Influence of digital hypothermia on lamellar events related to IL-6/gp130 signalling in equine sepsis-related laminitis.

BACKGROUND: Interleukin-6 (IL-6) is consistently increased in the digital lamellae in different studies of sepsis-related laminitis (SRL). IL-6 signalling through the gp130 receptor activates similar signalling (i.e. mTORC1-related signalling) previously reported to be activated in models of endocrinopathic laminitis. OBJECTIVES: To assess the activation state of signalling proteins downstream of IL-6/gp130 receptor complex activation in an experimental model of SRL. STUDY DESIGN: Randomised experimental study. METHODS: Lamellar phospho-(P) protein concentrations downstream of the IL-6/gp130 receptors were assessed in the oligofructose (OF) model of SRL. Fifteen Standardbred horses were administered water (CON, n = 8) or oligofructose (OF, n = 7) via a nasogastric tube. At 12 h post-OF/water administration, one randomly assigned forelimb was exposed to continuous digital hypothermia (CDH) by placement in ice water (ICE, maintained at <7°C); the other forelimb was maintained at ambient temperature (AMB). Lamellar tissue samples were collected after 24 h of CDH from both ICE and AMB forelimbs and immediately snap-frozen. Lamellar proteins of interest were assessed by immunoblotting and immunofluorescence. RESULTS: Immunoblotting revealed increase (P<0.05) in the phosphorylation states of Akt (Ser 473), RPS6 (Ser235/236), RPS6 (Ser240/244), STAT3 (Ser727) and STAT3 (Tyr705) in lamellar tissue from OF-treated animals (AMB OF vs. AMB CON limbs); CDH resulted in decreased (P<0.05) lamellar concentrations of phosphorylated Akt, p70S6K, RPS6 (235/236), RPS6 (240/244) and STAT3 (S727) in OF-treated animals (AMB OF vs. ICE OF). Immunofluorescence showed that activated/phosphorylated forms of RPS6 and STAT3 were primarily localised to lamellar epithelial cells. MAIN LIMITATIONS: The nature, sequence and timing of sub-cellular events in this experimental model may differ from those that accompany naturally occurring sepsis. CONCLUSIONS: There were increased lamellar concentrations of activated signalling proteins downstream of the IL-6/Gp130 receptor complex in OF-treated horses; CDH inhibited this activation for the majority of the proteins assessed. These results demonstrate similar lamellar signalling (e.g. mTORC1-related signalling) and, therefore, possible therapeutic targets occurring in sepsis-related laminitis as previously reported in models of endocrinopathic laminitis.

Effect of Continuous Digital Hypothermia on Lamellar Inflammatory Signaling When Applied at a Clinically-Relevant Timepoint in the Oligofructose Laminitis Model.

BACKGROUND: Although continuous digital hypothermia (CDH) protects lamellae from injury in the oligofructose (OF) model of sepsis-related laminitis (SRL), conflicting results exist from these studies regarding effects of CDH on lamellar inflammatory events. HYPOTHESIS/OBJECTIVES: To determine the effect of CDH on lamellar inflammatory events in normal and OF-treated horses when instituted at a clinically relevant time point (onset of clinical signs of sepsis in this model). ANIMALS: Standardbred geldings (n = 15) aged 3-11 years were used. METHODS: In a randomized, controlled discovery study, animals were administered either OF (OF group, n = 8) or water (CON group, n = 8) by nasogastric tube and CDH was initiated in one forelimb (ICE) 12 hours later. Lamellar tissue samples were collected 24 hours after initiation of CDH (ICE and ambient [AMB] forelimbs). Lamellar mRNA concentrations of inflammatory mediators and lamellar leukocyte numbers were assessed using qPCR and immunohistochemistry, respectively; values from four sample groups (CON AMB, OF AMB, CON ICE, and OF ICE) were analyzed using mixed model linear regression. RESULTS: Although lamellar mRNA concentrations of multiple inflammatory mediators (IL-1ß, IL-6, CXCL1, MCP2, COX-2) were increased after OF administration (OF AMB group versus CON AMB; P < 0.05), only 2 inflammatory mediators (IL-6 and COX-2) and lamellar leukocyte numbers were decreased with CDH (OF ICE versus OF AMB; P < 0.05). CONCLUSIONS AND CLINICAL IMPORTANCE: Continuous digital hypothermia initiated at a time point similar to that commonly used clinically (clinical onset of sepsis) resulted in a more focused inhibition of inflammatory signaling.

Effect of Delayed Digital Hypothermia on Lamellar Inflammatory Signaling in the Oligofructose Laminitis Model.

