HMGB1 acts as an agent of host defense at the gut mucosal barrier.

Mucosal barriers provide the first line of defense between internal body surfaces and microbial threats from the outside world. 1 In the colon, the barrier consists of two layers of mucus and a single layer of tightly interconnected epithelial cells supported by connective tissue and immune cells. 2 Microbes colonize the loose, outer layer of colonic mucus, but are essentially excluded from the tight, epithelial-associated layer by host defenses. 3 The amount and composition of the mucus is calibrated based on microbial signals and loss of even a single component of this mixture can destabilize microbial biogeography and increase the risk of disease. 4-7 However, the specific components of mucus, their molecular microbial targets, and how they work to contain the gut microbiota are still largely unknown. Here we show that high mobility group box 1 (HMGB1), the prototypical damage-associated molecular pattern molecule (DAMP), acts as an agent of host mucosal defense in the colon. HMGB1 in colonic mucus targets an evolutionarily conserved amino acid sequence found in bacterial adhesins, including the well-characterized Enterobacteriaceae adhesin FimH. HMGB1 aggregates bacteria and blocks adhesin-carbohydrate interactions, inhibiting invasion through colonic mucus and adhesion to host cells. Exposure to HMGB1 also suppresses bacterial expression of FimH. In ulcerative colitis, HMGB1 mucosal defense is compromised, leading to tissue-adherent bacteria expressing FimH. Our results demonstrate a new, physiologic role for extracellular HMGB1 that refines its functions as a DAMP to include direct, virulence limiting effects on bacteria. The amino acid sequence targeted by HMGB1 appears to be broadly utilized by bacterial adhesins, critical for virulence, and differentially expressed by bacteria in commensal versus pathogenic states. These characteristics suggest that this amino acid sequence is a novel microbial virulence determinant and could be used to develop new approaches to diagnosis and treatment of bacterial disease that precisely identify and target virulent microbes.

Transgenic expression of the coxsackie/adenovirus receptor enables adenoviral-mediated gene delivery in naive T cells.

The inability to easily and efficiently introduce genes into primary T cells has hampered the investigation of the pathways controlling T cell fate. To enable adenoviral-mediated gene transfer into normal naive T cells, transgenic (Tg) mice expressing the coxsackie/adenovirus receptor (CAR) in their T cell compartment were constructed. Whereas naive T cells are resistant to adenoviral infection, Tg expression of CAR on T cells greatly facilitates adenoviral-mediated gene expression ex vivo, in vivo, and in differentiated T helper cells. Thus we have developed a technology for efficient gene delivery to naive T cells. By using adenoviral vectors encoding specific inhibitors, we show that G1 cyclin-dependent kinase, NF-kappaB, and caspase activities are required for the proliferation of primary T cells. In addition, by expressing Bcl-x(L) protein at a level that closely approximates mitogen-induced levels, we demonstrate that Bcl-x(L) expression is sufficient to account for mitogen-mediated survival of primary T cells. Thus, adenoviral-mediated gene delivery to CAR Tg T cells should be useful for the analysis of many genes controlling T cell fate.

Spontaneous multiple cholecystoenteric fistulas--a case report.

Spontaneous multiple cholecystoenteric fistulas are relatively rare complications of chronic cholecystitis. One cholecystoduodenal and two cholecystocolonic fistulas were observed in a 65-year-old woman whose symptoms included fever, chills, jaundice, diarrhea, and prolonged right upper quadrant pain. Pneumobilia, which is a pathognomonic sign of bilioenteric fistula, was also detected by her plain abdomen X-ray on admission. Both types of fistulas were correctly diagnosed preoperatively by barium enema, upper GI series and endoscopic retrograde cholangiopancreaticography. The patient was referred for surgery and fistulas were identified during laparotomy. Cholecystectomy, division of these fistulas, and primary repair of these bowel defects were successfully performed. The postoperative course was unremarkable. We report this unusual case and briefly review the hypothesized pathogenesis, typical symptomatology, radiographic diagnosis, complications and therapeutic modalities of this condition.

Chylomicron-sized lipid particles are formed in the setting of apolipoprotein B deficiency.

