myc-related proteins and DNA sequences in Trypanosoma brucei.

The cAMP content of Trypanosoma brucei increases in parallel with ascending mammalian parasitemia to very high levels just before differentiation of the long-slender to the short-stumpy bloodstream form. Because expression of myc oncogenes is required for vertebrate cells to interpret proliferation signals and declines in response to cAMP mediated differentiation, we investigated whether T. brucei also harbored myc-like proteins and genes. Accordingly, we probed lysates of long-slenders, short-stumpies and procyclics (insect midgut stage) with antibody to myc proteins and also hybridized myc gene family sequences to procyclic DNA. We found that antibody to myc-family proteins of mammals reacts with 40 kDa and 55 kDa proteins in all three life cycle stages, and that procyclic DNA contains three EcoRI fragments that are homologous to a v-myc probe. One of these fragments also hybridizes to a synthetic 25-mer oligonucleotide deduced from a consensus sequence in the second exon of the myc family and expresses a 3.2 kb mRNA transcript in Northern blots of procyclic RNA. The conservation of myc-family homologous across the broad phylogenetic gap between mammals and trypanosomes illustrates ancient evolutionary relationships and raises the possibility of stage-specific expression of myc genes during the life cycle of T. brucei.

Effect of theophylline on differentiation of Trypanosoma brucei.

Differentiation of Trypanosoma brucei in the mammal limits the degree of parasitemia and prepares the trypanosome for passage back into the tsetse fly. In an attempt to define the signals that control differentiation, we found that theophylline, in contrast to indomethacin, blocked differentiation, prolonged parasitemia, elevated prostaglandin and cyclic AMP concentrations of rat plasma, and depressed intratrypanosomal cyclic AMP. Relatively nontoxic drugs that alter differentiation are powerful tools for elucidating the events that control this important process.

Growth and antigenic variation of Trypanosoma brucei, T. rhodesiense and T. gambiense in subcutaneous millipore chambers.

The inability to cultivate infective bloodstream forms of the African trypanosomes in cell-free media has complicated studies of the biology of trypanosomes and the pathogenesis of trypanosomiasis. We attempted to overcome this problem by subcutaneous implantation in mice of Millipore chambers that isolate trypanosomes from cells but permit diffusion of soluble substances across their membranes. Chambers were inoculated with 5 X 10(4) to 5 X 10(5) per ml Trypanosoma brucei, T. rhodesiense or T. gambiense; the trypanosomes multiplied rapidly, persisted for as long as five weeks, and remained infective, even when the original inocula were freed of donor cells by ion-exchange. The presence of anti-trypanosomal IgG and IgM in the sera and chambers of recipient mice proved that trypanosomal and mammalian products crossed the membranes. Chamber trypanosomes also expressed two important aspects of normal in vivo biological behaviour: (i) differentiation from long slender to short stumpy bloodstream forms and (ii) antigenic variation. Death of trypanosomes was associated with the presence of IgM antibody in the chambers. This model provides a system for study of an entire population of trypanosomes in an extravascular, cell-free environment.

Cultured human endothelial cells elaborate neutrophil chemoattractants.

