Cytokine gene transfer in cancer therapy.

New strategies based on gene transfer technology are employed in cancer therapy. Cytokines are polypeptides involved in immunity and inflammation, and essentially control the magnitude of the immune response. Genetically modified tumor cells releasing various cytokines have been shown to enhance tumor immunogenicity and to induce the regression of preexisting tumors. In some instances, immunological memory has been generated to resist the subsequent challenge with unmodified, parental tumor cells. Cytokine gene transfer into antitumor effector cells, as well as antigen presenting cells, is also being investigated to augment antitumor immune responses.

Cloning and sequence analysis of the immediate promoter region and cDNA of porcine granulocyte colony-stimulating factor.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine that stimulates the proliferation and differentiation of hematopoietic progenitor cells committed to the neutrophil/granulocyte lineage. Recombinant G-CSF (rG-CSF) is routinely used in the prevention of chemotherapy-induced neutropenia and in the setting of bone marrow transplantation. Chronic idiopathic and congenital neutropenic disorders also show improvement after rG-CSF injections. Applications of either rG-CSF or G-CSF gene transfected cells into mice give rise to leukocytosis, which can be measured easily. This makes G-CSF a versatile tool for studying systemic effects of therapeutic proteins delivered by genetically modified cells in vivo. Although the biological activity of G-CSF is not species-specific, studies on long-term expression would require the use of species-identical proteins in order to avoid host immune reactions against the foreign gene product. Because of the physiological and immunological similarity of pigs and human, the pig has become an important large-animal model for biomedical research. We have therefore cloned porcine G-CSF cDNA from RNA isolated from pig PBLs. Pig G-CSF is a 195-amino-acid polypeptide that shares a high degree of homology to human (78%), murine (71%) as well as rat (68%) G-CSF. In contrast to human and murine, but not to rat G-CSF, a different ATG translation start codon is used, resulting in a shorter, but still functional signal sequence.

Mutations of polyomavirus VP1 allow in vitro growth in undifferentiated cells and modify in vivo tissue replication specificity.

Mutants of polyomavirus with altered host specificities were isolated in undifferentiated C2 myoblast cells (L. Ricci, R. Maione, C. Passananti, A. Felsani, and P. Amati, J. Virol. 66:7153-7158, 1992). The mutations responsible for this phenotype belonged to either of the two classes: a large duplication of the enhancer region or a 6-bp deletion in the VP1 coding region. Since both classes of mutations enabled the virus to grow in undifferentiated myoblast cells, we investigated their ability to replicate in embryonal carcinoma cells and in various tissues in newborn mice. Our results show that both kinds of mutations confer the ability to replicate in vitro in embryonal carcinoma F9 cells; the VP1 mutants acquired an in vivo host range of replication which is different from that of their original wild-type strain, whereas the mutation in the regulatory region did not alter the in vivo growth spectrum.

Serogroups of Campylobacter jejuni from man and animals.

A total of 186 campylobacter strains from aborted calf and sheep fetuses, from scouring dogs, rabbits and man, and from retailed poultry were isolated and examined biochemically and serologically for heat stable antigens. Immune sera were produced in rabbits against Penner reference strains from 1 to 60, and against two field isolates. Out of 186 biochemically tested strains 179 (96.2%) proved C. jejuni and only 6 (3.2%) C. coli. One strain has been identified as C. laridis. In cattle and sheep 3.2 and 21.7% respectively of all campylobacter abortions were due to C. jejuni infection. The same agent caused 12.7% of diarrhoea of dogs. The campylobacter infection rate of freshly slaughtered and dressed chicken varied between 25 and 64.3%. Out of the serologically examined 140 C. jejuni strains 118 (84.3%) could be assigned to 16 Penner serogroups and 13 (9.3%) to 2 further serogroups. Serogroups 8 (31.4%), 1 (19.3%) and 2 (12.1%) occurred most frequently. The human isolates represented the widest serotype distribution, as 32 tested strains belonged to 12 serogroups. All those serogroups which caused abortion or diarrhoea in animals or were isolated from poultry carcases were isolated also from man with diarrhoea, but some serogroups were found only in man.

Serogroups of Campylobacter fetus and Campylobacter jejuni isolated in cases of ovine abortion.

Of 38 aborted ovine fetuses from 23 sheep flocks 29 C. fetus subsp. fetus and 22 C. jejuni were isolated and examined biochemically and serologically for heat-stable antigens. Serologic examinations were carried out by passive haemagglutination test. In case of C. fetus subsp. fetus strains alkaline antigen extraction was used. Antisera to two serogroups of C. fetus and to Penner serotype reference strains 1 to 60 were produced in rabbits. Abortion was caused in 18 (78.3%) flocks by C. fetus subsp. fetus and in 5 (21.7%) flocks by C. jejuni. Six C. fetus subsp. fetus strains grew well at both 43 and 25 degrees C. With one exception all C. fetus subsp. fetus were resistant, whereas all 29 C. fetus subsp. venerealis strains were sensitive to 30 micrograms/ml cefoxitin and cefamandole. These two cephalosporins can be used to differentiate the two subspecies of C. fetus. Passive haemagglutination test using alkaline antigen extraction is a proper method for the examination of heat-stable antigens of both C. fetus subspecies. Out of 24 C. fetus subsp. fetus strains 13 belonged to serogroup A(01), and 11 to serogroup B(02). C. jejuni strains examined belonged to Penner serogroup 1 (6 strains), to serogroup 5 (4 strains) and to serogroup 8 (4 strains).