The mechanisms for packaging large quantities of neutral lipids into apolipoprotein (apo) B-containing lipoproteins (chylomicrons or VLDL) are incompletely understood. However, several lines of evidence have suggested that the addition of core lipids to apoB to form a lipoprotein particle within the endoplasmic reticulum (ER) may involve two steps: first, the addition of small amounts of core lipids to membrane-bound apoB, generating a lipid-poor, small apoB-containing particle, and second, the fusion of that particle with a larger, independently formed triglyceride-rich and apoB-free "lipid particle." We sought to test this two-step hypothesis of apoB core lipidation by using electron microscopy to compare chylomicron assembly in mice that are genetically deficient in the ability to synthesize apoB in the intestine to control mice. In 19-day gestational mice (fasting setting) that were deficient in intestinal apoB synthesis, chylomicron-sized lipid particles in the lumen of the enterocyte ER were even more abundant and were 2- to 3-fold larger than those in the enterocytes of normal control mice. However, there were fewer lipid-staining particles in the Golgi apparatus, and many fewer particles in the extracellular space, compared with normal control mice. In both types of newborn suckling mice, much larger lipid particles were assembled within the lumen of the ER. They were however, less abundant and rarely reached the Golgi apparatus in fatty enterocytes of intestines deficient in apoB synthesis. These observations provide in vivo evidence that chylomicron formation could involve the synthesis of apoB-free triglyceride-rich particles within the endoplasmic reticulum (ER) lumen, and that the transport of these lipid particles out of the ER to Golgi apparatus and interstitium is facilitated by the acquisition of apoB.

Dual mechanisms for the low plasma levels of truncated apolipoprotein B proteins in familial hypobetalipoproteinemia. Analysis of a new mouse model with a nonsense mutation in the Apob gene.

Familial hypobetalipoproteinemia (FHbeta), a syndrome characterized by low plasma cholesterol levels, is caused by mutations in the apo-B gene that interfere with the synthesis of apo-B100. FHbeta mutations frequently lead to the synthesis of a truncated form of apo-B, which typically is present in plasma at < 5% of the levels of apo-B100. Although many FHbeta mutations have been characterized, the basic mechanisms causing the low plasma levels of truncated apo-B variants have not been defined. We used gene targeting to create a mutant allele that exclusively yields a truncated apo-B, apo-B83. In mice heterozygous for the Apob83 allele, plasma levels and the size and density distribution of apo-B83-containing lipoproteins were strikingly similar to those observed in humans with FHbeta and an apo-B83 mutation. Analysis of mice carrying the Apob83 mutation revealed two mechanisms for the low plasma levels of apo-B83. First, Apob83 mRNA levels and apo-B83 secretion were reduced 76 and 72%, respectively. Second, apo-B83 was removed rapidly from the plasma, compared with apo-B100. This mouse model provides a new level of understanding of FHbeta and adds new insights into apo-B metabolism.

Transgenic mice expressing human ApoB95 and ApoB97. Evidence that sequences within the carboxyl-terminal portion of human apoB100 are important for the assembly of lipoprotein.

The structural features of apolipoprotein (apo) B that are important for its covalent linkage to apo(a) to form lipoprotein(a) (Lp(a)) are incompletely understood. Although apoB100 cysteine 4326 is required for the disulfide linkage with apo(a), other structural features, aside from a single free cysteine residue, must be important for apoB's initial interaction with apo(a) and for facilitating the formation of the disulfide bond. To determine if sequences carboxyl-terminal to cysteine 4326 affect the efficiency of Lp(a) formation, we used "pop-in, pop-out" gene targeting in a human apoB yeast artificial chromosome to introduce nonsense mutations into exon 29 of the apoB gene. The mutant yeast artificial chromosomes, which coded for the truncated versions of human apoB, apoB95, and apoB97, were then used to express these mutant forms of apoB in transgenic mice. As judged by in vitro assays of Lp(a) formation, apoB95 (4330 amino acids) formed a small amount of Lp(a) but did so slowly. In contrast, apoB97 (4397 amino acids) formed Lp(a) rapidly, although not quite as rapidly as the full-length apoB100 (4536 amino acids). These results were supported by an analysis of double-transgenic mice expressing both human apo(a) and either apoB95 or apoB97. In mice expressing both apoB95 and apo(a), there was only a trace amount of Lp(a) in the plasma, and most of the apo(a) was free, whereas in mice expressing both apoB97 and apo(a), virtually all of the apo(a) was bound to apoB97 in the form of Lp(a). These results show that sequences carboxyl-terminal to apoB95 (amino acids 4331-4536) are not absolutely required for Lp(a) formation, but this segment of the apoB molecule, particularly residues 4331-4397, is necessary for the efficient assembly of Lp(a).

