. Because of the difficulty of purifying the hybrid cells from the fusion, fusion mixtures were used in these studies. Recently, we developed a technique using fluorescent-dye staining and fluorescence-activated cell sorting that enabled the hybrid cells to be instantly purified from the fusion mixture. In the present study, the hybrid cells were purified from a fusion between mouse DCs and B16F0 melanoma tumor cells using the new technique. The purified cells, named instant dendritomas (IDs) were then compared with fusion mixtures in stimulating antitumor immune responses. The results from cytotoxicity assays, interferon-gamma production and in vivo lung tumor metastasis demonstrated that IDs are more effective than fusion mixture in stimulating antitumor immunity. Meanwhile, there was no significant difference in the antitumor immunities activated by IDs from allogenic fusion or IDs from syngenic fusion.

Murine tyrosinase expressed by a T7 vector in bone marrow-derived dendritic progenitors effectively prevents and eradicates melanoma tumors in mice.

Dendritic cell (DC)-mediated cancer immunotherapy is a very promising alternative approach to cancer treatment. In a previous study, we successfully transfected bone marrow-derived dendritic progenitors (BMDDPs) with a T7 vector--a nonviral, cytoplasmic-based autogene expression system--encoding a model tumor antigen, firefly luciferase, and subsequently stimulated the transfected cells to differentiate into DCs. When injected into experimental mice, those DCs generated a strong immune response against tumor cells bearing luciferase, which not only prevented occurrence of metastasis but also eradicated existing tumors. In the present study, we constructed a T7 vector encoding mouse tyrosinase, a well--known melanoma associated tumor antigen, and used it to transfect BMDDPs. Reverse transcriptase polymerase chain reaction and Western analysis confirmed the expression of tyrosinase by DCs differentiated from transfected BMDDPs. Two immunizations of these DCs at a dose of 2 x 10(6) of each successfully prevented tumor growth. More importantly, one injection of 2 x 10(6) of these DCs into mice followed by five doses of recombinant human interleukin-2 administration effectively eradicated existing tumors as indicated by pulmonary metastasis assay.

Rapid generation and flow cytometric analysis of stable GFP-expressing cells.

Expression of green fluorescent protein (GFP) represents a unique method for the fluorescent labeling of viable mammalian cells, with many potential applications. The studies detailed in this report examine the detection of GFP expression in murine cells using flow cytometry. Direct comparison of NIH 3T3 cells transiently expressing GFP or GFPS65T, a mutant form of GFP, showed that GFPS65T fluorescence (using 488 nm excitation) was detected more readily than fluorescence from wildtype GFP. Efficient generation of cell lines that stably expression GFPS65T was achieved using a plasmid vector that encoded a hygromycin phosphotransferase/GFPS65T fusion protein. Flow cytometric detection of NIH 3T3 cells expressing this fusion protein was improved using a 510/20 band pass filter instead of the standard filter setup for fluorescein detection. Additionally, staining the surface of these cells with phycoerythrin, RED 670, or allophycocyanin did not interfere with the detection of GFPS65T fluorescence, indicating that multiparameter analyses using GFPS65T fluorescence are possible. Importantly, we also observed that GFPS65T expression could be detected in NIH 3T3, BW5147, or freshly cultured Thy1lo CD3- murine bone marrow cells transduced with a retroviral vector encoding the fusion protein, suggesting that the potential applications of this system may be quite broad.

[Evaluation of 5-year surgical treatment outcome in patients with vulvar tumors]. / Ocena 5-letnich wyników leczenia chirurgicznego chorych na raka sromu.

A retrospective study of all patients with carcinoma of the vulva treated by surgery and/or radiation therapy, between 1980 and 1985, is reported. Sixty eight patients were analyzed for survival, recurrence patterns, complications and clinical features. The stages of disease were as follows: Stage I or II 49 pts, Stage III 13 pts and Stage IV 6 pts. The 5-year survival rate was 45.2%. Recurrences were diagnoses in 25% of the patients. With the less aggressive surgical approach used, combined with radiation therapy to eradicate subclinical disease, the morbidity rate was acceptable and the survival rate comparable to the reported after more aggressive surgery.

Reliable method for the simultaneous detection of cytoplasmic and surface CD3 epsilon expression by murine lymphoid cells.

During their development, T-cell precursors (pre-T cells) undergo a variety of changes with respect to their expression of specific surface proteins. Among the most critical of the surface markers acquired by developing T cells is the T-cell receptor (TCR)/CD3 complex. Prior to the assembly and transport of complete TCR/CD3 multimeric complexes to the plasma membrane, the individual constituent subunits are expressed in the cytoplasm (ER-Golgi). In order to study the expression of the T-cell receptor TCR/CD3 complex during pre-thymic T-cell differentiation, we have developed a flow cytometric technique for the simultaneous detection of surface (sCD3 epsilon) and cytoplasmic CD3 epsilon (cCD3 epsilon). This technique, which employs fixation in 1% paraformaldehyde and permeabilization with 1% saponin and 0.025% digitonin, features reliable internalization and low nonspecific binding of anti-CD3 epsilon in murine lymphoid cells, as well as tissue culture cell lines. The combination of saponin and digitonin treatment was also compatible with the staining of sCD3 and other lymphocyte surface antigens such as Thy1, CD4, CD8, B220, and IgM. In contrast, permeabilization of cells with the detergents Tween 20 and Triton X-100 was shown to remove surface-bound anti-CD3 epsilon. The present technique permitted the detection of discernible sCD3 epsilon and cCD3 epsilon double and single positive lymphocytes and may prove useful in defining bone marrow-resident pre-T cells.

