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Multixenobiotic resistance (MXR) is an important mechanism of cellular efflux mediated by ATP binding cassette (ABC) transporters that bind and actively remove toxic substrates from the cell. This study was the first to identify ABC transporter P-glycoprotein (P-gp/ABCB1) as a representative of the MXR phenotype in earthworm (Eisenia fetida). The identified partial cDNA sequence of ABCB1 overlapped with ABCB1 homologues of other organisms from 58.5 % to 72.5 %. We also studied the effect of five modulators (verapamil, cyclosporine A, MK571, probenecid, and orthovanadate) on the earthworm's MXR activity by measuring the accumulation of model substrates rhodamine B and rhodamine 123 in whole body tissue of the adult earthworm. MK571, orthovanadate, and verapamil significantly inhibited MXR activity, and rhodamine 123 turned out to better reflect MXR activity in that species than rhodamine B. Our results show that E. fetida can serve well as a test organism for environmental pollutants that inhibit MXR activity.

The immunological structure of the porcine jejunal lamina propria in germ-free piglets was compared with that of their counterparts associated with two strains of commensal Escherichia coli, A0 34/86 serotype O83:K24:H31 and the O86 E. coli strain, up to 20 days post-colonization. In the antigen-presenting compartment, both dendritic cells (DC) and cells expressing CD163, probably macrophages were investigated. In addition we also assessed the number of CD2+/CD3+ (T) cells. In contrast to some previous reports, we show a total lack of both DC and T cells for germ-free animals in the diffuse lymphoid tissue of villi and crypts of the jejunum. Association with either strain of commensal E. coli had a profound effect on the immune structure and resulted in extensive recruitment of DC to the lamina propria and of T cells to epithelium and lamina propria. The data suggest that the earliest immigrant cells were monocytes, which soon acquired the phenotype of mucosal DC. T cells migrated in at a slightly slower rate. Nevertheless, the response could be extremely rapid: within 3 days of colonization with O83, the magnitude of this response was comparable to that observed 20 days post-colonization.

Many dietary constituents are chemopreventive in animal models, and experiments with cultured cells are revealing various potential mechanisms of action. Compounds classified as blocking agents can prevent, or greatly reduce, initiation of carcinogenesis, or suppressing agents can act on cell proliferation. Caffeic acid (CA) and caffeic acid phenethyl ester (CAPE), members of the polyphenolic compounds, are present in high concentrations in medicinal plants and propolis, a natural beehive product. A water-soluble extract of propolis (WSDP) and two components of propolis, CA and CAPE were investigated for direct antitumor activity in vivo and in vitro. The local presence of CA and CAPE in the tissue caused a significant delay in tumor formation and increased life span 29.30 to 51.73%, respectively. CA and CAPE, but not WSDP, significantly suppressed human HeLa cervical carcinoma cell proliferation in vitro. Based on these results, we postulate that the antitumor activity of polyphenolic compounds includes direct cytotoxic effects on tumor cells.

Polyphenolic compounds are widely distributed in the plant kingdom and display a variety of biological activities, including chemoprevention and growth inhibition of tumours. Propolis contains a conglomerate of polyphenolic compounds. We investigated the effect of propolis and polyphenolic compounds, components of propolis, on the growth and metastatic potential of a transplantable mammary carcinoma (MCa) of the mouse. Metastases in the lung were generated by 2 x 10(5) tumour cells injected intravenously (i.v.). A water-soluble derivative of propolis (WSDP) and the polyphenolic compounds (caffeic acid (CA) and caffeic acid phenethyl ester (CAPE)) were given to mice perorally before or after tumour cell inoculation. WSDP, CA and CAPE reduced the number of metastases in the lung. This implies that the antitumour activities of the compounds used in these studies are mostly related to the immunomodulatory properties of the compounds, their cytotoxicity to tumour cells, and their ability to induce apoptosis and/or necrosis.

