Angela Berndt

Chicken Cecum Immune Response to Salmonella enterica Serovars of Different Levels of Invasiveness

ABSTRACT Day-old chicks are very susceptible to infections with Salmonella enterica subspecies. The gut mucosa is the initial site of host invasion and provides the first line of defense against the bacteria. To study the potential of different S. enterica serovars to invade the gut mucosa and trigger an immune response, day-old chicks were infected orally with Salmonella enterica serovar Enteritidis, S. enterica serovar Typhimurium, S. enterica serovar Hadar, or S. enterica serovar Infantis, respectively. The localization of Salmonella organisms in gut mucosa and the number of immune cells in cecum were determined by immunohistochemistry in the period between 4 h and 9 days after infection. Using quantitative real-time reverse transcription (RT)-PCR, mRNA expression of various cytokines, chemokines, and inducible nitric oxide synthase (iNOS) was examined in cecum. As a result, all S. enterica serovars were able to infect epithelial cells and the lamina propria. Notably, serovar Enteritidis showed the highest invasiveness of lamina propria tissue, whereas serovars Typhimurium and Hadar displayed moderate invasiveness and serovar Infantis hardly any invasion capabilities. Only a limited number of bacteria of all serovars were found within intestinal macrophages. Elevated numbers of granulocytes, CD8+ cells, and TCR1+ cells and mRNA expression rates for interleukin 12 (IL-12), IL-18, tumor necrosis factor alpha factor, and iNOS in cecum correlated well with the invasiveness of serovars in the lamina propria. In contrast, changes in numbers of TCR2+ and CD4+ cells and IL-2 mRNA expression seemed to be more dependent on infection of epithelial cells. The data indicate that the capability of Salmonella serovars to enter the cecal mucosa and invade lower regions affects both the level and character of the immune response in tissue.

Gamma/delta T cell response of chickens after oral administration of attenuated and non-attenuated Salmonella typhimurium strains

Poultry represents an important source of Salmonella infection in man. Despite intensive research on immunity, little is known about the involvement of T cell sub-populations in the immunological response of chickens against infection with non-host-adapted Salmonella (S.) serovars. In this study, the T cell composition of blood lymphocytes (CD4(+)CD8(+); CD4(+)CD8(-); CD4(-)CD8(+); CD8(+)TcR1(+); CD8(-)TcR1(+), CD8(+)TcR1(-)) after oral administration of the non-attenuated S. typhimurium wild-type strain 421 (infection) or the attenuated vaccine strain Salmonella vac((R)) T (immunization) to day-old chicks was investigated and compared with non-treated chickens by flow cytofluorometry. Additionally, the occurrence of T cell sub-populations (CD4(+); CD8(+); TcR1(+)(gammadelta); TcR2(+)(alphabeta(1))) in ceca, spleen and bursa of Fabricius of the birds was studied immunohistologically. Blood samples and tissues were examined between days 1 and 12 of age. Chicks inoculated with S. typhimurium 421 or Salmonella vac((R)) T showed significantly elevated percentages of CD8(+)TcR1(+) in blood on days 7, 8 and 9, or on day 8 in comparison to control animals. The CD4 to CD8 cell ratio was about 3:1 in infected animals on day 5 of age. In the organs of treated chicks the numbers of CD8(+)(gammadelta) and TcR1(+)(gammadelta) cells had markedly increased on days 4 and 5 in ceca, 8 and 9 in the bursa and 9 and 12 in the spleen. Moreover, infected or vaccinated birds revealed larger quantities of CD4(+) and TcR2(+) T cells in ceca on days 4 and 5. As shown by double staining, the TcR1(+) cells in the organs of infected animals additionally carried the CD8 antigen. In conclusion, immunization of day-old chicks with the attenuated Salmonella live vaccine strain resulted in the same changes in T cell composition as seen after infection with the non-attenuated Salmonella wild-type strain, but at a lower level. The remarkable increase of CD8(+)TcR1(+)(gammadelta) double positive cells in treated birds indicates an important role of this cell sub-population in the immunological defense of chickens against Salmonella exposure.

Circulating γδ T Cells in Response to Salmonella enterica Serovar Enteritidis Exposure in Chickens

ABSTRACT γδT cells are considered crucial to the outcome of various infectious diseases. The present study was undertaken to characterizeγδ (T-cell receptor 1+ [TCR1+]) T cells phenotypically and functionally in avian immune response. Day-old chicks were orally immunized with Salmonella enterica serovar Enteritidis live vaccine or S. enterica serovar Enteritidis wild-type strain and infected using the S. enterica serovar Enteritidis wild-type strain on day 44 of life. Between days 3 and 71, peripheral blood was examined flow cytometrically for the occurrence of γδ T-cell subpopulations differentiated by the expression of T-cell antigens. Three different TCR1+ cell populations were found to display considerable variation regarding CD8α antigen expression: (i) CD8α+high TCR1+ cells, (ii) CD8α+dim TCR1+ cells, and (iii) CD8α− TCR1+ cells. While most of the CD8α+high TCR1+ cells expressed the CD8αβ heterodimeric antigen, the majority of the CD8α+dim TCR1+ cells were found to express the CD8αα homodimeric form. After immunization, a significant increase of CD8αα+high γδ T cells was observed within the CD8α+high TCR1+ cell population. Quantitative reverse transcription-PCR revealed reduced interleukin-7 receptor α (IL-7Rα) and Bcl-x expression and elevated IL-2Rα mRNA expression of the CD8αα+highγδ T cells. Immunohistochemical analysis demonstrated a significant increase of CD8α+ and TCR1+ cells in the cecum and spleen and a decreased percentage of CD8β+ T cells in the spleen after Salmonella immunization. After infection of immunized animals, immune reactions were restricted to intestinal tissue. The study showed that Salmonella immunization of very young chicks is accompanied by an increase of CD8αα+high γδ T cells in peripheral blood, which are probably activated, and thus represent an important factor for the development of a protective immune response to Salmonella organisms in chickens.

