Differences in arterial and mixed venous IL-6 levels: the lungs as a source of cytokine storm in sepsis.

BACKGROUND: Several investigators have shown that blood levels of interleukin 6 (IL-6) correlate with the severity of illness in critically ill or injured patients. However, little is known about differential arterial and venous blood levels of the cytokine, especially across the lungs. METHODS: We measured differences in IL-6 levels in pulmonary and systemic arterial blood and then documented the production or elimination of IL-6 by the lungs in 19 patients with severe illness. Prospective data were obtained from multiple, simultaneous systemic arterial (ART) and mixed venous (MV) blood samples that were drawn for IL-6 analysis from systemic arterial and pulmonary artery catheters in 7 patients awaiting vascular operation and in 12 trauma patients being treated in the intensive care unit. RESULTS: A lung disorder was present in 5 patients (pneumonia [n = 1], lung trauma [n = 4]) and absent in the remaining 14 patients. The following data were obtained (mean +/- SD) from the highest MV IL-6 levels (pg/mL) in each patient. In patients with a lung disorder (n = 5) compared with those with no disorder (n = 14), ART IL-6 was 9309 +/- 12,521 versus 134 +/- 128 (P =.010), MV IL-6 was 5516 +/- 7420 versus 137 +/- 129 (P =.011), the absolute difference was 3793 +/- 5271 versus -3 +/- 15 (P =.011), and the percentage difference was 37.4% +/- 29.8% versus 1.5% +/- 12.3% (P =.001). The ART and MV IL-6 levels tended to be much higher in the 5 patients with pneumonia (n = 1) and lung injuries (n = 4) than in the patients without apparent pulmonary problems. In addition, the patients with a primary lung disorder demonstrated a net increase in IL-6 levels across the lungs, whereas there was no increase, but rather, a net reduction of IL-6 levels across the lungs in patients without a lung disorder. CONCLUSIONS: The lung appears to be a major producer of IL-6 in patients with an inflammatory lung process. There is a 39% increase in the level of IL-6 as it passes through inflamed lung, producing a marked difference in ART and MV IL-6 levels. Normal lung demonstrated little effect on either ART or MV IL-6 levels.

Surgical reconstruction of the extracranial vertebral artery: management and outcome.

PURPOSE: The purpose of this study was to identify the risk and outcome of reconstruction of the extracranial vertebral artery (ECVA). METHOD: The study was conducted as a retrospective review of 369 consecutive ECVA reconstructions. RESULTS: The clinical presentations consisted of hemispheric symptoms alone in 4% of the cases, hemispheric and vertebrobasilar symptoms in 30%, and vertebrobasilar symptoms alone in 60%. The cause of the lesion was atherosclerosis (n = 300), extrinsic compression (n = 42), dissection (n = 7), radiation arteritis (n = 5), intimal hyperplasia (n = 3), fibromuscular dysplasia (n = 2), previous surgical ligation (n = 3), aneurysm (n = 2), and other (n = 5). All the patients underwent preoperative arteriography. There were 252 proximal ECVA reconstructions (218 transpositions, 42 bypass grafting procedures, and two other) and 117 distal ECVA reconstructions (85 bypass grafting procedures, 25 transpositions, and seven other). In 83 patients, the ECVA operation was performed concomitant with a carotid or supraaortic trunk reconstruction. This series was analyzed in two separate sets: before 1991 (n = 215), when changes in indications and management were occurring; and after 1991 (n = 154), when we acquired a dedicated anesthesia team and digital arteriography in the operating room and established uniform protocols for the management of ECVA disease. The stroke, death, and stroke/death rates for the period before 1991 were, respectively, 4. 1%, 3.2% and 5.1%. The stroke, death, and stroke/death rates for the period after 1991 were, respectively, 1.9%, 0.6% and 1.9%. The patency rate at 5 years was 80%. The survival rate at 5 years was 70%. Most of the deaths during the follow-up period were caused by cardiac disease. Among the survivors, the protection rate from stroke was 97%. CONCLUSION: The changes in operative selection and management have improved the results of ECVA reconstruction. The data reported for ECVA reconstruction in patients who underwent operation since 1991 reflect the outcome of ECVA reconstruction today. In our experience, a reconstruction of the ECVA is less risky than a carotid reconstruction.