Susceptibility to atherosclerosis in mice expressing exclusively apolipoprotein B48 or apolipoprotein B100.

All classes of lipoproteins considered to be atherogenic contain apo-B100 or apo-B48. However, there is a distinct paucity of data regarding whether lipoproteins containing apo-B48 or apo-B100 differ in their intrinsic ability to promote the development of atherosclerosis. To address this issue, we compared the extent of atherosclerosis in three groups of animals: apo-E-deficient mice (apo-B+/+apo-E-/-) and apo-E-deficient mice that synthesize exclusively either apo-B48 (apo-B48/48apo-E-/-) or apo-B100 (apo-B100/100apo-E-/-). Mice (n = 25 in each group) were fed a chow diet for 200 days, and plasma lipid levels were assessed throughout the study. Compared with the levels in apo-B+/+apo-E-/- mice, the total plasma cholesterol levels were higher in the apo-B48/48apo-E-/- mice and were lower in the apo-B100/100apo-E-/- mice. However, the ranges of cholesterol levels in the three groups overlapped. Compared with those in the apo-B+/+apo-E-/- mice, atherosclerotic lesions were more extensive in the apo-B48/48apo-E-/- mice and less extensive in the apo-B100/100apo-E-/- mice. Once again, however, there was overlap among the three groups. The extent of atherosclerosis in each group of mice correlated significantly with plasma cholesterol levels. In mice from different groups that had similar cholesterol levels, the extent of atherosclerosis was quite similar. Thus, susceptibility to atherosclerosis was dependent on total cholesterol levels. Whether mice synthesized apo-B48 or apo-B100 did not appear to have an independent effect on susceptibility to atherosclerosis.

Phenotypic analysis of mice expressing exclusively apolipoprotein B48 or apolipoprotein B100.

Apolipoprotein (apo)-B is found in two forms in mammals: apo-B100, which is made in the liver and the yolk sac, and apo-B48, a truncated protein made in the intestine. To provide models for understanding the physiologic purpose for the two forms of apo-B, we used targeted mutagenesis of the apo-B gene to generate mice that synthesize exclusively apo-B48 (apo-B48-only mice) and mice that synthesize exclusively apo-B100 (apo-B100-only mice). Both the apo-B48-only mice and apo-B100-only mice developed normally, were healthy, and were fertile. Thus, apo-B48 synthesis was sufficient for normal embryonic development, and the synthesis of apo-B100 in the intestines of adult mice caused no readily apparent adverse effects on intestinal function or nutrition. Compared with wild-type mice fed a chow diet, the levels of low density lipoprotein (LDL)-cholesterol and very low density lipoprotein- and LDL-triacylglycerols were lower in apo-B48-only mice and higher in the apo-B100-only mice. In the setting of apo-E-deficiency, the apo-B100-only mutation lowered cholesterol levels, consistent with the fact that apo-B100-lipoproteins can be cleared from the plasma via the LDL receptor, whereas apo-B48-lipoproteins lacking apo-E cannot. The apo-B48-only and apo-B100-only mice should prove to be valuable models for experiments designed to understand the purpose for the two forms of apo-B in mammalian metabolism.

A genetic model for absent chylomicron formation: mice producing apolipoprotein B in the liver, but not in the intestine.

The formation of chylomicrons by the intestine is important for the absorption of dietary fats and fat-soluble vitamins (e.g., retinol, alpha-tocopherol). Apo B plays an essential structural role in the formation of chylomicrons in the intestine as well as the VLDL in the liver. We have developed genetically modified mice that express apo B in the liver but not in the intestine. By electron microscopy, the enterocytes of these mice lacked nascent chylomicrons in the endoplasmic reticulum and Golgi apparatus. Because these mice could not form chylomicrons, the intestinal villus enterocytes were massively engorged with fat, which was contained in cytosolic lipid droplets. These mice absorbed D-xylose normally, but there was virtually no absorption of retinol palmitate or cholesterol. The levels of alpha-tocopherol in the plasma were extremely low. Of note, the absence of chylomicron synthesis in the intestine did not appear to have a significant effect on the plasma levels of the apo B-containing lipoproteins produced by the liver. The mice lacking intestinal apo B expression represent the first genetic model of defective absorption of fats and fat-soluble vitamins and provide a useful animal model for studying nutrition and lipoprotein metabolism.