Probes for INS

Cleft palate is one of the most common craniofacial congenital defects in humans. It is associated with multiple genetic and environmental risk factors, including mutations in the genes encoding signaling molecules in the sonic hedgehog (Shh) pathway, which are risk factors for cleft palate in both humans and mice. However, the function of Shh signaling in the palatal epithelium during palatal fusion remains largely unknown. Although components of the Shh pathway are localized in the palatal epithelium, specific inhibition of Shh signaling in palatal epithelium does not affect palatogenesis. We therefore utilized a hedgehog (Hh) signaling gain-of-function mouse model, K14-Cre;R26SmoM2, to uncover the role of Shh signaling in the palatal epithelium during palatal fusion. In this study, we discovered that constitutive activation of Hh signaling in the palatal epithelium results in submucous cleft palate and persistence of the medial edge epithelium (MEE). Further investigation revealed that precise downregulation of Shh signaling is required at a specific time point in the MEE during palatal fusion. Upregulation of Hh signaling in the palatal epithelium maintains the proliferation of MEE cells. This may be due to a dysfunctional p63/Irf6 regulatory loop. The resistance of MEE cells to apoptosis is likely conferred by enhancement of a cell adhesion network through the maintenance of p63 expression. Collectively, our data illustrate that persistent Hh signaling in the palatal epithelium contributes to the etiology and pathogenesis of submucous cleft palate through its interaction with a p63/Irf6-dependent biological regulatory loop and through a p63-induced cell adhesion network.

During a 19-month period, 5 smooth green snakes ( Opheodrys vernalis) maintained as an ex situ conservation colony presented with rapid clinical progression of locally invasive oropharyngeal squamous cell carcinoma. All 5 originated from the same wild source and were housed together or in close proximity. An infectious cause was considered likely, and nested conventional polymerase chain reaction (PCR) and in situ hybridization confirmed the presence of a novel alphaherpesvirus, Opheodrys herpesvirus 1, in the neoplastic tissue in 4 of the 5 snakes. Retrospective screening of previously submitted smooth green snakes by in situ hybridization did not detect virus in prior submissions from the colony. This report documents molecular characterization of an ophidian herpesvirus as well as colocalization of its viral nucleic acid with neoplastic transformation in snakes.

Abstract

Background

Toll-like receptor (Tlr) 9 is capable of recognizing exogenous and/or endogenous nucleic acids and plays a crucial role in innate and adaptive immunity. Recently, we showed that Tlr9 is overexpressed in podocytes, a component of the blood–urine barrier (BUB), in glomeruli of autoimmune glomerulonephritis (AGN) model mice. This study investigated the activation of peritubular capillary (PTC) endothelial cells (ECs), a component of the BUB in the tubulointerstitium, through overexpressing Tlr9, and the subsequent development of tubulointerstitial lesions (TILs) in AGN model mice.

Methods

Lupus-prone BXSB/MpJ-Yaa (Yaa) and BXSB/MpJ (BXSB) mice were used as an AGN model and control, respectively. In addition to histopathological and ultrastructural techniques, protein and mRNA levels were also evaluated. The relationship between Tlr9 and TIL indices was analyzed by statistical correlation analysis.

Results

Yaa mice developed TILs and showed strong Tlr9 mRNA expression in PTC ECs at 24 weeks (wks) of age. However, BXSB mice showed no TIL but faint expression of Tlr9 mRNA at 8 and 24 wks of age. Tlr9 protein localization on PTC was almost absent in BXSB mice at both ages but intense expression was found in Yaa mice only at 24 wks of age. Relative mRNA expression of Tlr9 and its putative downstream cytokines, including interleukin 1 beta (Il1b), Il6, interferon gamma (Ifng), and tumor necrosis factor alpha (Tnf) was markedly increased in isolated tubulointerstitium from Yaa mice at 24 wks of age. Furthermore, electron microscopy examination revealed PTC injury and TIL in Yaa mice at 24 wks. The expression level of Tlr9 in the tubulointerstitium was correlated with inflammatory cells in TILs, injured PTC, Ilb and Tnf expression, and damaged tubules (P < 0.05 and 0.01).

Conclusion

Induced expression of Tlr9 in ECs correlates with PTC injury and the development of TILs in lupus-prone AGN model mice.

Juvenile eastern water dragons ( Intellagama lesueurii lesueurii) are highly susceptible to infection with Bohle iridovirus (BIV), a species of ranavirus first isolated from ornate burrowing frogs in Townsville, Australia. To investigate the progression of BIV infection in eastern water dragons, 11 captive-bred juveniles were orally inoculated with a dose of 104.33 TCID50 and euthanized at 3, 6, 8, 10, 12, and 14 days postinfection (dpi). Viral DNA was detected via polymerase chain reaction (PCR) in the liver, kidney, and cloacal swabs at 3 dpi. Mild lymphocytic infiltration was observed in the submucosa and mucosa of the tongue and liver at 3 dpi. Immunohistochemistry (IHC) first identified viral antigen in foci of splenic necrosis and in hepatocytes with intracytoplasmic inclusion or rare single-cell necrosis at 6 dpi. By 14 dpi, positive IHC labeling was found in association with lesions in multiple tissues. Selected tissues from an individual euthanized at 14 dpi were probed using in situ hybridization (ISH). The ISH labeling matched the location and pattern detected by IHC. The progression of BIV infection in eastern water dragons, based on lesion severity and virus detection, appears to start in the spleen, followed by the liver, then other organs such as the kidney, pancreas, oral mucosa, and skin. The early detection of ranaviral DNA in cloacal swabs and liver and kidney tissue samples suggests these to be a reliable source of diagnostic samples in the early stage of disease before the appearance of clinical signs, as well as throughout the infection.

Previous investigations have indicated that RNAs are mostly present in the minor population of the youngest platelets, whereas translation in platelets could be biologically important. To attempt to solve this paradox, we studied changes in the RNA content of reticulated platelets, i.e., young cells brightly stained by thiazole orange (TObright), a fluorescent probe for RNAs. We provoked in mice strong thrombocytopenia followed by dramatic thrombocytosis characterized by a short period with a vast majority of reticulated platelets. During thrombocytosis, the TObright platelet count rapidly reached a maximum, after which TOdim platelets accumulated, suggesting that most of the former were converted into the latter within 12 h. Experiments on platelets, freshly isolated or incubated ex vivo at 37°C, indicated that their "RNA content", here corresponding to the amounts of extracted RNA, and the percentage of TObright platelets were positively correlated. The "RNA Content" normalized to the number of platelets could be 20 to 40 fold higher when 80-90% of the cells were reticulated (20-40 fg/platelet), than when only 5-10% of control cells were TObright (less than 1fg/platelet). TObright platelets, incubated ex vivo at 37°C or transfused into mice, became TOdim within 24 h. Ex vivo at 37°C, platelets lost about half of their ribosomal and beta actin RNAs within 6 hours, and more than 98% of them after 24 hours. Accordingly, fluorescence in situ hybridization techniques confirmed the presence of beta actin mRNAs in most reticulated-enriched platelets, but detected them in only a minor subset of control platelets. In vitro, constitutive translation decreased considerably within less than 6 hours, questioning how protein synthesis in platelets, especially in non-reticulated ones, could have a biological function in vivo. Nevertheless, constitutive transient translation in young platelets under pathological conditions characterized by a dramatic increase in circulating reticulated platelets could deserve to be investigated.