The effect of polyphenolic compounds isolated from propolis and propolis itself was investigated on the growth and metastatic potential of a transplantable mammary carcinoma (MCa) of CBA mouse. Metastases in the lung were generated by intravenous injection of tumor cells (2 x 10(5)). A water-soluble derivative of proplis (WSDP), caffeic acid (CA), caffeic acid phenethyl ester (CAPE) and quercetin (QU) were given to mice per os before tumor cells inoculation. Tested compounds significantly decreased the number of tumor nodules in the lung. According to the results obtained the antitumor activity of tested compounds can be related to the immunomodulatory properties of the compounds, their cytotoxicity to tumor cells, and their capacity to induce apoptosis and necrosis. The experimental data support that WSDP, CA, CAPE and QU could be potentially useful in the control of tumor growth in experimental models.

Total white blood cell (WBC) counts and percentages of CD4a+, CD8a+, CD5a+, CD45RA+, CD45RC+, wCD21+ and SWC3a+ cells in the peripheral blood of pigs were analysed in this study. Blood samples were collected before and on days 4, 10, 21 and 28 after vaccination. Group 1 pigs were vaccinated with a subunit E2 vaccine (gp E2 32 microg/dose), and Group 2 received a subunit vaccine combined with an attenuated ORF virus strain D1701 10(6.45) TCID50/dose. Control pigs received a placebo. The total WBC count and percentage of particular cell types were within the normal range in vaccinated and control pigs. Although the mechanism of attenuated ORF virus activity is not clear, changes were observed in CD4a+, CD5a+, CD8a+, CD45RA+ and CD45RC+ cells in pigs that received the combination of a subunit vaccine and ORF virus. However, the percentage of wCD21+ and SWC3a+ did not differ significantly from that recorded in pigs given only the subunit vaccine. At days 4 and 10 the number of pigs positive to E2 antibodies was higher in the group that received the subunit vaccine and ORF virus than in pigs vaccinated with the subunit vaccine only. A higher percentage of memory cells (CD45RC+) as well as Th and Tc lymphocytes in pigs that received the ORF virus and the subunit vaccine could be ascribed to a nonspecific influence of the ORF virus on the development (through cognate interactions between T and B cells) and the duration (presumed according to the finding of the clonal expression of memory cells) of humoral immunity (assessed by a higher number of seropositive pigs in this group). This seems likely since the proportion of these cells was found to be lower in the pigs that received E2 vaccine only.

The effect of propolis [it is a water-soluble derivative (WSDP)] and related polyphenolic compounds of propolis (caffeic acid, caffeic acid phenethyl ester and quercetin), honey, royal jelly and bee venom on tumour growth, metastasizing ability and induction of apoptosis and necrosis in murine tumour models (mammary carcinoma and colon carcinoma) was investigated. WSDP and related polyphenolic compounds showed significant anti-metastatic effect (P < 0.01 and P < 0.001) given either before or after tumour-cell inoculation. Oral or systemic application of WSDP or caffeic acid significantly reduced subcutaneous tumour growth and prolonged the survival of mice. Honey also exerted pronounced anti-metastatic effect (P < 0.05) when applied before tumour-cell inoculation (peroral 2 g kg(-1) for mice or 1 g kg(-1) for rats, once a day for 10 consecutive days). Royal jelly did not affect metastasis formation when given intraperitoneally or subcutaneously. However, intravenous administration of royal jelly before tumour-cell inoculation significantly (P < 0.05) inhibited metastasis formation. When mice were given 10(5) tumour cells intravenously immediately after bee venom injection, the number of tumour nodules in the lung was significantly lower (P < 0.001) than in untreated mice or mice treated with bee venom subcutaneously. Local presence of bee venom in the tissue caused significant delay in subcutaneous tumour formation. These findings clearly demonstrate that anti-tumour and anti-metastatic effects of bee venom are highly dependent on the route of injection and on close contact between components of the bee venom and tumour cells. These data show that honey bee products given orally or systemically may have an important role in the control of tumour growth and tumour metastasizing ability.

