Ivica Valpotić

A royal jelly as a new potential immunomodulator in rats and mice

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.

ANALYSES OF MONOCLONAL ANTIBODIES REACTING WITH PORCINE WCD6 : RESULTS FROM THE SECOND INTERNATIONAL SWINE CD WORKSHOP

Among the 57 monoclonal antibodies analyzed within the T-cell group of the Second International Swine CD Workshop, one mAb fell within cluster T14a that included the CD6 standard a38b2 (No. 175). The new mAb MIL8 (No. 082) and a38b2 both precipitated from activated T-cells a 150 kDa monomeric protein. Staining patterns on the various cell types were similar. There was no inhibition of binding of either mAb to peripheral blood T-cells with the opposite mAb. The new mAb, MIL8, reacts with a separate epitope on porcine wCD6.

Analyses of mAb reactive with porcine CD8

Among all mAb submitted to the first porcine CD workshop, based on FCM analyses six mAb could be identified to recognize the porcine CD8 analogue (workshop Nos. 004, 051, 052, 053, 108 and 109). In immunoprecipitation studies three mAb (Nos. 004, 108 and 109) recognized an antigen with an apparent molecular mass of about 35 kDa under reducing conditions and about 70 kDa under non-reducing conditions. The molecular masses of the antigens recognized by the three other mAb (Nos. 051, 052 and 053) are still unknown. Epitope analyses performed by blocking experiments led to the determination of two CD8 epitopes: CD8a and CD8b. CD8a is recognized by mAb Nos. 004, 051 and 052, and CD8b by Nos. 053, 108 and 109.

Summary of workshop findings for antibodies reacting with porcine T-cells and activation antigens: results from the Second International Swine CD Workshop

After initial evaluation of the 176 new and 19 control monoclonal antibodies (mAb) submitted to the Second International Swine CD Workshop, 57 were assigned to the T-cell/activation marker subgroup. These 57 mAb were further analyzed using flow cytometry on whole blood lymphocytes, splenocytes, Peyers patch lymphocytes, in vitro cell lines, broncho-alveolar lavage cells, Con A and PHA blasts, fetal cell populations, and by 2-color flow cytometry against mAb to porcine CD2, CD4, and CD8. Finally, the molecular weights of the target antigens were characterized when possible. As a result of these analyses, 23 mAb were distributed into 7 CD clusters. Newly confirmed mAb assignments included: two CD2; one CD4; two CD5; one wCD6; and one wCD25. Three new mAb were found that reacted with wCD8, one of which defined a new epitope, wCD8c. For the first time, mAb against porcine CD3 were identified, including 6 mAb that reacted with three different epitopes. Several new mAb reacted with antigens whose expression varied depending on the activation state of the test cell. These will require further characterization in order to assign a CD number.

Report on the analyses of mAb reactive with porcine CD8 for the second international swine CD workshop.

Based on an analysis of their reactivity with porcine peripheral blood lymphocytes (PBL), only three of the 57 mAbs assigned to the T cell/activation marker group were grouped into cluster T9 along with the two wCD8 workshop standard mAbs 76-2-11 (CD8a) and 11/295/33 (CD8b). Their placement was verified through the use of two-color cytofluorometry which established that all three mAbs (STH101, #090; UCP1H12-2, #139; and PG164A, #051) bind exclusively to CD8+ cells. Moreover, like the CD8 standard mAbs, these three mAbs reacted with two proteins with a MW of 33 and 35 kDa from lymphocyte lysates and were, thus, given the wCD8 designation. Because the mAb STH101 inhibited the binding of mAb 76-2-11 but not of 11/295/33, it was given the wCD8a designation. The reactivity of the other two new mAbs in the T9 cluster with the various subsets of CD8+ lymphocytes were distinct from that of the other members in this cluster including the standards. Although the characteristic porcine CD8 staining pattern consisting of CD8low and CD8high cells was obtained with the mAb UCP1H12-2, a wider gap between the fluorescence intensity of the CD8low and CD8high lymphocytes was observed. In contrast, the mAb PG164A, not only exclusively reacted with CD4-/CD8high lymphocytes, but it also failed to recognize CD4/CD8 double positive lymphocytes. It was concluded that this mAb is specific for a previously unrecognized CD8 epitope, and was, thus, given the wCD8c designation. A very similar reactivity pattern to that of PG164A was observed for two other mAbs (STH106, #094; and SwNL554.1, #009). Although these two mAbs were not originally positioned in the T cell subgroup because of their reactivity and their ability to inhibit the binding of PG164A, they were given the wCD8c designation. Overall, five new wCD8 mAbs were identified. Although the molecular basis for the differences in PBL recognition by these mAbs is not yet understood, they will be important in defining the role of CD8+ lymphocyte subsets in health and disease.

