<i>P. dolloi</i> gut epithelial cells (GEC) express J chain.

<p>A) P. <i>dolloi</i> gut cryosection stained with the nuclear stain DAPI (blue) showing the characteristic nuclear morphology of epithelial cells B) <i>P. dolloi</i> gut cryosection stained with Cy-3 J chain Stellaris FISH probe. Epithelial cells contained numerous J chain transcripts (dots). Ep: epithelial cells, Lu: lumen, Bm: basal membrane C) Gut epithelial cells were dissected from control <i>P. dolloi</i> gut cryosections using LCM. Total RNA from <i>P. dolloi</i> pre-pyloric spleen, LCM gut epithelial cells (GEC) and muscle was extracted and used as template for RT-PCR using J chain (J), IgM, IgW and cytokeratin 8 (CK-8) specific primers. EF-1α served as cDNA control. “–” indicates J chain negative control. M (marker) indicates the DNA ladder. The sequences of all PCR products were confirmed by cloning and sequencing.</p

Experimental bacterial infection model in <i>P. dolloi</i>.

<p>A) Delivery of <i>E. ictaluri</i> to <i>P. dolloi</i> intranasally. B) Skin ulcers (black arrows) produced by <i>E. ictaluri</i> in an infected <i>P. dolloi</i> individual. No ulcers were observed in control specimens.</p

Discovery of J Chain in African Lungfish (<i>Protopterus dolloi</i>, Sarcopterygii) Using High Throughput Transcriptome Sequencing: Implications in Mucosal Immunity

<div><p>J chain is a small polypeptide responsible for immunoglobulin (Ig) polymerization and transport of Igs across mucosal surfaces in higher vertebrates. We identified a J chain in dipnoid fish, the African lungfish (<i>Protopterus dolloi</i>) by high throughput sequencing of the transcriptome. <i>P. dolloi</i> J chain is 161 aa long and contains six of the eight Cys residues present in mammalian J chain. Phylogenetic studies place the lungfish J chain closer to tetrapod J chain than to the coelacanth or nurse shark sequences. J chain expression occurs in all <i>P. dolloi</i> immune tissues examined and it increases in the gut and kidney in response to an experimental bacterial infection. Double fluorescent in-situ hybridization shows that 88.5% of IgM⁺ cells in the gut co-express J chain, a significantly higher percentage than in the pre-pyloric spleen. Importantly, J chain expression is not restricted to the B-cell compartment since gut epithelial cells also express J chain. These results improve our current view of J chain from a phylogenetic perspective.</p></div

Fold change of J chain expression in pre-pyloric spleen, post-pyloric spleen, gut, kidney and lung of Nigerian spotted lungfish infected with <i>Edwardsiella ictaluri</i> compared to samples from PBS mock control fish.

<p>Bars represent means ± standard error of four fish. Different letters represent statistically significant groups after Tukey's test (p<0.05).</p

Expression of J chain by B cells of Nigerian spotted lungfish (<i>P. dolloi</i>).

<p>A) Mean percentage of IgM⁺ and IgM⁺ J chain⁺ B cells and B) Mean percentage of IgW⁺ and IgW⁺ J chain⁺ B cells in the pre-pyloric spleen and gut of control <i>P. dolloi</i> (n = 3) studied by double FISH staining. Cells were counted from 20 different fields (×60). Differences were statistically significant when p<0.05. n.s: non-significant differences. C–E) Examples of representative FISH staining showing IgM⁺ J chain⁺ lymphocytes (white asterisks) from a gut cryosection. C) Cy5-IgM FISH probe D) Cy3-Jchain FISH probe E) DAPI nuclear stain. F–H) Examples of representative FISH staining showing a IgW⁺ J chain⁻ lymphocyte (yellow asterisk) from a pre-pyloric spleen cryosection. F) Cy5-IgW FISH probe G) Cy3-Jchain FISH probe H) DAPI nuclear stain.</p

Tissue distribution of J chain expression in control Nigerian spotted lungfish (<i>P. dolloi</i>) (n = 6).

<p>The relative expression of each gene was normalized first with the house keeping genes, and then divided by the average expression level in the pre-pyloric spleen, which had the lowest value. Bars represent means ± standard error of four fish. Different letters represent statistically significant groups after Tukey's test (p<0.05).</p

[Lola] Albright... [Oleg] Vidov

Additional file 3: Figure 2. Ingenols do not induce pro-inflammatory cytokine release in purified resting CD4+ T cell cultures. Concentrations of pro-inflammatory cytokines TNFα, IFNγ, IL-1β and IL-6 were not significantly elevated in the presence of ingenol-3,20-dibenzoate (ingenol DB) or ingenol B compared to media alone control at 72 h in purified resting CD4+ T cell cultures. Mean values and standard deviation of seven independent experiments using resting CD4+ T cells from aviremic ART-treated HIV positive participants are shown. Pro-inflammatory cytokine concentrations were significantly elevated in positive control cultures (T cell receptor stimulation via CD3 and CD28 antibodies). ** P value <0.01; *** P value <0.001