The recruitment of circulating neutrophils to acutely injured lungs might be regulated by vascular endothelial cells. Conditioned media from confluent monolayers of endothelial cells derived from human umbilical veins were assayed for neutrophil chemotactic activity by the Boyden chamber technique. Among 20 confluent monolayers tested, 14 (70%) elicited chemotactic activity. The generation of chemotactic activity was time dependent. In four experiments, the chemotactic index at 6 hours of incubation was 3.7 +/- 0.4, at 24 hours was 5.0 +/- 0.5, and at approximately 48 hours was 6.6 +/- 0.6. Chemotactic activity was detectable in dilutions of 1:4 to 1:8. To characterize the factors in conditioned media responsible for enhanced neutrophil migration, pooled conditioned media from confluent endothelial cell monolayers eliciting maximal chemotactic activity (chemotactic index = 7.46 on pooled sample) were chromatographed on a Biogel A-0.5m column. Two column fractions corresponding to molecular weights of 35,000 and approximately 1500 daltons enhanced neutrophil migration. No migration-enhancing column effluents were observed from unconditioned medium. Both of the active column fractions from conditioned media were found to be chemotactic by checkerboard analysis. Activity in the 35,000-dalton peak was abolished by either treatment with Pronase or heat (56 degrees C X 30 minutes) but was not extracted into chloroform-methanol. Conversely, chemotactic activity in the 1500-dalton peak resisted heat and proteolysis but was extractable into chloroform-methanol. These data indicate that cultured human endothelial cells generate neutrophil chemoattractants, and that at least two chemoattractants, a 35,000-dalton protein and a 1500-dalton lipid, are generated. Our findings suggest that endothelial cells have the capacity to recruit neutrophils to vascular surfaces and may thereby initiate or modulate the inflammatory response.

Immunotherapy of a murine colon cancer with syngeneic spleen cells, immune RNA and tumor antigen.

In combination with xenogeneic immune RNA (l-RNA) and tumor antigen (TA), syngeneic spleen cells inhibited the growth of a N-methyl-N-nitrosourethane-induced colon carcinoma (CT-26) in BALB/c mice. The sequential administration of 1 X 10(7) spleen cells, l mg of anti-CT-26 l-RNA and 0.4 mg of CT-26 TA resulted in a 20% survival rate of mice bearing established CT-26 tumors. On the other hand, administration of the spleen cells only, l-RNA only or TA only was not effective in inhibiting tumor growth. Similarly, when any one of the three agents (l-RNA, TA and spleen cells) was omitted, no anti-tumor effect was obtained. A higher dose (1 X 10(8) and a lower dose(1 X 10(6) of spleen cells decreased the anti-tumor effect of this combination therapy. A higher dose (2 mg) and a lower dose (0.5 mg) of l-RNA, as well as a higher dose of TA (0.8 mg) had no influence on the anti-tumor effect. However, a lower dose of TA (0.2 mg) in combination with spleen cells and l-RNA decreased the anti-tumor effect. When all three agents were administered at higher or lower doses, no anti-tumor effect was obtained. When mice bearing CT-26 tumors were treated with spleen cells, plus l-RNA directed against a syngeneic but antigenically different tumor (BP/B/5) and BP/B/5/TA, with spleen cells plus CT 26 I-RNA and BP/B/5TA, or with spleen cells plus BP/B/5 l-RNA and CT-26 TA, no anti-tumor effect was observed. These results indicate that the anti-tumor responses observed were due to tumor-specific immune responses. In conclusion, in order to obtain growth retardation or regression of established CT-26 tumor transplants, the sequential administration of all three agents (spleen cells, l-RNA and TA) was required. The optimal doses of each agent were found to be 1 X 10(7) spleen cells, 0.5 mg or 1 mg of l-RNA and 0.4 mg of tumor antigen. Higher or lower doses decreased the anti-tumor effect. Tumor-specific immune reactions appeared to be involved.

Enhancement of metastasis development by BCG immunotherapy.

When BALB/c mice bearing growing transplants of a syngeneic colon carcinoma--Colon Tumor 26 (CT 26)--were treated with Bacillus Calmette-Guerin (BCG), no effect on tumor growth rate or survival time was observed compared to untreated controls. However, after excision of primary tumor transplants, enhanced development of lung metastases was noted in the BCG-treated mice, resulting in both as increased mortality rate (from metastatic disease) and a shorter survival time.

Immunogenicity of chemically induced murine colon cancers.

The antigenicity and immunogenicity of three colorectal carcinomas induced in BALB/c mice by 1,2-dimethylhydrazine or N-methyl-N-nitrosourethan were studied. All tumors were readily transplantable. Two of these tumors metastasized when transplants reached sufficient size. All tumors were found to be immunogenic in the strain of origin, and all tumors were shown to contain unique tumor-specific transplantation antigens in cross-protection experiments. The use of these tumors as an animal model for studies of adjuvant immunotherapy and chemoimmunotherapy is suggested.