Analysis of monoclonal antibodies reactive with the porcine CD2 antigen

As result of the First International Swine CD Workshop, six monoclonal antibodies (mAbs) (numbers 014, 023, 024, 057, 128, and 130) clustered closely to the internal standard anti-porcine CD2 mAb, MSA4. Despite the close clustering, the cluster was split into two subgroups. To further characterize the relationship between these mAbs, they were used in flow cytometry to inhibit binding of MSA4 to porcine lymphocytes. mAbs 014 (1038-8-31), 023 (MAC83), 024 (MAC80), 057 (PG168), and 128 (MSA4) completely inhibited the binding of MSA4, mAb 130 (MSA2) failed to inhibit MSA4 binding. On dual parameter flow cytometry comparing MSA2 with MSA4, all MSA2+ cells were MSA4+, two thirds of the MSA4+ cells were MSA2-. We conclude that five of the mAbs bind to the same or a closely related epitope on porcine lymphocytes. mAb 130 appears to have aberrantly clustered with the CD2 group of mAb.

Summary of workshop findings for porcine T-lymphocyte antigens.

Fifty-four mAb preselected in the first round of the first porcine CD workshop for their possible reactivity with T-lymphocyte specific antigens and/or activation antigens were further analysed in a second round. PBMC, thymocytes and nylon-wool purified T lymphocytes derived from peripheral blood, mesenteric lymph nodes and spleen served as target cells for flow cytometric analyses. For the classification of activation antigens several experiments were performed with activated, mitogen-stimulated T lymphocytes and long-term T-lymphocyte cultures. Out of the 54 mAb, 35 mAb could be distributed to six different CD clusters and two swine workshop clusters (SWC). Five mAb could be distributed to the porcine CD2, four mAb to the CD4. Six mAb seemed to recognize the porcine CD5 and two mAb the porcine CD6 analogue. Six mAb were directed against the porcine CD8, whereas two different epitopes could be defined. One mAb was directed against the porcine CD25 analogue. Nine mAb could be clustered to the SWC1, defining an antigen on T lymphocytes and cells of the myeloic linage. Two mAb with high T-cell specificity were clustered to the SWC2.

Analysis of mAb reactive with the porcine SWC1

Among 54 mAb determined to be reactive with porcine T lymphocytes and/or activation antigens, eight mAb (workshop Nos. 005, 031, 080, 091, 092, 093, 094 and 110) derived from different laboratories grouped together in the T11 cluster and were ordered into the SWC1. One mAb (No. 111) which belong also to this group was lost during the workshop. The SWC1 antigen is a molecule expressed on the majority of leukocytes, resting T lymphocytes, monocytes and granulocytes, but not on B lymphocytes. On T lymphocytes it is down-regulated after activation. The molecular mass of the antigen is unknown. Epitope analyses revealed that seven out of the nine mAb recognized similar epitopes on the SWC1 molecule.

Analyses of monoclonal antibodies reactive with porcine CD6

Amongst the monoclonal antibodies (mAbs) submitted to the first porcine CD workshop, two mAbs (workshop numbers 055 and 120) could be identified to recognize the porcine CD6 analogue. Both mAbs seemed to be highly T-cell specific and showed neither reactivity with cells of the myeloic lineage nor with B lymphocytes. The observed molecular mass of the antigen precipitated by mAb 120 of 110 kDa confirmed this classification. Without molecular analyses of the antigen recognized by mAb 055, but similar staining pattern in FCM compared with 120, mAb 055 was allocated to the wCD6 subcluster.

Recruitment of intestinal CD45RA+ and CD45RC+ cells induced by a candidate oral vaccine against porcine post-weaning colibacillosis

To assess the influence of a live attenuated oral vaccine against porcine post-weaning colibacillosis (PWC) induced by enterotoxigenic Escherichia coli (ETEC) on mucosal lymphoid cell CD45 isoforms expression, experimental group of weaned pigs (n=6) was immunized orally with F4ac+ non-ETEC strain (day 0) and challenged with F4ac+ ETEC strain 7 days latter. Non-immunized ETEC-infected pigs (n=6) served as control. All pigs were killed on post-challenge day 7. The small intestine was excised for isolation of jejunal lamina propria (JLP) and ileal Peyers patch (IPP) lymphocytes and immunohistochemical studies. The results obtained by immunophenotyping of isolated cells show that the proportion of CD45RA+ and CD45RC+ JLP, but not IPP, cells were higher in the non-ETEC-immunized ETEC-infected pigs versus non-immunized infected. Additionally, while CD45RA+ JLP cells increased only slightly, the expression of CD45RC isoform on the JLP cells was significantly higher (P< or =0.01) in the experimental than in the control group. The results of the quantitative phenotypic analysis of isolated lymphocytes were not confirmed by immunohistochemical in situ staining. The majority of intestinal immune cells was found to express CD45RA antigen in situ, but no differences were observed between the two groups of weaned pigs neither in CD45RA+ nor in CD45RC+ cells. Our overall evidence indicates that the increased expression of CD45RC isoform was in fact induced in a limited number of JLP T cells in the vaccinated pigs. This was accompanied with the impaired protection of the vaccinated pigs from challenge-induced PWC.