Hermann-Josef Rothkötter

Site-specific expression of CD11b and SIRPα (CD172a) on dendritic cells: implications for their migration patterns in the gut immune system

Dendritic cells (DC) in the intestinal tract play a major role in directing the mucosal immune system towards tolerance or immunity. We analyzed whether different mucosal DC subsets in pigs have specific functions, localizations, or migration patterns in vivo. Therefore, we collected physiologically migrating DC by pseudo‐afferent cannulation of the intestinal duct in eight Göttingen minipigs. Lymph DC were phenotypically and functionally characterized and compared to DC found on histological sections of porcine small intestine and mesenteric lymph nodes (MLN). Four different DC subpopulations were detected. Lamina propria (LP) DC were mainly CD11b+ signal regulatory protein α (SIRPα)+, DC in Peyers patches were mainly CD11b–/SIRPα+ in subepithelial domes and CD11b–/SIRPα– in interfollicular regions, whereas MLN DC were largely CD11b+/SIRPα–. Of these four subsets, only the CD11b+/SIRPα+ DC and the CD11b+/SIRPα– DC were present in lymph. This suggests that DC migration to MLN largely originates from the LP. Lymph DC expressed high levels of MHC class II and costimulatory molecules and had a low capacity for FITC‐dextran uptake, indicating a mature phenotype. However, lymph DC did not induce PBMC proliferation in MLR, and migration was not significantly influenced by mucosal antigen application.

Vulnerability of polarised intestinal porcine epithelial cells to mycotoxin deoxynivalenol depends on the route of application.

Background and Aims Deoxynivalenol (DON) is a Fusarium derived mycotoxin, often occurring on cereals used for human and animal nutrition. The intestine, as prominent barrier for nutritional toxins, has to handle the mycotoxin from the mucosa protected luminal side (apical exposure), as well as already absorbed toxin, reaching the cells from basolateral side via the blood stream. In the present study, the impact of the direction of DON exposure on epithelial cell behaviour and intestinal barrier integrity was elucidated. Methods A non-transformed intestinal porcine epithelial cell line (IPEC-J2), cultured in membrane inserts, serving as a polarised in vitro model to determine the effects of deoxynivalenol (DON) on cellular viability and tight junction integrity. Results Application of DON in concentrations up to 4000 ng/mL for 24, 48 and 72 hours on the basolateral side of membrane cultured polarised IPEC-J2 cells resulted in a breakdown of the integrity of cell connections measured by transepithelial electrical resistance (TEER), as well as a reduced expression of the tight junction proteins ZO-1 and claudin 3. Epithelial cell number decreased and nuclei size was enlarged after 72 h incubation of 4000 ng/mL DON from basolateral. Although necrosis or caspase 3 mediated apoptosis was not detectable after basolateral DON application, cell cycle analysis revealed a significant increase in DNA fragmentation, decrease in G0/G1 phase and slight increase in G2/M phase after 72 hours incubation with DON 2000 ng/mL. Conclusions Severity of impact of the mycotoxin deoxynivalenol on the intestinal epithelial barrier is dependent on route of application. The epithelium appears to be rather resistant towards apical (luminal) DON application whereas the same toxin dose from basolateral severely undermines barrier integrity.

Mycotoxin deoxynivalenol (DON) mediates biphasic cellular response in intestinal porcine epithelial cell lines IPEC-1 and IPEC-J2

The Fusarium derived mycotoxin deoxynivalenol (DON) is frequently found in cereals used for human and animal nutrition. We studied effects of DON in non-transformed, non-carcinoma, polarized epithelial cells of porcine small intestinal origin (IPEC-1 and IPEC-J2) in a low (200 ng/mL) and a high (2000 ng/mL) concentration. Application of high DON concentrations showed significant toxic effects as indicated by a reduction in cell number, in cellular reduction capacity measured by MTT assay, reduced uptake of neutral red (NR) and a decrease in cell proliferation. High dose toxicity was accompanied by disintegration of tight junction protein ZO-1 and increase of cell cycle phase G2/M. Activation of caspase 3 was found as an early event in the high DON concentration with an initial maximum after 6-8 h. In contrast, application of 200 ng/mL DON exhibited a response pattern distinct from the high dose DON toxicity. The cell cycle, ZO-1 expression and distribution as well as caspase 3 activation were not changed. BrdU incorporation was significantly increased after 72 h incubation with 200 ng/mL DON and NR uptake was only transiently reduced after 24 h. Low dose effects of DON on intestinal epithelial cells were triggered by mechanisms different from those responsible for the high dose toxicity.