Generation of monoclonal antibodies specific for mouse apolipoprotein B-100 in apolipoprotein B-48-only mice.

Over the past 10 years, many laboratories have investigated lipid metabolism and atherogenesis with a variety of transgenic and gene knockout mouse models. Although many of these studies have yielded valuable insights, some have been hampered by a paucity of useful antibodies against mouse proteins. For example, many laboratories have analyzed genetic and dietary interventions affecting lipoprotein metabolism without useful antibodies against mouse apolipoprotein (apo) B. In this study, we sought to develop highly specific monoclonal antibodies against mouse apoB-100. To achieve this goal, gene-targeted mice that synthesize exclusively apoB-48 (apoB-48-only mice) were immunized with mouse apoB-100. The immune response against apoB-100 was robust, as judged by high titers of antibodies against mouse apoB-100. After fusing the splenic lymphocytes of the apoB-48-only mice with a myeloma cell line, we identified and cloned hybridomas that produced mouse apoB-100-specific monoclonal antibodies. Those antibodies were useful for developing sensitive and specific immunoassays for mouse apoB-100. This study illustrates the feasibility and utility of using gene-targeted mice to develop monoclonal antibodies against mouse proteins.

Knockout of the abetalipoproteinemia gene in mice: reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes.

Abetalipoproteinemia, an inherited human disease characterized by a near-complete absence of the apolipoprotein (apo) B-containing lipoproteins in the plasma, is caused by mutations in the gene for microsomal triglyceride transfer protein (MTP). We used gene targeting to knock out the mouse MTP gene (Mttp). In heterozygous knockout mice (Mttp+/- ), the MTP mRNA, protein, and activity levels were reduced by 50%, in both liver and intestine. Compared with control mice (Mttp+/+), chow-fed Mttp+/- mice had reduced plasma levels of low-density lipoprotein cholesterol and had a 28% reduction in plasma apoB100 levels. On a high-fat diet, the Mttp+/- mice exhibited a marked reduction in total plasma cholesterol levels, compared with those in Mttp+/+ mice. Both the livers of adult Mttp+/- mice and the visceral endoderm of the yolk sacs from Mttp+/- embryos manifested an accumulation of cytosolic fat. All homozygous embryos (Mttp-/-) died during embryonic development. In the visceral endoderm of Mttp-/- yolk sacs, lipoprotein synthesis was virtually absent, and there was a marked accumulation of cytosolic fat droplets. In summary, half-normal MTP levels do not support normal levels of lipoprotein synthesis and secretion, and a complete deficiency of MTP causes lethal developmental abnormalities, perhaps because of an impaired capacity of the yolk sac to export lipids to the developing embryo.

Principles for assessment of patient outcomes in mental health care.

With the dramatic changes that are occurring in mental health and substance abuse treatment systems, it is imperative that the field keep its focus on the patient and the patient's outcomes of care. Outcomes management systems that measure the processes of care, the patient's characteristics, and the patient's outcomes of care can be helpful in maintaining this focus. To facilitate the development of these systems, the Outcomes Roundtable, a group of mental health consumer, professional, service, and policy-making organizations, has articulated a set of 12 broadly applicable principles of outcomes assessment. The principles call for outcomes assessments that are appropriate to the question being answered, that use tools with demonstrated validity and reliability and sensitivity to clinically important changes over time, and that always include the consumer perspective. In addition, the principles recommend outcomes assessments that create minimal burden for respondents and are adaptable to different health care systems, that include general health status as well as mental health status, and that include consumers' evaluation of treatment and outcomes. Outcomes assessment tools should quantify the type and extent of treatment, should include generic and disorder-specific information, and should measure areas of personal functioning affected by the disorder. Outcomes should be reassessed at clinically meaningful points in time. Outcomes assessment should use appropriate scientific design and representative samples and should examine outcomes of consumers who prematurely leave treatment as well as those who continue in treatment.