Effects of Salmonella enterica serovar Enteritidis on cellular recruitment and cytokine gene expression in caecum of vaccinated chickens

Although vaccination of poultry is a suitable method to limit human food borne gastroenteritis caused by Salmonella (S.), the immune mechanisms responsible for a longer lasting protection against Salmonella infection in birds are not completely understood. To reveal unique protection-related immune parameters, day-old chicks were vaccinated with a commercial live S. Enteritidis vaccine and challenged with wild-type S. Enteritidis 147N at day 56 of life. The bacterial cell count was determined in gut and liver, while the immune cell composition and cytokine gene expression patterns were analysed by immunohistochemistry and quantitative real-time RT-PCR in caecum samples. The presented data suggest that the vaccine-elicited immune protection against the Salmonella wild-type infection was rather related to the bacterial count in gut mucosa and liver than to the colonisation in gut lumen. The higher number of Salmonella wild-type organisms found in caecal wall and liver of the non-immunised compared to immunised birds after challenge correlated with a more pronounced gene expression rate for IL-8, LITAF, iNOS, IL-12 and IFN-gamma. In contrast, immunised birds exhibited higher amounts of CD8(+) T cells as well as IgA than the non-immunised chickens after S. Enteritidis 147N infection in caecum. The results demonstrated a distinctive immune reaction pattern of previously vaccinated compared to non-vaccinated chickens upon S. Enteritidis wild-type challenge.

Studies of the influence of ochratoxin A on immune and defence reactions in weaners.

Even in subtoxic amounts, the mycotoxin, ochratoxin A, produced immunomodulation in weaner pigs in a dose‐dependent mode. In addition to increased counts of total leukocytes and neutrophils in the blood, reduced lymphocyte levels were observed. There was a striking increase in the counts of eosinophils and of apoptotic phagocytes. Functionally, there was a predominance of the production of reactive oxygen radicals in whole blood, reduced phagocytosis performance and reduced expression of a swine‐specific surface marker (SWC1) on lymphocytes. In a few single experiments, clinical manifestations could be demonstrated. Lung clearance and the degree of severity of experimental pneumonia as well as cutaneous hypersensitization may be influenced by ochratoxin A.

Isolation of a new chlamydial agent from infected domestic poultry coincided with cases of atypical pneumonia among slaughterhouse workers in France

Three cases of atypical pneumonia in individuals working at a poultry slaughterhouse prompted an epidemiological survey in 10 poultry farms that had supplied birds. Using a Chlamydiaceae-specific real-time PCR assay, chlamydial agents were detected in 14 of 25 investigated flocks. Rather unexpectedly, Chlamydophila psittaci was identified only in one of the positive flocks, whereas ArrayTube DNA microarray testing indicated the presence of a new, so far unclassified member of the genus Chlamydophila. For further characterization of the agent involved, positive cloacal swabs were used to inoculate embryonated chicken eggs and isolates were obtained from 6 different flocks. Sequencing of 16S rRNA genes revealed nearly identical sequences of all samples. Alignment with representative sequences of Chlamydiaceae showed the separate position of the present strains outside the currently recognized species of Chlamydophila, but clearly within this genus. In contrast, partial ompA gene sequences displayed considerable diversity among the isolates, which had already been observed in restriction enzyme analysis of ompA PCR products. These data suggest that each farm had been infected with a different strain of this new chlamydial agent, the zoonotic potential and the exact taxonomic status of which have yet to be defined.

Expression of Snail is associated with myofibroblast phenotype development in oral squamous cell carcinoma

