Tam N. Pham

Involvement of CD14 and toll-like receptor 4 in the acute phase response of serum amyloid A proteins and serum amyloid P component in the liver after burn injury.

Acute phase proteins such as serum amyloid A proteins (SAAs) and serum amyloid P component (SAP) are induced in the liver after various insults (e.g., infection, injury). The cellular and molecular mechanisms controlling the expression of these acute phase proteins may be specifically designed for different insults. The roles of two central molecules of the lipopolysaccharide (LPS)-mediated inflammation pathway (CD14 and toll-like receptor 4 [Tlr4]) were investigated for the regulation of SAAs and SAP in the liver of mice after an 18% total body surface area burn injury. RT-PCR analysis revealed a subtype- and time-dependent induction of SAA mRNAs between 3 h and 3 days, while there was a peak induction of SAP mRNA at day 1. Marked elevations of SAA and SAP protein levels at day 1 supported the mRNA data. Furthermore, a differential regulation of SAAs and SAP mRNAs was noted between CD14 knockout (KO) and their control mice after injury. SAA protein was induced to a lesser degree after injury in C3H/HeJ (Tlr4-defective) mice than in their control mice. In addition, in both CD14 KO and C3H/HeJ mice, the induction of SAP protein was significantly reduced compared with respective controls. These data provide evidence that CD14 and Tlr4 participate, at least in part, in a cascade of signaling events that control the immediate-early and differential induction of SAAs and SAP in the liver after injury. They also suggest that LPS may be one of the initial inducing agents associated with these acute phase responses in the liver after injury.

Sodium azide burn: a case report

Chemical burn injuries commonly occur at the workplace and can be caused by a variety of agents. Sodium azide is a volatile compound used in the industrial setting and it is also a constituent of car airbags. The known toxic effects of sodium azide include hypotension, bradycardia, and headaches. At the cellular level, it inhibits of ATP production by blocking the respiratory oxidation cascade. In the burn literature only one previous report documents a sodium azide hand burn caused by airbag malfunction. We report a case of massive exposure and resultant systemic toxicity from a sodium azide canister explosion.

CD14-dependent modulation of transcriptional activities of endogenous retroviruses in the lung after injury.

Lipopolysaccharide (LPS) plays a central role in the pathogenesis of distant organs after burn. Recent studies demonstrated the regulation of mouse endogenous retroviruses (MuERVs) in several organs after burn. In this study, the role of CD14, a LPS receptor, in burn-mediated regulation of MuERV expression in the lung was investigated. CD14 knockout (KO) and wild type (WT) mice were subjected to burn followed by RT-PCR analysis of alterations in the MuERV expression in the lung 1 day after injury. Even without injury, CD14 KO mice had a unique profile of MuERV expression compared to WT. Three bands (Lung-1, Lung-2, and Lung-3) in CD14 KO were downregulated after injury. Lung-2 and Lung-3 transcripts were almost identical to 2 previously described defective env transcripts of MuERVs, respectively. The Lung-1-1 transcript was a double spliced message generated by the env and a set of novel splicing signals, whereas the Lung-1-2 transcript was a defective env transcript. Only the Lung-1-1 transcript had a significant ORF capable of encoding a gag-pol fusion polypeptide. Putative proviral sequences of Lung-1-1 and Lung-1-2 transcripts were mapped to chromosomes 4 and 11, respectively. The results from this study suggest that the absence of CD14 expression in CD14 KO mice contributes to the transcriptional regulation of MuERVs in the lung after injury.

Regulation of murine endogenous retroviruses in the thymus after injury

We recently reported the induction of murine endogenous retroviruses (murine AIDS-related) in several distant organs of mice after burn injury. The regulation of endogenous retroviruses in response to burn injury was further investigated in the thymus. Female C57BLKS/J mice were subjected to 18% total body surface area flame burn injury. Thymus tissues collected at several time points (3 h to 7 days) were analyzed for the expression of subgenomic transcripts of murine endogenous retroviruses by RT-PCR. Interestingly, a novel 1.7-Kb subgenomic transcript and a recently described 1.1 Kb subgenomic transcript of murine endogenous retroviruses were transiently down-regulated in the thymus at day 1 after injury. The 1.7 Kb transcript has a coding potential for a truncated form of the envelope protein (total 214 amino acids) with a deletion of 418 amino acids near the C-terminus. The second transcript of 1.1 Kb has an open reading frame for the C-terminal transmembrane domain of the envelope protein including the p2E protein (R peptide). These data suggest the pathophysiologic effects of burn injury on the differential expression of murine endogenous retroviruses in the thymus after injury.