Anatomical particularities of the porcine immune system - a physician's view.

In this article the anatomical structure of the porcine immune organs is described. The focus is on their particularities that are related to the use of pigs as an animal model. Key issues of the intrauterine development of the lymphoid organs are presented, such as the specific epithelio-chorial placenta, the appearance of the thymic tissue and the initial development of B cells. The role of the thymus for the development of alpha/beta and gamma/delta T cells and the location of tonsillar tissue in the naso-pharynx, in the oral cavity and at the basis of the tongue are described. The porcine spleen is of interest for surgical techniques to treat splenic trauma adequately. The observation of the inverted lymph node structure of pigs is puzzling and it remains unclear why only few species have this distinct morphological organisation. Based on the functional differences in lymphocyte recirculation observed in pigs, specific lymph cannulation experiments are possible in the porcine immune system. The porcine intestinal lymphoid tissue and the lymphocytes in the mucosal epithelium and lamina propria are of interest for studying the gut immune responses. For use as a model the fact that the pig is a monogastric omnivorous animal represents an advantage, although the porcine ileal Peyers patch has no obvious anatomical equivalent in man. Based on the detailed knowledge of porcine immune morphology the pig is suitable as model animal for immunology--in addition to the various experimental approaches in physiology, pharmacology, surgery, etc. that are applicable to human medicine.

Lymphocyte migration in the intestinal mucosa: entry, transit and emigration of lymphoid cells and the influence of antigen.

Lymphocyte migration is important to transport immunological information between the different compartments of the intestinal immune system. Large numbers of lymphocytes emigrate from the Peyers patches and reach the blood circulation after expansion and maturation within the mesenteric lymph nodes. So far the frequency of antigen specific lymphocytes emigrating from the Peyers patches after oral stimulation is not known. After mesenteric lymph node resection those cells emigrating from the intestinal wall are accessible by calculating the major intestinal lymph duct. The first antigen specific cells draining from the intestines are obviously not lymphocytes but dendritic cells, thus the antigen is rapidly trapped in the parenchyma of the lymph nodes in vivo. When lymphocytes were taken from intestinal lymph, labeled in vitro and retransfused, marked numbers of B-cells were re-detected in intestinal lymph. Later preferentially T-cells recirculated through the gut wall. After immigration into the intestinal lamina propria the lymphocytes may enter the space between epithelial cells, where they are present as intraepithelial lymphocytes. Lymphoid cells in the S-phase of the cell cycle have been detected in all compartments of the intestinal wall. Apoptosis is probably a further important mechanism for the regulation of intestinal immunity in removing cells reacting against harmless dietary antigens to maintain oral tolerance.

Natural alternatives to in-feed antibiotics in pig production: can immunomodulators play a role?

As a result of the European ban of in-feed growth-promoting antibiotics, new strategies are being developed to increase the resistance to disease in farm animals. In pig production, this is of particular importance during the weaning transition when piglets are subjected to major stressful events, making them highly sensitive to digestive disorders. At this time, the development of both innate and adaptive immunity at the mucosal surface is critical in preventing the potential harmful effects of intestinal pathogenic agents. Strategies aiming at stimulating natural host defences through the use of substances able to modulate immune functions have gained increasing interest in animal research, and different bioactive components a priori sharing those properties have been the subject of in vivo nutritional investigations in pig. Among these, yeast derivates (β-glucans and mannans) are able to interact with immune cells, particularly phagocytic cells. However, studies where they have been fed to pigs have shown inconsistent results, suggesting that their ability to target the sensitive immune cells through the oral route is questionable. The plant extracts, which would benefit from a positive image in the public opinion, have also been tested. However, due to a lack of data on the bioactive components of particular plants and the large diversity of species, it has proved difficult to prepare extracts of equivalent potency and thus, the literature on their influence on pig immunity remains inconclusive. In considering piglet immunity and health benefits, the most promising results to date have been obtained with spray-dried animal plasma, whose positive effects would be provided by specific antibodies and non-specific competition of some plasma components with bacteria for intestinal receptors. The major positive effect of spray-dried animal plasma is in reducing the infiltration of gut-associated lymphoid tissue by immune cells, which is likely to be the result of a decreased colonisation by potentially harmful bacteria. This review also highlights the limitations of some of the published in vivo studies on the immunomodulatory activity of certain feed additives. Among those, the lack of standardisation of extracts and the heterogeneity of piglet-rearing conditions (e.g. exposure to pathogens) are likely the most limiting.