Snail is a regulator of epithelial–mesenchymal transition (EMT) and considered crucial to carcinoma metastasis, myofibroblast transdifferentiation, and fibroblast activation. To investigate the role of Snail in oral squamous cell carcinoma (OSCC), its immunohistochemical expression was analysed in 129 OSCC samples and correlated to nodal metastasis, histological grade, E-cadherin, and alpha smooth-muscle-actin (αSMA). The results were compared to findings in 23 basal cell carcinomas (BCC). Additionally, the influence of TGFβ1 and EGF on Snail, E-cadherin, vimentin, and αSMA expression was analysed in two OSCC cell lines. As a result, Snail-positive cells were mainly found in the stroma of the OSCC invasive front without statistically significant correlation to histological grade or nodal metastasis. Snail was co-localised to αSMA but not to E-cadherin or cytokeratin and showed a significant correlation to the loss of membranous E-cadherin. All BCCs were Snail negative. In OSCC culture, the growth-factor-mediated EMT-like phenomenon was accompanied by αSMA down-regulation. In summary, Snail expression in OSCC is a stromal phenomenon associated with the myofibroblast phenotype and not related to growth-factor-mediated transdifferentiation of the carcinoma cells themselves. Consequently, Snail immunohistochemistry cannot contribute to the prediction of the metastatic potential. Furthermore, stromal Snail expression is suggested to be the result of mutual paracrine interaction of fibro-/myofibroblasts and dedifferentiated carcinoma cells leading to the generation of a special type of carcinoma-associated fibroblasts.

Heterogeneity of avian γδ T cells

gammadelta T cells are distinct with respect to tissue localisation, phenotype and biological functions and similarities between species are not very apparent. To elucidate local and functional heterogeneity of non-stimulated avian gammadelta T cells, the CD8-characterised gammadelta T cell subsets [CD8alpha(+high) (CD8alphaalpha(+) and CD8alphabeta(+)); CD8alpha(+dim); CD8(-)] of blood, spleen and caecum were flow cytometrically quantified and analysed for proliferation state as well as sorted for determination of immune-relevant gene expression by quantitative real-time RT-PCR. The number of avian CD8-characterised gammadelta T cell subsets differed in dependence on tissue and age of bird. Compared to blood and spleen, caecum showed the highest percentage of gammadelta T cells as well as of the CD8alpha(+high) gammadelta T cell subset in 7-week-old birds. Generally, the CD8alphabeta(+) cells significantly outnumbered the CD8alphaalpha(+) lymphocytes within the CD8alpha(+high) gammadelta T cell population of all organs. Additionally, the splenic CD8alphabeta(+) subpopulation revealed the highest proliferation activity. By RT-PCR, mRNA expression of immune-relevant genes was proved in non-stimulated gammadelta T cell subsets, but on different levels. Generally, both CD8alpha(+high) cell subsets (CD8alphaalpha(+) and CD8alphabeta(+)) of blood and spleen showed elevated expression levels for Fas ligand (FasL), XCL1 (lymphotactin) and interferon-gamma (IFNgamma) compared to the CD8alpha(-) gammadelta T cell subset. In contrast, all caecal gammadelta T cell subsets showed similar high levels of these transcripts. Notably, the CD8alphaalpha(+) cells of all locations showed unique expression of TLR4 and interleukin (IL)-2. The results demonstrated that avian gammadelta T cells are not only heterogeneous concerning their CD8 antigen characteristics and tissue localisation, but also with regard to functional features such as proliferation and mRNA expression.

Studies of the influence of ochratoxin A on immune and defence reactions in the mouse model

Summary. In the mouse model, the mycotoxin ochratoxin A has a non‐selective suppressive effect on various immune and defence reactions. Apart from weight depression, lymphopenia, neutrophilia and eosinophilia, antibody‐producing cells, antibody titres in blood serum and phagocytosis of Escherichia coli by blood phagocytes become suppressed. Moreover, immunized animals show a lower survival rate after experimental infection with Pasteurella multocida as well as an increase in oxygen radicals in blood cells.

EGF/TGFβ1 co-stimulation of oral squamous cell carcinoma cells causes an epithelial-mesenchymal transition cell phenotype expressing laminin 332.

Epithelial-mesenchymal transition (EMT) is suggested to be crucial for the development of an invasive and metastatic carcinoma cell phenotype. Therefore, the definition of this phenotype is of great clinical interest. We recently evidenced vimentin positive cells in oral squamous cell carcinoma (OSCC) invasive front expressing laminin γ2 chain mRNA implicating an EMT origin of these cells. To further elucidate the nature of these cells, we have investigated the relation between EMT criteria and laminin-332 expression in a cell culture model of transforming growth factor beta-1 (TGFβ1)/epithelial growth factor (EGF) long time co-stimulation. We demonstrate that in contrast to TGFβ1 or EGF alone, co-stimulation induces phenotype transition in OSCC cells which fulfils the criteria of EMT in terms of vimentin up-regulation and E-cadherin down-regulation on protein level as well as cell scattering. Furthermore, cells displayed a strongly enhanced invasiveness and adhesion to type I-IV collagens. Phenotype transition is accompanied by an enhanced expression of laminin-332, especially of its γ2 chain. We further analyse the expression of extracellular matrix related genes by RT-PCR profiling. With respect to strongly enhanced proteins, data confirm the EMT phenotype of co-stimulated OSCC cells and expression of laminin-332. Furthermore, alpha catenin, collagen type 16, the integrin α7 and β1 chains, and MMP11 are suggested as candidates with potential role in EMT in OSCC. In summary we are able to show that EMT in OSCC is mediated by multiple growth factors and is accompanied by laminin γ2 chain up-regulation evidencing the existence of an intermediate Vim(+) /Ln332(+) EMT phenotype as seen in situ.