First mouse model for combined osteogenesis imperfecta and Ehlers-Danlos syndrome.

By using a genome-wide N-ethyl-N-nitrosourea (ENU)-induced dominant mutagenesis screen in mice, a founder with low bone mineral density (BMD) was identified. Mapping and sequencing revealed a T to C transition in a splice donor of the collagen alpha1 type I (Col1a1) gene, resulting in the skipping of exon 9 and a predicted 18-amino acid deletion within the N-terminal region of the triple helical domain of Col1a1. Col1a1(Jrt) /+ mice were smaller in size, had lower BMD associated with decreased bone volume/tissue volume (BV/TV) and reduced trabecular number, and furthermore exhibited mechanically weak, brittle, fracture-prone bones, a hallmark of osteogenesis imperfecta (OI). Several markers of osteoblast differentiation were upregulated in mutant bone, and histomorphometry showed that the proportion of trabecular bone surfaces covered by activated osteoblasts (Ob.S/BS and N.Ob/BS) was elevated, but bone surfaces undergoing resorption (Oc.S/BS and N.Oc/BS) were not. The number of bone marrow stromal osteoprogenitors (CFU-ALP) was unaffected, but mineralization was decreased in cultures from young Col1a1(Jrt) /+ versus +/+ mice. Total collagen and type I collagen content of matrices deposited by Col1a1(Jrt) /+ dermal fibroblasts in culture was ∼40% and 30%, respectively, that of +/+ cells, suggesting that mutant collagen chains exerted a dominant negative effect on type I collagen biosynthesis. Mutant collagen fibrils were also markedly smaller in diameter than +/+ fibrils in bone, tendon, and extracellular matrices deposited by dermal fibroblasts in vitro. Col1a1(Jrt) /+ mice also exhibited traits associated with Ehlers-Danlos syndrome (EDS): Their skin had reduced tensile properties, tail tendon appeared more frayed, and a third of the young adult mice had noticeable curvature of the spine. Col1a1(Jrt) /+ is the first reported model of combined OI/EDS and will be useful for exploring aspects of OI and EDS pathophysiology and treatment.

CD14-dependent alterations in c-Jun expression in the liver after burn injury.

BACKGROUND: Burn injury-triggered activation of lipopolysaccharide signaling via the CD14 pathway alters the expression of a variety of downstream genes contributing to pathogenic changes in distant organs. The regulation of CD14 and its role in the immediate-early response of c-Jun in the liver after burn injury were investigated in this study. MATERIALS AND METHODS: An incidental identification of the differential induction of CD14 mRNA after an approximately 18% TBSA burn injury in mice was confirmed by RT-PCR and immunohistochemical analyses of CD14 expression. Subsequently, CD14's role in the immediate-early regulation of c-Jun expression in the liver after injury was examined by Western blot analysis using CD14 knockout (KO) mice. RESULTS: RT-PCR analysis demonstrated a rapid and transient induction of CD14 mRNA in the liver and lungs of mice after injury. Immunohistochemical analysis revealed a peak induction of CD14 reactivity in cells appearing to be Kupffer cells at day 1 after injury. Furthermore, an augmented and delayed induction of c-Jun mRNA was observed in the liver of CD14 KO mice after injury compared to wild-type controls. The induction of phosphorylated (serine 63 or serine 73) forms of c-Jun after injury was lower in the livers of CD14 KO mice than that in WT controls. CONCLUSIONS: This study provides evidence that injury elicits CD14 induction as well as hyperphosphorylation of the c-Jun N-terminus activation domain and that CD14 is involved in the modulation of c-Jun's transactivation potential via phosphorylation, which may be associated with hepatic pathogenesis after injury.

Alterations in the levels of metallothionein and metals in the liver, and unique serum liver enzyme response in metallothionein knock-out mice after burn injury.

OBJECTIVES: Metallothionein (MT) is a small cysteine-rich protein that sequesters and distributes metal ions. Its overexpression stimulates cell proliferation and inhibits apoptosis. We investigated the effects of burn injury on MT expression and metal localization. We also sought to determine roles of MT in the pathophysiologic alterations in the liver after injury. METHODS: Mice (C57BLKS/J, MT-I/II knock-out, KO, and wild-type control mice) were subjected to an 18% burn. Liver tissues harvested after injury were analyzed for the MT expression and the levels of zinc, copper, manganese, and iron. Levels of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase were measured in serum samples from MT-I/MT-II KO mice and controls after injury. RESULTS: Transient induction of MT-I and MT-II mRNAs was observed 3-6 h after injury, while MT-I/MT-II protein peaked on day 1. The induction was localized to hepatocytic nuclei. The intrahepatic levels of zinc, copper, and iron were transiently elevated on day 1, when a downregulation of manganese was evident. Interestingly, only the serum levels of aspartate aminotransferase were significantly augmented in MT-I/MT-II KO mice compared to controls after injury. CONCLUSIONS: These data suggest that MT and metals may participate in the pathogenesis of the liver after burn injury.

Burn injury induces an inhibitory signal in the lung Smad pathway.

