Synthesis and in vitro study of the anticancer activity of new analogs of octreotide.

Based on the structure of Octreotide (SMS 201-995) some modified at positions 5 with Dap (diaminopropanoic acid), Dab (diaminobutanoic acid) and Orn new C-amide analogs were synthesized. The Thr6 was replaced by unnatural amino acids Tle (t-leucine). The cytotoxic effects of the novel compounds were tested in vitro against a panel of human tumor cell lines. All investigated compounds exhibited different concentration-dependent antiproliferative effects against the HT-29, MDA-MB-231, HepG2 and HeLa cell lines after 24 h treatment. The compound 2 (D-Phe-c(Cys-Phe-D-Trp- Dap-Tle-Cys)-Thr-NH2) had antiproliferative effects on MDA-MB-231 cells with the IC50 0.03 mM. The HeLa and HepG-2 cells were most sensitive towards tested compounds at various concentrations. Results demonstrated that the peptide analogs 3 (D-Phe-c(Cys-Phe-D-Trp-Lys-Tle-Cys)-Thr-NH2), 4 (D-Phe-c(Cys-Phe-D-Trp-Orn-Tle-Cys)-Thr-NH2) and 5 (RC-102) exert the most pronounced inhibition of the cell vitality up to 77% at higher concentrations and were not toxic to the normal Lep-3 cells.

Influence of purified saponin mixture from Astragalus corniculatus Bieb. on phagocytic cells in Graffi-tumor bearing hamsters.

A purified saponin mixture (PSM) from Astragalus corniculatus Bieb. protected significantly hamsters against the experimental Graffi myeloid tumor. The application of PSM increased the survival rates, prolonged mean survival time and the tumor growth was markedly reduced. A purified saponin mixture (PSM) of Astragalus corniculatus Bieb. was evaluated for its immunostimulating potentials on the phagocytic cells in Graffi-tumor bearing hamsters. The number, migration and phagocytic indexes of peritoneal macrophages (pMøs) and of blood polymorphonuclear leukocytes (PMNs) were evaluated in healthy and Graffi-tumor bearing hamsters (G-TBH) treated with PSM. It was established that the Graffi myeloid tumor induced suppression of the phagocytic abilities of pMøs and PMNs. The number and migration of pMøs was significantly decreased during the whole period of observation. All tested parameters-number, migration and phagocytic activities of pMøs, as well as phagocytic ability of PMNs increased significantly in healthy and G-TBH after i.p. application of the 50 mg/kg body weight PSM. The PMS extracts from Astragalus corniculatus Bieb. are isolated and examined and their immunostimulating and immunorestorating impact on phagocitic cells was proven for the first time. This effect could be due to their high content of purified saponins.

Pretreatment of guinea-pigs with iron or desferal influences the course of Yersinia enterocolitica infections.

The effects of iron excess and desferrioxamine in pretreated guinea-pigs on the immune response (production of Yops) and on the histological changes in infections with Yersinia enterocolitica 0:3 and Y. enterocolitica 0:8 were investigated. The prior overload of the guinea pigs with Dextrofer or treatment with Desferal increased the pathogenic activity of Y. entercolitica 0:3 and led to a generalized infection. Immunoblot analysis showed that in conditions of iron overload the expression of outer membrane proteins (Yops) of Y. enterocolitica 0:8 was blocked. This was accompanied by weak changes in the tissues. The iron limited conditions stimulated production of a low molecular weight protein (17 kDa) on day 6 and easier proliferation of the bacterium. This in vivo study intends to show that in Y. enterocolitica infections a leading role is played not only by iron itself but also by the bacterial strain.

Arthritis after experimental infection with Yersinia enterocolitica 0:3 in rabbits.

Arthritis in rabbits was caused after experimental oral infection with Yersinia enterocolitica (serotype 0:3, biotype 4, pYV+). Clinical and laboratory signs, bacterial dissemination to the viscera, immune response and morphological findings were studied from day 1 to day 40 post-infection (p.i.). Augmentation of body temperature and erythrocyte sedimentation rate occurred on day 1, and on day 8 p.i. was accompanied by leucopenia. The number of alveolar macrophages was increased up to the 15th day p.i., in contrast to peritoneal macrophage numbers. Extensive bacterial colonization of the internal organs was detected at necropsy until the end of the experiment. Analysis of the cell immune response revealed activation of B cells in peripheral blood, spleen and thymus as well as augmentation of T-cell number in the lymphoid organs examined on days 15, 28 and 40 p.i. Histological changes typical of a generalized infection, such as purulent meningoencephalitis, catarrhal pneumonia and lymphadenitis, were observed. Clinical and morphological manifestations of arthritis were also established. The results obtained show that Y. enterocolitica (serotype 0:3, pYV+) induces a generalized, non-lethal infection in Chinchilla rabbits, complicated by arthritis.