NAMI'S managed care report card. National Alliance for the Mentally Ill.

This article reports on the National Alliance for the Mentally Ill (NAMI) national report card to examine quality of care under managed care. The national report card will provide a summary of state policies underpinning the move of managed care into the public mental health system, and directly probe consumer and family member perceptions and experiences of managed care and the policies and practices of managed care organizations.

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.

Transgenic mice that overexpress mouse apolipoprotein B. Evidence that the DNA sequences controlling intestinal expression of the apolipoprotein B gene are distant from the structural gene.

An 87-kilobase (kb) P1 bacteriophage clone (p649) spanning the mouse apolipoprotein (apo) B gene was used to generate transgenic mice that express high levels of mouse apoB. Plasma levels of apoB, low density lipoprotein cholesterol, and low density lipoprotein triglycerides were increased, and high density lipoprotein cholesterol levels were decreased in the transgenic mice, compared with nontransgenic littermate controls. Although p649 contained 33 kb of 5'-flanking sequences and 11 kb of 3'-flanking sequences, the tissue pattern of transgene expression was different from that of the endogenous apoB gene. RNA slot blots and RNase protection analysis indicated that the transgene was expressed in the liver but not in the intestine, whereas the endogenous apoB gene was expressed in both tissues. To confirm the absence of transgene expression in the intestine, the mouse apoB transgenic mice were mated with the apoB knockout mice, and transgenic mice that were homozygous for the apoB knockout mutation were obtained. Because of the absence of transgene expression in the intestine, those mice lacked all intestinal apoB synthesis, resulting in a marked accumulation of fats within the intestinal villus enterocytes. The current studies, along with prior studies of human apoB transgenic animals, strongly suggest that the DNA sequence element(s) controlling intestinal expression of the apoB gene is located many kilobases from the structural gene.

Mutagenesis of the human apolipoprotein B gene in a yeast artificial chromosome reveals the site of attachment for apolipoprotein(a).

Lipoprotein(a) [Lp(a)] is a lipoprotein formed by the disulfide linkage of apolipoprotein (apo) B100 of a low density lipoprotein particle to apolipoprotein(a). Prior studies have suggested that one of the C-terminal Cys residues of apo-B100 is involved in the disulfide linkage of apo-B100 to apo(a). To identify the apo-B100 Cys residue involved in the formation of Lp(a), we constructed a yeast artificial chromosome (YAC) spanning the human apo-B gene and used gene-targeting techniques to change Cys-4326 to Gly. The mutated YAC DNA was used to generate transgenic mice expressing the mutant human apo-B100 (Cys4326Gly). Unlike the wild-type human apo-B100, the mutant human apo-B100 completely lacked the ability to bind to apo(a) and form Lp(a). This study demonstrates that apo-B100 Cys-4326 is required for the assembly of Lp(a) and shows that gene targeting in YACs, followed by the generation of transgenic mice, is a useful approach for analyzing the structure of large proteins coded for by large genes.

Transgenic mice expressing high levels of human apolipoprotein B develop severe atherosclerotic lesions in response to a high-fat diet.