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.

Disease progression in heterosexual patients infected with closely related subtype B strains of HIV type 1 with differing coreceptor usage properties.

Previously we described a heterosexual outbreak of HIV-1 subtype B in a town in the north of England (Doncaster) where 11 of 13 infections were shown to be linked by phylogenetic analysis of the env gp120 region. The 11 infections were related to a putative index case, Don1, and further divided into two groups based on the patients' disease status, their viral sequences, and other epidemiological information. Here we describe two further findings. First, we found that viral isolates and gp120 recombinant viruses derived from patients from one group used the CCR5 coreceptor, whereas viruses from the other group could use both the CCR5 and CXCR4 coreceptors. Patients with the X4/R5 dual tropic strains were symptomatic when diagnosed and progressed rapidly, in contrast to the other patient group that has remained asymptomatic, implying a link between the tropism of the strains and disease outcome. Second, we present additional sequence data derived from the index case, demonstrating the presence of sequences from both clades, with an average interclade distance of 9.56%, providing direct evidence of a genetic link between these two groups. This new study shows that Don1 harbored both strains, implying he was either dually infected or that over time intrahost diversification from the R5 to R5/X4 phenotype occurred. These events may account for/have led to the spread of two genetically related strains with different pathogenic properties within the same heterosexual community.

Injury-associated differential regulation of histone expression and modification in the thymus of mice.

One of the key events in the regulation of gene expression is chromatin remodeling involving histone regulation. We investigated the effects of burns on the expression of histone that might be associated with altered molecular and pathological profiles in the thymus. A markedly decreased expression of histone variant H2A.1 mRNA was identified in the thymus after burn during a differential display experiment. Subsequently, we examined the histone expression (mRNA and protein) and posttranslational modification in the thymus after burn. Also, changes in proliferating cell nuclear antigen (PCNA), a central molecule in chromatin assembly, was examined. Reverse-transcription polymerase chain reaction analysis revealed a transient decrease in the expression of several histone variants (H2A.1, H1(r1), H3-B, H3-1, and H4-D) mRNAs in the thymus at 1 day after burn. A decrease in histone subtypes H2A, H2B, H3, and H4, but not H1, was demonstrated 1 and 3 days after burn according to the results of Western blot. Furthermore, there were different levels of decreases in acetylated and dimethylated forms of histone H3 1 and 3 days after burn. In addition, decreased levels of PCNA were evident in the thymus 1 day after burn. Changes in the expression of histones and PCNA may reflect mere decrease in proliferating cells and/or a reorganization of the chromatin structure associated with altered transcriptional activities, eventually contributing to the phenotypic changes in the thymus after burn.

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.

Alterations in the expression and modification of histones in the liver after injury.

Chromatin remodeling plays a key role in the transcriptional activation of regulatory factors in the liver in response to a variety of stress signals. The effects of burn injury on histone expression and its modification were investigated in this study. Liver tissues collected after a flame burn injury were subjected to RT-PCR and Western blot analyses of histone regulation. There was a marked induction of histone H3-D variant mRNA at 3 and 6 h. In contrast, histone H2A.2 variant mRNA had a downregulation at 3 days. No apparent changes were noted in other histone variants examined. Western blot analysis revealed a downregulation of all 5 histone subtypes (H1, H2A, H2B, H3, and H4) at 1 day and there was a subsequent induction of H1 and H2A subtypes at 3 days after injury. There was an induction of modified forms (phospho-, acetyl-, and dimethyl-) of histone H3 subtype at day 3. Furthermore, a transient elevation in PCNA (proliferating cell nuclear antigen) levels was apparent in the liver at day 3, which parallels the induction of phospho-histone H3, which is a mitosis marker. These findings suggest that histones participate in a cascade of events associated with phenotypic alterations in the liver after injury.

Burn injury-mediated alterations in cell cycle progression in lymphoid organs of mice.

A key event in the cellular and molecular pathogenesis of multiple organ failure (MOF) after burn injury may be the change in profiles of the cell cycle progression in affected organs. We investigated the effects of burn injury on cell cycle progression in immune organs. Cell cycle analysis in the lymphoid tissues of mice after 18% burn injury revealed that S phase entry was temporarily arrested in the thymus 1 day after injury, whereas the spleen had substantially increased S phase entry at day 8. This mode of cell cycle regulation was reproduced in different age groups and strains of mice. Furthermore, the reactivity to the Ki-67 antibody (indicative of proliferation) was markedly reduced in the thymic cortex at day 1. There was a distinct pattern of hematopoietic foci formation and increased reactivities to the Ki-67 antibody in myelogenous cells in the red pulp of spleen at day 7, consistent with the elevated S phase entry. These data suggest that differential regulation of cell cycle progression may play a crucial role in the phenotypic changes in immune organs after burn injury.

Identification of truncated form of mouse HAX-1s gene (HAX-1xs) and characterization of its expression in small intestine and thymus of mice after burn injury.