BACKGROUND: In the oligofructose (OF) model of sepsis-related laminitis (SRL), digital hypothermia ("cryotherapy") initiated before the onset of clinical signs is reported not only to limit lamellar injury, but also to cause marked inhibition of lamellar inflammatory signaling. HYPOTHESIS/OBJECTIVES: Because hypothermia also has been reported to be protective when not initiated until the onset of lameness in the OF model of SRL, we hypothesized that the lamellar protection conferred by hypothermia is caused by local lamellar inhibition of inflammatory signaling as described when hypothermia was initiated earlier in the disease process. ANIMALS: Eight Standardbred geldings aged 3-11 years with no lameness and no abnormalities of the feet detectable by gross or radiographic examination. METHODS: Using the OF model of SRL, lamellar mRNA concentrations of proinflammatory cytokines, chemokines, and endothelial adhesion proteins were compared between samples from treated limbs (CRYO, submerged in ice water for 36 hour starting at the onset of lameness), untreated limbs (NON-CRYO, opposite limb from CRYO limbs maintained at ambient temperature), and untreated limbs from normal horses in which laminitis was not induced (CON). RESULTS: Although OF administration resulted in increases in lamellar mRNA concentrations of several inflammatory mediators in NON-CRYO limbs (vs CON), digital hypothermia had no significant effect on these increases. CONCLUSIONS AND CLINICAL IMPORTANCE: The lack of inflammatory inhibition in lamellar tissue samples in our study indicates that the protective effects of digital hypothermia instituted at the onset of clinical signs of laminitis do not arise from inhibition of inflammatory pathways.

Inhibition of HIV type 1 replication by simultaneous infection of peripheral blood lymphocytes with human immunodeficiency virus types 1 and 2.

A productive infection of peripheral blood lymphocytes by HIV-1 was severely inhibited by the simultaneous infection of these cells with HIV-2. A similar reciprocal effect on HIV-2 infection was not observed. The extent of virus replication was determined by virus-specific antigen capture assays of the supernatants of the infections. The inhibitory effect was observed with T cell-tropic, dual-tropic, as well as with primary HIV-1 isolates from different subtypes (A, B, C, E, F, and O). Infection of PBLs with different subtypes of HIV-2 (A and B) as well as with SIV(mac) resulted in the inhibition of HIV-1. However, the inhibitory effect was limited to PBLs; similar results were not observed in a T cell line. The inhibition of HIV-1 replication was independent of HIV-2 concentration; however, the infection by HIV-2 had to take place within 24 hr after PBLs were infected by HIV-1 for inhibition of HIV-1 replication to occur. The inhibition could be reversed by the addition of PHA. Analysis of HIV-1 RNA and DNA demonstrated that the inhibition was not at uptake or reverse transcription and that equal amounts of PBLs were infected by HIV-1 in single infections and coinfections. Immunocytochemical analysis of HIV-1 proteins demonstrated that equal numbers of cells were infected and that equal amounts of intracellular HIV-1 Env and Gag proteins were produced throughout the culture period. Therefore we conclude that HIV-2 can potently inhibit the productive infection of PBLs by HIV-1 and that the mechanism of this inhibition appears to prevent HIV-1 assembly or release from PBLs.

T cell activation-induced and HIV tat-enhanced CD95(APO-1/Fas) ligand transcription involves NF-kappaB.

CD95(APO-1 / Fas) ligand (CD95L) gene expression is critically involved in activation-induced T cell apoptosis. We and other have previously shown that HIV-1 Tat which is essential for efficient HIV gene expression sensitizes CD95-mediated apoptosis and up-regulates CD95L expression in T cells. In the present study we have investigated the regulatory mechanism for CD95L expression. Two NF-kappaB binding sites are localized at - 537 to - 521 and - 57 to - 47 (relative to the transcription start site) of the human CD95L promoter. We show that both elements bind to NF-kappaB and SP-1 transcription factors and NF-kappaB is involved in transactivation of the CD95L promoter upon T cell activation. Mutations at each NF-kappaB site by two base pair substitutions resulted in 30 - 70 % reduction of the promoter activity. The effect of Tat on the human CD95L promoter activity was mapped to the same sites. Mutation of each NF-kappaB site also impaired the effect of Tat on CD95L promotor activity. We also show that ectopic expression of Tat protein in Jurkat T cells greatly increases NF-kappaB binding to its target DNA. Our studies provide evidence that Tat-enhanced CD95L expression is regulated at least in part by the NF-kappaB sites of the promoter.