In fish, bacterial pathogens can enter the host by one or more of three different routes: (a) skin, (b) gills and (c) gastrointestinal tract. Bacteria can cross the gastrointestinal lining in three different ways. In undamaged tissue, bacteria can translocate by transcellular or paracellular routes. Alternatively, bacteria can damage the intestinal lining with extracellular enzymes or toxins before entering. Using an in vitro (Ussing chamber) model, this paper describes intestinal cell damage in Atlantic salmon (Salmo salar L.) caused by the fish pathogen Aeromonas salmonicida ssp. salmonicida, the causative agent of furunculosis. The in vitro method clearly demonstrated substantial detachment of enterocytes from anterior region of the intestine (foregut) upon exposure to the pathogen. In the hindgut (posterior part of the intestine), little detachment was observed but cellular damage involved microvilli, desmosomes and tight junctions. Based on these findings, we suggest that A. salmonicida may obtain entry to the fish by seriously damaging the intestinal lining. Translocation of bacteria through the foregut (rather than the hindgut) is a more likely infection route for A. salmonicida infections in Atlantic salmon.

In situ zymography is a method for the detection and localization of enzymatic activity in tissue sections. This method is used with frozen sections because routine fixation of tissue in neutral-buffered formalin inhibits enzyme activity. However, frozen sections present with poor tissue morphology, making precise localization of enzymatic activity difficult to determine. Ethanol- and zinc-buffered fixative (ZBF) are known to preserve both morphological and functional properties of the tissue well, but it has not previously been shown that these fixatives preserve enzyme activity. In the present study, we show that in situ zymography can be performed on ethanol- and ZBF-fixed paraffin-embedded tissue. Compared with snap-frozen tissue, ethanol- and ZBF-fixed tissue showed stronger signals and superior morphology, allowing for a much more precise detection of gelatinolytic activity. Gelatinolytic enzymes could also be extracted from both ethanol- and ZBF-fixed tissue. The yield, as analyzed by SDS-PAGE gelatin zymography and Western blotting, was influenced by the composition of the extraction buffer, but was generally lower than that obtained from unfixed tissue.

Tertiary lymphoid structures (TLS) develop in the kidneys of lupus-prone mice and systemic lupus erythematosus (SLE) patients with lupus nephritis (LN). Here we investigated the presence of mesenchymal stem cells (MSCs) in the development of TLS in murine LN, as well as the role of human MSCs as lymphoid tissue organizer (LTo) cells on the activation of CD4+ T cells from three groups of donors including Healthy, SLE and LN patients. Mesenchymal stem like cells were detected within the pelvic wall and TLS in kidneys of lupus-prone mice. An increase in LTβ, CXCL13, CCL19, VCAM1 and ICAM1 gene expressions were detected during the development of murine LN. Human MSCs stimulated with the pro-inflammatory cytokines TNF-α and IL-1β significantly increased the expression of CCL19, VCAM1, ICAM1, TNF-α, and IL-1β. Stimulated MSCs induced proliferation of CD4+ T cells, but an inhibitory effect was observed when in co-culture with non-stimulated MSCs. A contact dependent increase in Th2 and Th17 subsets were observed for T cells from the Healthy group after co-culture with stimulated MSCs. Our data suggest that tissue-specific or/and migratory MSCs could have pivotal roles as LTo cells in accelerating early inflammatory processes and initiating the formation of kidney specific TLS in chronic inflammatory conditions.

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Immune aggregates organized as tertiary lymphoid structures (TLS) are observed within the kidneys of patients with systemic lupus erythematosus and lupus nephritis (LN). Renal TLS was characterized in lupus-prone New Zealand black  New Zealand white F1 mice analyzing cell composition and vessel formation. RNA sequencing was performed on transcriptomes isolated from lymph nodes, macrodissected TLS from kidneys, and total kidneys of mice at different disease stages by using a personal genome machine and RNA sequencing. Formation of TLS was found in antiedouble-stranded DNA antibodyepositive mice, and the structures were organized as interconnected large networks with distinct T/B cell zones with adjacent dendritic cells, macrophages, plasma cells, high endothelial venules, supporting follicular dendritic cells network, and functional germinal centers. Comparison of gene profiles of whole kidney, renal TLS, and lymph nodes revealed a similar gene signature of TLS and lymph nodes. The up-regulated genes within the kidneys of lupus-prone mice during LN development reflected TLS formation, whereas the down-regulated genes were involved in metabolic processes of the kidney cells. A comparison with human LN gene expression revealed similar up-regulated genes as observed during the development of murine LN and TLS. In conclusion, kidney TLS have a similar cell composition, structure, and gene signature as lymph nodes and therefore may function as a kidney-specific type of lymph node.

Squamous cell carcinomas (SCCs) arising in the oral cavity are associated with poor survival, mainly due to metastatic disease. In contrast, skin SCCs rarely metastasize and are usually curable. To study influence of tongue and skin stroma on cancer growth and induction of lymphangiogenesis, xenograft tumors of human carcinoma cells were established either in tongue or skin of BALB/c nude mice. Two oral and two skin SCC cell lines were used, as well as an endometrial adenocarcinoma cell line. Tongue tumors established from all cell lines were larger than corresponding skin tumors. Peritumoral lymphatic vessel density was up to five times higher in tongue than in corresponding skin tumors, and mRNA level of the lymphangiogenic growth factor vascular endothelial growth factor (VEGF)-C was twice as high in tongue tumors compared with corresponding skin tumors. Contrary to lymphatic vessel density, blood vessel density was higher in skin tumors than in tongue tumors. In a cohort of patient samples, lymphatic vessel density was found to be higher in tongue SCCs compared with skin SCCs, supporting a clinical relevance of our findings. Our results show that the tumor stroma has a profound impact on cancer growth and induction of lymphangiogenesis and angiogenesis. The difference in lymphatic vessel density between tongue and skin tumors may be important in directing metastatic potential of tumors arising in these organs.

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