Burn injury often leads to distant organ injury such as acute respiratory distress syndrome. We hypothesize that the pathophysiologic changes in distant organs result from orchestrated regulation of multiple genes in response to bum injury. Differential display was performed to identify genes regulated in distant organs in response to burn injury. Initial characterization of differentially amplified products demonstrated that HAX-1s mRNA was regulated in several distant organs after 18% total body surface area (TBSA) full-thickness flame burn injury in mice. Further characterization of HAX-1s mRNA revealed a novel transcript variant, which is rapidly and transiently induced in multiple tissues of mice within 6 h after burn injury. This novel HAX-1s transcript variant, called HAX-1xs, has an internal deletion of 252 nucleotides and single point mutation, resulting in reading frame intact. Western blot and immunohistochemical analyses of multiple tissues of mice using rabbit antibody raised against a 15-mer synthetic peptide clearly revealed the presence of HAX-1xs protein in the duodenum, and suggested that expression of HAX-1xs and/or HAX-1s was tissue- and cell type-specific. The expression of HAX-1xs and/or HAX-1s was distinctively regulated in Paneth cells of the duodenum and macrophages of the thymus after burn injury. These findings suggest that HAX-1xs has a different biological activity from HAX-1s and participates in a cascade of immediate-early cellular events in response to burn injury.

Burn-induced thymic apoptosis corresponds with altered TGF-beta(1) and Smad 2/3.

Immune suppression is a common complication of injury. Transforming growth factor-beta(1) (TGF-beta(1)), a cytokine with diverse anti-inflammatory and anti-apoptotic effects, may play an important role. Smad 2 and Smad 3 are transcription factors that mediate the effects of TGF-beta(1). We hypothesized that burn-induced immunosuppression would be accompanied by increased apoptosis in lymphoid organs, a change likely associated with changes in TGF-beta(1) and Smad 2/3 expression. Mice were subjected to 18% body surface area flame burn. Lymph nodes, spleen, and thymus were harvested at multiple time points after injury. TGF-beta(1) and Smad 2/3 expression and levels of apoptosis were determined in whole tissue and in sorted T-cells by flow cytometry, RT-PCR, ELISA, and Western blot. TGF-beta(1) protein expression in the thymus increased from 1 to 7 days. Smad 2/3 protein expression was decreased at the same time points. This down-regulation was more dramatic in the non-T-cells than in the T-cells themselves. RT-PCR confirmed down-regulation of Smad 3 mRNA in the thymus from 3 to 6 h. Apoptosis in the thymus doubled at 1 day (6.4% control vs 12.8% burned), which corresponded with a marked decrease in thymus mass on subjective assessment. No changes were observed in other lymphoid organs. Burn injury in mice increases TGF-beta(1) expression in the thymus, while suppressing expression of its intracellular mediator, Smad 2/3. This response is most pronounced in the non-T-cell tissue, which suggests the thymic epithelial cells may be responsible for the increased thymic apoptosis. This TGF-beta(1) and Smad 2/3 counterregulation in response to injury may represent a potential mechanism for postinjury immune suppression.

Induction of murine AIDS virus-related sequences after burn injury.

To better understand the molecular signaling events leading to systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF), changes in gene expression profiles after burn injury were investigated by differential display. C57BLKS/J mice were subjected to 18% total body surface area (TBSA) full-thickness burn and various tissues were harvested at multiple time points after injury. Initial differential display revealed that retroviral transcripts similar to the envelope sequence of murine AIDS (MAIDS) virus were rapidly and transiently up-regulated after injury. Subsequent RT-PCR and DNA sequencing analyses confirmed the transient up-regulation of retroviral sequences similar to those of the MAIDS virus. In addition, the presence and induction of the subgenomic envelope transcripts of these MAIDS virus-related sequences, including a novel double spliced message, were identified after burn injury. These data suggest that the transcriptional efficiency of the integrated retroviral DNA and reactivation of defective MAIDS virus-related sequences may be affected by pathophysiological signals, such as burn injury. The elevated expression of these MAIDS virus-related retroviral sequences may affect the transcriptional activities of the flanking genes at the integration sites and may be a cause of altered local and systemic immune responses to burn-related stress.

Direct quantification of autologous serum albumin leakage after burn injury in mice.

The current technique determining the extent of capillary leakage after injury is to measure the leakage of dye-labeled foreign albumin. A recent report, however, demonstrated that albumin leakage is dependent upon the type of fluorescent dye used for labeling. We chose to develop and test a technique for determining the extent of vascular albumin leakage after burn injury without the use of dyes. Skin and blood samples were harvested at 3h and 7 days after burn injury in mice. Total skin lysates and extracts as well as sera were analyzed for albumin leakage. Coomassie staining and Western blot analyses of skin preparations followed by the densitometric measurement revealed increased levels of albumin, suggesting that the leakage of serum albumin started within 3h after burn injury. Instead of employing dye-labeled foreign albumin, the use of SDS-polyacrylamide gel electrophoresis of tissue extracts followed by Coomassie staining will allow for a simple and direct quantification of autologous albumin leakage due to burn as well as other types of injury.