We previously generated transgenic mice expressing human apolipoprotein (apo-) B and demonstrated that the plasma of chow-fed transgenic animals contained markedly increased amounts of LDL (Linton, M. F., R. V. Farese, Jr., G. Chiesa, D. S. Grass, P. Chin, R. E. Hammer, H. H. Hobbs, and S. G. Young 1992. J. Clin. Invest. 92:3029-3037). In this study, we fed groups of transgenic and nontransgenic mice either a chow diet or a diet high in fat (16%) and cholesterol (1.25%). Lipid and lipoprotein levels were assessed, and after 18 wk of diet, the extent of aortic atherosclerotic lesions in each group of animals was quantified. Compared with the female transgenic mice on the chow diet, female transgenic mice on the high-fat diet had higher plasma levels of cholesterol (312 +/- 17 vs 144 +/- 7 mg/dl; P < 0.0001) and human apo-B (120 +/- 8 vs 84 +/- 3 mg/dl; P < 0.0001). The higher human apo-B levels were due to increased plasma levels of human apo-B48; the human apo-B100 levels did not differ in animals on the two diets. In mice on the high-fat diet, most of the human apo-B48 and apo-B100 was found in LDL-sized particles. Compared with nontransgenic mice on the high-fat diet, the transgenic animals on the high-fat diet had significantly increased levels of total cholesterol (312 +/- 17 vs 230 +/- 19 mg/dl; P < 0.0001) and non-HDL cholesterol (283 +/- 17 vs 193 +/- 19 mg/dl; P < 0.0001). The extent of atherosclerotic lesion development within the ascending aorta was quantified by measuring total lesion area in 60 progressive sections, using computer-assisted image analysis. Neither the chow-fed transgenic mice nor the chow-fed nontransgenic mice had significant atherosclerotic lesions. Nontransgenic animals on the high-fat diet had relatively small atherosclerotic lesions (< 15,000 microns 2/section), almost all of which were confined to the proximal 400 microns of the aorta near the aortic valve. In contrast, transgenic animals on the high-fat diet had extensive atherosclerotic lesions (> 160,000 microns 2/section) that were widely distributed throughout the proximal 1,200 microns of the aorta. Thus, human apo-B expression, in the setting of a diet rich in fats, causes severe atherosclerosis in mice.

Knockout of the mouse apolipoprotein B gene results in embryonic lethality in homozygotes and protection against diet-induced hypercholesterolemia in heterozygotes.

Apolipoprotein B is synthesized by the intestine and the liver in mammals, where it serves as the main structural component in the formation of chylomicrons and very low density lipoproteins, respectively. Apolipoprotein B is also expressed in mammalian fetal membranes. To examine the consequences of apolipoprotein B deficiency in mice, we used gene targeting in mouse embryonic stem cells to generate mice containing an insertional disruption of the 5' region of the apolipoprotein B gene. Mice that were heterozygous for the disrupted apolipoprotein B allele had an approximately 20% reduction in plasma cholesterol levels, markedly reduced plasma concentrations of the pre-beta and beta-migrating lipoproteins, and an approximately 70% reduction in plasma apolipoprotein B levels. When fed a diet rich in fat and cholesterol, heterozygous mice were protected from diet-induced hypercholesterolemia; these mice, which constitute an animal model for hypobetalipoproteinemia, should be useful for studying the effects of decreased apolipoprotein B expression on atherogenesis. The breeding of heterozygous mice yielded no viable homozygous apolipoprotein B knockout mice. Most homozygous embryos were resorbed by midgestation (before gestational day 11.5); several embryos that survived until later in gestation exhibited exencephalus. The embryonic lethal phenotype was rescued by complementation with a human apolipoprotein B transgene--i.e., human apolipoprotein B transgenic mice that were homozygous for the murine apolipoprotein B knockout mutation were viable. Our findings indicate that apolipoprotein B plays an essential role in mouse embryonic development.

Asymptomatic homozygous hypobetalipoproteinemia associated with apolipoprotein B45.2.

Familial hypobetalipoproteinemia is caused by apolipoprotein (apo) B gene mutations and is frequently associated with a truncated apo-B protein in the plasma. Homozygosity for mutations yielding a truncated apo-B is extremely rare; fewer than five true homozygotes have been described in the world's literature. These patients typically have normal levels of triglycerides and virtually absent low density lipoprotein (LDL) cholesterol. The clinical status of these patients is variable, ranging from asymptomatic in two homozygotes who synthesized a truncated apo-B (apo-B87) to severe neurological disease resulting from vitamin E deficiency in a homozygote who synthesized a shorter apo-B (apo-B50). In this report, we describe a 48-year-old female homozygous for a nonsense mutation resulting in an even shorter apo-B, apo-B45.2. Although this individual had virtually no LDL cholesterol, she was asymptomatic and had normal plasma levels of vitamin E. This case demonstrates that homozygosity for an apo-B mutation associated with a relatively short apo-B truncation can be completely asymptomatic.