Fig 2: Representative ?uorescent micrographs of whole-mount preparations of myenteric ganglia from the duodenum of a control and a diabetic rat after TNFR1-HuCD or TNFR2-HuCD double-labeling immunohistochemistry. HuCD as a pan-neuronal marker was applied to label myenteric neurons. A: TNFR1-HuCD; B: TNFR2-HuCD. Arrows indicate myenteric neurons. Scale bar: 20 µm. CD: Control duodenum; DD: Diabetic duodenum.

Fig 3: R. mucosa alters surface TNF-related marks in human keratinocyte cultures and patients with AD. (A) Representative images for immunofluorescent staining of tumor necrosis factor alpha (TNFa; red), TNFa converting enzyme (TACE; green) and both (merge; yellow) for human primary keratinocytes stimulated with R. mucosa from healthy volunteers or media alone. White lines represent scale of 200 micrometers. (B, C) Quantification of signal for indicated marker in replicate wells. (D, E) Representative image (D) and quantification in replicate wells (E) for immunofluorescent staining of tumor necrosis factor receptor 2 (TNFR2). White lines (D) represent scale of 200 micrometers. (F–H) 14 pediatric patients with atopic dermatitis (AD) were treated with topical R. mucosa for a total of four months. (F, G) Levels of soluble tumor necrosis factor receptor 1 (sTNFR1) and sTNFR2 in the serum at enrollment (Pre) or after 16 weeks of active treatment (Post) are shown. Red dots indicate the two patients that did not achieve at least a 50% improvement in symptoms during treatment. (H) Percent change in sTNFR1 and sTNFR2 levels are contrasted against improvement in SCORing AD are shown along with a simple linear regression line. Data represent three independent experiments and are displayed as mean ± SEM. ***p<0.001, **p<0.01, *p<0.05 as determined by Student T test.

Fig 4: Representative electron micrographs of portions of the ganglionic neuropil or perikaryon of myenteric neurons from different gut segments of control, diabetic and insulin-treated diabetic rats after TNFR1 or TNFR2 post-embedding immunohistochemistry. A: TNFR1; B: TNFR2. Arrows indicate 18 nm gold particles labeling TNFR1/TNFR2. Scale bar: 500 nm. CD: Control duodenum; CI: Control ileum; CC: Control colon; DD: Diabetic duodenum; DI: Diabetic ileum; DC: Diabetic colon; IDD: Insulin-treated diabetic duodenum; IDI: Insulin-treated diabetic ileum; IDC: Insulin-treated diabetic colon.

Fig 5: Tissue levels of TNFR1 and TNFR2 in intestinal smooth muscle layer homogenates including the myenteric plexus from the different gut segments of control, diabetic and insulin-treated diabetic rats. A: TNFR1; B: TNFR2. Both the TNFR1 and TNFR2 levels decreased remarkably in the duodenum of diabetics, but they remained unchanged in other segments. Data are expressed as means ± SE. aP < 0.01, bP < 0.001, cP < 0.0001 (relative to the controls). dP < 0.01, eP < 0.001, fP < 0.0001 (between diabetics and insulin-treated diabetics). C: Controls; D: Diabetics; ID: Insulin-treated diabetics.