The possible tumor growth- and metastasis-inhibiting effects of bee venom in mice and in tumor cell cultures were studied. The tumor was a transplantable mammary carcinoma (MCa) of CBA mouse. Intravenous administration of bee venom to mice significantly reduced the number of metastases in the lung. However, subcutaneous administration of bee venom did not reduce the number of lung metastases, indicating that the antitumor effect of the venom could be highly dependent on the route of injection as well as close contact between the components of the venom and the tumor cells, as was shown by in vitro studies on MCa cells. We also observed variations in immunological parameter induced by bee venom. We proposed that bee venom has an indirect mechanism of tumor growth inhibition and promotion of tumor rejection that is based on stimulation of the local cellular immune responses in lymph nodes. Apoptosis, necrosis, and lysis of tumor cells are other possible mechanisms by which bee venom inhibits tumor growth.

Ten pigs, aged 85 days, were vaccinated with a subunit vaccine containing 32 microg of classical swine fever virus glycoprotein E2 (gp E2) (group 1), and a further 10 pigs were vaccinated with a C strain vaccine (10(4+/-0.15) TCID50/ml), produced by amplification in minipig kidney (MPK) cell culture (group 2). Nine non-vaccinated pigs served as a control group (group 3). Serum samples were collected before (day 0) and at 4, 10, 21 and 28 days after vaccination and were analysed by two commercially available enzyme immunoassays and by a neutralizing peroxidase-linked assay (NPLA). At the same times, peripheral blood was taken for determining the total leukocyte count and the body temperature was taken daily. Antibodies were not detected in serum samples collected before vaccination (day 0), and no side-effects that could be connected with vaccination were observed during the trial. Ten days after vaccination 6/10 pigs vaccinated with the subunit vaccine were seropositive. On days 21 and 28, the ratios of serologically positive to vaccinated pigs were 9/10 and 10/10, respectively. Four of the ten pigs that were vaccinated with the C strain vaccine were positive on day 21 and 9/10 on day 28. However, the results of the NPLA showed that only 4/10 pigs had an antibody titre > 1:32 at the end of the trial in both the vaccinated groups, even though the subunit vaccine initiated an earlier and higher level of neutralizing antibodies than the vaccine produced from the C strain. Challenge was performed 28 days after vaccination on four randomly selected pigs from both vaccinated groups. The pigs survived the challenge without showing any clinical signs of classical swine fever (CSF), while two nonvaccinated control pigs died on the 10th and 12th days after infection.

The influence of an attenuated classical swine fever virus C strain vaccine and a subunit E2 vaccine against classical swine fever on the peripheral blood leucocyte proportion and phenotypic expression in 12-week-old pigs was studied. The C strain was amplified in minipig kidney cell culture and final product contained 10(4 +/- 0.15) TCID50/ml, while the subunit vaccine contained 32 microg per dose of gp E2. Haematological findings showed that the vaccines did not cause leucopenia or lymphocytopenia and the number of neutrophils and eosinophils during the observation period was within physiological range. The results of the proportion of CD4a+, CD5a+, CD8a+, wCD21+, CD45RA+, CD45RC+ , non-T non-B, SWC3a+ and CD11b+ cells were gained by single-colour flow cytometry. At the end of the trial a significantly increase of percentage of CD4+, CD5a+, CD8+, wCD21+ cells has been found in pigs that received the subunit vaccine and the percentage of CD4+, CD5a+, CD8+, CD45RA+ and CD45RC+ cells was higher in pigs that received the attenuated vaccine. Twenty-eight days after vaccination the percentage of CD4+, CD45RA+ and CD45RC+ was significantly higher in pigs vaccinated with the C strain than in pigs vaccinated with the subunit vaccine. In contrary, the percentage of the wCD21- cells was higher in pigs that received the subunit vaccine. Statistically higher values of SWC3a+ and lower values of CD11b+ cells was observed in pigs that received the attenuated vaccine than in pigs vaccinated with the subunit vaccine. Taken altogether, our results showed that the subunit vaccine produced a better stimulation of B cells and CD11b+ monocytes/macrophages /granulocytes/NK cells, whereas the attenuated vaccine induced a higher response of Th cells, naive/memory cells and macrophages/neutrophils. Thus, both vaccines were able to influence the porcine immune system, by activating different subsets of the immune effector/accessory cells.