Cloning and characterization of five novel Dictyostelium discoideum rab-related genes.

Low-M(r) GTPases belonging to the Ras superfamily are known to regulate a wide range of cellular processes including cell proliferation, actin cytoskeleton organization, and vesicular trafficking along the secretory and endosomal/lysosomal pathway. We are studying the regulation of lysosomal and endosomal vesicular trafficking in the simple eukaryote, Dictyostelium discoideum. Using an oligodeoxyribonucleotide (oligo) encoding one of the most highly conserved amino acid (aa) regions found in the low-M(r) GTPases (important in GTP binding), we have cloned 18 new cDNAs encoding proteins belonging to the Ras superfamily. In this report, we describe the characterization of five of these cDNAs coding for proteins belonging to the Ypt1/Sec4/Rab family; mammalian members of this family have been shown to function in the regulation of vesicular trafficking. Two of the cDNAs, rab1A and rab1B, code for proteins highly homologous to mammalian Rab1. An additional cDNA, rabA, codes for a protein that is only 60% identical to Rab1 at the aa sequence level and probably represents a new member of the rab gene family. Finally, two cDNAs, rabB and rabC, code for novel proteins belonging to the Rab gene family that are no greater than 50% identical in aa sequence to any previously described member. Southern blot analysis indicated that rab1A and and rab1B belong to a small Dictyostelium family of at least five related genes, while rabA belongs to a different and smaller family of related genes. In contrast, rabB and rabC appear to be represented by single genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and characterization of seven novel Dictyostelium discoideum rac-related genes belonging to the rho family of GTPases.

Cellular processes including proliferation, organization of the actin cytoskeleton, vesicular traffic and secretion of proteins comprising the lysosomal/endosomal system are regulated by low-molecular-weight GTP-binding proteins of the Ras superfamily. However, to date only three Dictyostelium discoideum ras-like genes and two ypt-1/sec4-like genes have been identified and characterized. We report here the identification (using an oligodeoxyribonucleotide probe) of seven additional cDNAs coding for members highly related to the Rac proteins (Ras-related-C3 botulinum toxin substrate) which belong to the Rho (Ras homologous) family of GTPases. Three of these rac-related genes (rac1A, rac1B and rac1C) predict proteins with > 90% amino acid (aa) sequence identity with each other and > 80% identity to the human rac1 gene product, whereas the other members (racA, racB, racC and racD) predict proteins with 46-74% identity to the rac1 and rhoA gene products and to each other. The D. discoideum proteins were entirely conserved over the four regions known to be important for GTP binding and all contained the C-terminal CAAX aa motifs shared by other Rho proteins. Interestingly, the D. discoideum rac-related genes revealed unique patterns of expression during growth and development. For instance, the steady-state level of rac1 mRNA, encoded by three highly related genes, increased transiently during aggregation and then rapidly decreased. In contrast, the cellular abundance of mRNAs encoded by the other rac-like genes decreased at different rates and to different levels during development from the peak levels observed during growth. This suggests that the GTP-binding proteins encoded by these genes may play unique roles during the different stages of the D. discoideum life cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Etiological factors in invasive corpus uteri carcinoma.

The case-control method was applied in order to test how various types of diet as well as past diseases, tobacco smoking and occupational exposure may affect the risk of incidence of corpus uteri cancer in the population of natives and among immigrant women. The highest risk of incidence was noted in the group of natives persistently using a diet rich in meat, animal fat, amylum meals and sugar but lacking raw vegetables. Such a high risk was not observed in the group of immigrant women what might be caused by more frequent change of the type of diet. Some past diseases (arterial hypertension, diabetes, diseases of organs of reproduction and urinary system) do affect a relatively high risk of corpus uteri carcinoma in both populations. However, no noteworthy results have been obtained in the risk of corpus uteri carcinoma as far as tobacco smoking and occupational exposure are concerned.

Cysteine proteinase changes during macrocyst formation in Dictyostelium mucoroides.

Cysteine proteinases were detected in vegetative myxamoebae of Dictyostelium mucoroides DM7 using chromogenic substrates and by electrophoretic analysis (gelatin-SDS-PAGE) which revealed three enzymes, dmCP30, dmCP35 and dmCP46 (a minor form). During the initial stages of macrocyst formation the cysteine proteinaes were secreted and disappeared almost completely from the cells. High extracellular levels of activity towards N-benzoyl-L-prolyl-L-phenylalanyl-L-arginine 4-nitroanilide and of dmCP30 persisted throughout macrocyst development. Three new intracellular proteinases, dmCP31, dmCP36 and dmCP40, were produced as macrocysts formed but their activity was only detected by gelatin-SDS-PAGE. Their appearance was specific to the developmental pathway leading to macrocyst formation. This is the first direct evidence for the accumulation of cysteine proteinases during a developmental process in a cellular slime mould.