Studies on the toxicity of deoxynivalenol (DON), sodium metabisulfite, DON-sulfonate (DONS) and de-epoxy-DON for porcine peripheral blood mononuclear cells and the Intestinal Porcine Epithelial Cell lines IPEC-1 and IPEC-J2, and on effects of DON and DONS on piglets.

The in vitro effects of deoxynivalenol (DON), de-epoxy-DON, DON-sulfonate (DONS) and sodium metabisulfite (Na(2)S(2)O(5), SBS) on porcine peripheral blood mononuclear cells (PBMC), and on the Intestinal Porcine Epithelial Cell lines IPEC-1 and IPEC-J2 were examined by using the MTT assay. In addition, an uncontaminated and a DON contaminated triticale were included in diets either untreated (CON, FUS) or SBS treated (CON-SBS, FUS-SBS) and fed to piglets for 28 d starting from weaning. The diet concentrations of DON and DONS amounted to 0.156, 2.312, 0.084 and 0.275 mg and to<0.05, <0.05, <0.05 and 1.841 mg/kg, respectively. PBMC of the so-exposed piglets were also subjected to the MTT assay. Neither DONS and SBS nor de-epoxy-DON affected the viability of PBMC, IPEC-1 and IPEC-J2 significantly up to concentrations of 17, 8 and 23 microM, respectively. For DON, IC(50) values were estimated at 1.2+/-0.1, 1.3+/-0.5 and 3.0+/-0.8 microM for PBMC, IPEC-1 and IPEC-J2, respectively. PBMC from piglets fed the SBS treated diets were characterized by a significantly decreased stimulation index and an increased IgA supernatant concentration with the SBS effect being significantly more pronounced after feeding the FUS-SBS diet. Further studies should clarify the possible impact of SBS on the porcine immune system.

Retrospective evaluation of undergraduate medical education by doctors at the end of their residency time in hospitals: Consequences for the anatomical curriculum

Reform of the undergraduate medical curriculum, including the debate on which of its parts or subjects are superfluous, is a topic of interest in many countries.

Parenteral long-acting amoxicillin reduces intestinal bacterial community diversity in piglets even 5 weeks after the administration

We investigated the long-term effects of a single intramuscular administration of amoxicillin (15 mg kg−1) 1 day after birth, on piglet intestinal microbiota. Animals received no creep feed before weaning on day 28 of age. For the next 11 days, the piglets received a wheat–barley-based diet. Colon digesta samples were collected on day 39 and subjected to denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments. DGGE fingerprint diversity indices differed between the group treated with amoxicillin and the untreated group (0.8±0.19 and 1.03±0.17, respectively, P=0.012). Reamplification and sequencing of two bands present in all samples revealed that a Roseburia faecalis-related population was strongly reduced in relative abundance (98% identity) in the treated group, while an enterobacterial population with 100% identity to Shigella spp., Escherichia coli and Salmonella enterica serovar Typhi was enriched. A band corresponding to Lactobacillus sobrius was present only in the control group. The protective effect of prophylactic antibiotic administration may be outweighed by the long-lasting disturbance of the gut ecosystem.

Contribution of serum IgA to intestinal lymph IgA, and vice versa, in minipigs.

Immune cells in pig gut lymph are rather well studied, but data on gut lymph immunoglobulins and their origin are nonexistent. Such data are important to understand the interplay between pig systemic and intestinal immunity as a basis for vaccination studies. In some species, gut lymph contributes much to plasma IgA, but apparently not in humans. To estimate the contributions of pig serum IgA to intestinal lymph IgA and vice versa, concentrations of IgA, IgG, IgM, albumin, haptoglobin, C3 and alpha 2-macroglobulin were measured by radial immunodiffusion in paired porcine intestinal lymph and serum samples. All proteins, except IgA, had lymph/serum ratios (< 1.0) inversely related to their size, depending on passive diffusion from serum. The mean lymph/serum ratio of IgA was 2.2 instead of an expected 0.50 or 0.65 (dimer or monomer, respectively), indicating that of the IgA in gut lymph, 22.7 or 29.5% came from serum, vs 77.3 or 70.5% from the intestine. Percentage of polymeric IgA, measured by gelfiltration and corrected radial immunodiffusion, was 64.3% in porcine mesenteric lymph and 47.3% in serum. As the pig plasma volume and daily gut lymph flow into circulation were known, it could be calculated that roughly 31% of the total plasma IgA originated daily from local intestinal synthesis, reaching blood via mesenteric lymph.