CD14-mediated alterations in transcription and splicing of endogenous retroviruses after injury

Summary.Increase in systemic levels of lipopolysaccharide (LPS) contributes to the pathogenesis of distant organ injury after burn. Stress signals elicited from burn influence transcriptional activities of mouse endogenous retroviruses (MuERVs) in various distant organs. The involvement of LPS pathways in the burn-mediated regulation of MuERVs in the spleen was investigated in this study. Spleen harbors substantial numbers of tissue macrophages, a key responder to LPS stimulation. Spleen tissues collected from CD14 (LPS receptor) knockout (KO) and wild type (WT) mice after burn were subjected to RT-PCR analysis of MuERV expression. There was a substantial induction of 2 bands and a marked downregulation of a band in CD14 KO mice compared to WT mice after burn. Sequence analysis of these CD14- and burn-dependent bands identified 3 new alternatively spliced and 2 defective env transcripts of MuERVs as well as novel splicing signals. Chromosomal loci of putative MuERVs sharing the unique U3 sequences of these transcripts were mapped by surveying the entire genome of C57BL/6J mice. In addition, coding potentials, transcriptional regulatory elements, and adjacent cellular genes of these putative MuERVs were analyzed. The results from these studies suggest that injury-triggered LPS/CD14 signaling events play roles in the transcriptional regulation of certain MuERVs carrying unique U3 promoter sequences.

A Potential Mechanism for Immune Suppression by Beta-Adrenergic Receptor Stimulation following Traumatic Injury

Background: β-Adrenergic agents suppress inflammation and may play an important role in posttraumatic infections. Mechanisms may include inhibition of MAP kinase signaling. We sought to determine whether MKP-1 contributed to catecholamine suppression of innate immunity and also wanted to know whether early catecholamine treatment after traumatic injury increases the risk of later nosocomial infection. Methods: We performed experiments using THP-1 cells and peripheral blood mononuclear cells from healthy individuals. We exposed cells to epinephrine and/or LPS and measured inflammatory gene transcription and MAP kinase activation. We inhibited MKP-1 activity to determine its role in catecholamine-induced immune suppression. Finally, we studied injured subjects to determine whether early catecholamine treatment was associated with nosocomial infection. Results: Epinephrine increases MKP-1 transcripts and protein and decreases LPS-induced p38 and JNK phosphorylation and TNF-α gene transcription. RNAi inhibition of MKP-1 at least partially restores LPS-induced TNF-α gene expression (p = 0.024). In the clinical cohort, subjects treated with β-adrenergic agents had an increased risk of ventilator-associated pneumonia (aOR = 1.9; 95% CI = 1.3–2.6) and bacteremia (aOR = 1.5; 95% CI = 1.1–2.3). Conclusions: MKP-1 may have a role in catecholamine-induced suppression of innate immunity, and exogenous catecholamines might contribute to nosocomial infection risk.

BURN INJURY INDUCES AN INHIBITORY SIGNAL IN THE LUNG SMAD PATHWAY.: 3

Smad signaling mediates the cellular response to transforming growth factor-beta (TGF-beta). We hypothesize that variations in Smad signaling modify the response to TGF-beta signaling in the lung after injury. C57BLKS/J mice were subjected to an 18% surface area burn injury, sacrificed at specific time points and their lung tissue was harvested. Lung TGF-beta1 expression, as determined by RT-PCR, ELISA and PAI/Luciferase assay, was not affected by injury. Western blots for Smad2/3 and Smad4 on nuclear fractions revealed decreased Smad2, Smad3, and Smad4 protein levels at 3h, while their total cellular levels did not differ from control mice. Smad7 protein increased transiently at 3 h. Correlating with Smad inhibition, transcription in type I alpha-2 collagen was also transiently depressed. By RT-PCR, Smad3 and Smad7 mRNAs decreased at 3 h, while Smad2 and Smad4 mRNA levels remained constitutive. Burn injury did not alter lung TGF-beta1 expression but caused Smad inhibition through decreased nuclear translocation of Smad2, Smad3, and Smad4, and upregulated Smad7. Transcription was not the key regulatory step in Smad protein expression, as transient decreases in Smad3 and Smad7 mRNA did not correlate with protein levels. It appears that Smad activity may in part attenuate TGF-beta activity after burn injury.