Thymectomy does not affect tumour rejection and cytophilic activity in a murine allogeneic lymphoma model.

An experimental model was devised in which an AKR lymphoma is conditioned to grow in BALB mice leading to reproducible tumor incidence which makes tumor-bearing (progressor) and tumor-rejecting (regressor) animals simultaneously available. The object of this paper was to determine the effect of neonatal thymectomy (xT) on allogeneic tumor incidence and on anti-tumor cytophilic activity. The latter was determined by the adherence of lymphoma cells to guinea pig peritoneal macrophages previously incubated with preheated mouse serum. The results obtained, in 2-3 month old animals, show no difference in lethal tumor incidence between xT and intact mice, 36% (14/39) vs. 39% (14/36). Neither did xT alter the significant increase in cytophilic antibodies detected in regressor serum, 115 +/- 15 (S.E.) vs. 106+/- 22 0/00 macrophages bearing tumor cells as compared to control values in either xT or normal serum, 53 +/- 3 vs 52 +/- 3. This background cytophilic activity was not significantly altered in progressor serum of either xT or intact mice, 36 +/- 5 vs 65 +/- 6. The specificity of the antitumor cytophilic antibodies was determined by the negative results obtained when a different tumor was used as target cell. It can be concluded that ant-tumor cytophilic antibodies are detectable in regressor but not in progressor serum. Thymectomy in this model does not alter either in vivo tumor incidence or humoral cytophilic activity. Since no thymic remnants were encountered at autopsy, it is postulated that AKR lymphoma cells, which proved to be neoplastic T cells, are capable of rendering T-immunocompetent a thymectomized allogeneic BALB host.

Antigenic differences between AKR lymphoma and thymus cells leading to detection of a tumor antigen associated with immunological enhancement.

In an experimental model conditioning for enhancement, an AKR lymphoma was made to grow in BALB/c mice, permitting the simultaneous comparison of tumor-bearing (progressor) and tumor-rejecting (regressor) animals. By immunofluorescence using as target AKR lymphoma and normal thymus cells, both acetone-fixed and unfixed, it was observed that the allogeneic progressor serum contained three antibodies, two of which could be asborbed by thymocytes while the other combined selectively with the acetone-fixed lymphoma target. This tumor-specific antibody could not be detected in regressor serum which, on the other hand, could be completely absorbed by thymocytes. The identification of this acetone-resistant tumor antigen led to the preparation of aceton-treated acellular lymphoma extracts: a precipitate was obtained which upon inoculation in BALB/c mice produced an antiserum that combined selectively with lymphoma targets. In vivo experiments showed that pretreatment with this antigen led to a significant increase in allogeneic tumor incidence, 76% as compared to 37% in the controls. It is concluded that in this allogeneic model, an acetone-resistant tumor-specific antigen and the corresponding antibody are involved in tumor enhancement.

Active immunological enhancement of sarcoma 180 in splenectomized Swiss mice.

Maximal enhancement of tumor growth was obtained in Swiss mice, with or without spleen, inoculated with S180 within a subcutaneously implanted glass cylinder and pretreated with soluble tumor antigen.

Conditions favoring immunological enhancement of a murine allogeneic lymphoma.

An in vivo model was used which permits the growth of AKR lymphoma allografts inoculated within a glass cylinder subcutaneously implanted in BALB mice. Pretreatment of the host with acellular tumor extracts or tumor cells enclosed within a diffusion chamber significantly increased tumor incidence. On the contrary, donor spleen extracts did not alter tumor incidence while viable spleen cells within a diffusion chamber even prevented tumor development. It can be concluded that in this model a condition of maximal tumor enhancement can be attained with soluble tumor antigen but not with normal spleen extracts.