Polyphenolic compounds are widely distributed in the plant kingdom and display a variety of biological activities, including chemoprevention and tumor growth inhibition. Propolis is made up of a variety of polyphenolic compounds. We compared how the routes of administration of polyphenolic compounds deriving from propolis and of propolis itself affect the growth and metastatic potential of a transplantable mammary carcinoma (MCa) of the CBA mouse. The influence of tested compounds on local tumor growth was also studied. Metastases in the lung were generated by 2 x 10(5) tumor cells injected intravenously (IV). A water-soluble derivative of propolis (WSDP) and polyphenolic compounds (caffeic acid, CA, and CA phenethyl ester, CAPE) were given to mice per os (PO) or intraperitoneally (IP) before or after tumor cell inoculation. Tested compounds significantly decreased the number of lung colonies. When mice were inoculated with 10(5) MCa cells in the exact site of subcutaneous injection of different doses of WSDP, CA, or CAPE, tumor growth was inhibited, and survival of treated mice was prolonged. Antitumor activity, according to the results obtained, is mostly related to the immunomodulatory properties of the compounds and their capacity to induce apoptosis and necrosis. In conclusion, results presented here indicate that WSDP, CA, and CAPE could be potential useful tools in the control of tumor growth in experimental tumor models when administrated PO; because PO administration is the easiest way of introducing a compound used for prevention and/or cure of any disease, it is likely that this article has reached the goal of the investigation.

The quantitative and distribution patterns of porcine peripheral blood and tonsillar lymphoid/myeloid cell subsets were assessed in order to establish the immune status of farm pigs prior to their transfer to fattening units. Peripheral blood and tonsillar samples were taken from clinically healthy, nonvaccinated, 12-week-old pigs, either ex vivo or following euthanasia. Single-colour flow cytometry, using monoclonal antibodies (mAbs) reactive with the swine leukocyte cluster of differentiation (CD) antigens, gave the proportions of lymphoid (9.7% CD4+, 8.0% CD8+, 36.9% CD5a+, 20.3% CD16+, 6.9% CD21+, 86.3% CD45+, 41.8% CD45RA+, 48.3% CD45RC+), null cells (6.9%) and myeloid cells (23.7% CD11b+ and 5.4% SWC3a+) in peripheral blood. In situ identification and distribution of lymphoid cells in the tonsils (CD3a+, CD21+, CD45RA+, CD45RC+) was performed with anti-CD mAbs using the avidin-biotin complex method. Most CD3a+ cells were in the parafollicular areas, with many cells in the follicles. CD21+ cells were scattered throughout the parafollicular area, with only a few cells inside lymphoid follicles. CD45RA+ cells were mostly concentrated in the follicles but many positive cells were present in the parafollicular area. Many CD45RC+ cells were visible in the parafollicular area, a few positive cells were in the crypt epithelium, and single cells were inside the follicles.

In order to study a possible immunomodulatory effect of the royal jelly (RJ) secreted by mandibular and hypopharingeal glands of the worker honeybee (Apis mellifera Linné.) we have used a well established rodent model. The CBA mice were given s.c. 0.1 ml of RJ, 7 days before, or immediately after, the immunization with sheep red blood cells (SRBC). The Y59 rats received i.m. 0.4 ml or i.v. 0.025 ml of RJ once or twice at 7 day intervals. Serum levels of total proteins and immunoglobulins in the rats that received RJ once or twice within a 2-week-period were significantly lower (P < or = 0.05) as compared with the nontreated animals. In mice which were immunized with 4 x 10(8) of SRBC 7 days after the application of RJ the number of plaque forming splenocytes was significantly higher (P < or = 0.05) than that in the controls. Both the weight of inguinal lymph node and the number of peripheral blood lymphocytes were increased (P < or = 0.05) in RJ-treated mice 3 or 5 days after the immunization, respectively. Neutrophils were decreased (P < or = 0.05) in the mice that were killed 5 or 10 days after the RJ treatment. Overall these results indicate that RJ exhibited immunomodulatory properties by stimulating antibody production and immunocompetent cell proliferation in mice or depressing humoral immune functions in rats. Both phenomena, though species-related in this model, could probably be reversed by changing the dose or the route of RJ application.