Investigations on the aminoacid content of tumor associated antigens of rat sarcoma cells induced by virus.

In our previous work (Alexandrov et al., 1996) was reported that the rat sarcoma cells induced by SR-RSV express two tumor associated antigens (TAA). The one TAA has a molecular weight of 52 kD and is detected by the help of a monoclonal antibody 2C2 only on the outer side of the plasma membrane of the sarcoma cells. The other antigen, with molecular weight of 28 kD, is expressed on the outher and inner side of the membrane. The antigens were isolated as a pure fraction by polyacrylamide electrophoresis and prepared for aminoacid analysis after that. The consisting 16 bound aminoacids were in different amounts. Both antigens are rich in glycine and poor in aromatic and sulphur-containing aminoacids. The presence of glucosamine and galactosamine in the antigens proves their glycoprotein nature. The received data show that the both TAA-s differ not only in molecular weights, place of expression and functional activity, but also in the amount of the bound aminoacids which constitute their proteins.

Experimental mixed infection with Yersinia enterocolitica and Listeria monocytogenes in guinea pigs.

The pathogenesis and the cell immune response (CIR) of guinea pigs after mixed infection with Y. enterocolitica and L. monocytogenes was investigated. The guinea pigs were infected per os with 1.1 x 10(9) CFU Y. enterocolitica 0:3, (pYV+) and four days later with 1.1 x 10(9) CFU L. monocytogenes 4B. Clinical, paraclinical and morphological findings attending the infectious process were followed in dynamics up to the 28th day post infection (p.i.) with L. monocytogenes. The phagocyting activity of alveolar macrophages (aMa) was suppressed against Y. enterocolitica, in contrast to peritoneal macrophages (pMa) engulfing yersiniae more actively at the end of the study. Moreover, the tendency of augmented entering in both phagocytes of L. monocytogenes cells was well demonstrated, starting at the earlier intervals of examination. Histopathological studies showed a purulent meningoencephalitis and a catarrhal pneumonie, non-reactive micronecroses in the spleen and lymphadenitis catarrhalis in the mesenteric lymph nodes. Analysis of the T-cell immune response (T-CIR) showed maximal values in the spleen lymphocytes after Y. enterocolitica and L. monocytogenes mixed infection. The B-CIR occurred early (at the 7th day p.i.) and was maximal at the 28th day p.i. in blood lymphocytes. The results obtained demonstrated that the mixed infection of guinea pigs with Y. enterocolitica and L. monocytogenes runs has a non lethal, generalized illness with a dominant role of L. monocytogenes cells.

Immunological and electronmicroscopic studies in pigs infected with Yersinia enterocolitica 0:3.

The cellular immune response after an experimental oral infection with Yersinia enterocolitica (serotype 0:3, biotype 4, harbouring the virulence plasmid-p YV) was studied in pigs. A maximal stimulation of the T cell population in the thymus, spleen and peripheral blood was stated on the 15th day post infection (p.i.) by the rosette forming cell (RFC) test. The hemolysins (produced by B cells and detected by the plaque forming cell test-PFC) were significantly increased on the 15th day p.i. among the thymus and spleen lymphocytes and on the 25th day p.i. among the blood lymphocytes. Blood and thymus lymphocytes were activated faster by the infectious agent in comparison to the spleen cells. The electronmicroscopic studies revealed an intracellular presence of the bacteria in alveolar macrophages (aMa) and peritoneal macrophages (pMa) as well as in Peyer's patches and tonsils as early as on the 4th day p.i. Extracellularly located bacteria were observed, too. The results have shown that inspite of the activation of T and B cell immune response, this infectious agent persisted in the porcine organism.

Amino acid investigations of tumor-associated antigen of Mc31 transformed cells.

There are numerous evidences (mainly indirect) for the presence of tumor-associated surface antigens (TASA) in cells transformed by avian oncornaviruses. Hayami, et al. (1977) and Ignjatovic et al. (1978) established that such antigens on avian leukosis virus (ALV) cells have neither virus, nor oncofetal origin. On leukosis virus strain Mc31-transformed turkey cells, Filchev and Ivanov (1986) and Wesselinova (1989, 1991) also describe a TASA. But there are only a few studies on the nature and properties of such antigens (with proven antigenic properties). A TASA, induced by avian tumor viruses with MW of about 42,000 Daltons have been established on hamster cells (Aupoix et al., 1974), but there has been no further examination of it. Bauer et al. (1979) only showed that a TSSA (tumor specific surface antigen) on avian virus-transformed cells is a glycoprotein. The protein fraction we isolated from Mc31-transformed turkey tumor cells (Wesselinova, 1994) is the first attempt to characterize such purified TASA on chicken cells. After we demonstrated that it is a protein with low MW (about 14,000 Daltons) possessing antigenic properties and that it is the specific for the tumor cells, we decided to investigate its amino acid content.