Targeting adipose tissue via systemic gene therapy.

Adipose tissue has a critical role in energy and metabolic homeostasis, but it is challenging to adapt techniques to modulate adipose function in vivo. Here we develop an in vivo, systemic method of gene transfer specifically targeting adipose tissue using adeno-associated virus (AAV) vectors. We constructed AAV vectors containing cytomegalovirus promoter-regulated reporter genes, intravenously injected adult mice with vectors using multiple AAV serotypes, and determined that AAV2/8 best targeted adipose tissue. Altering vectors to contain adiponectin promoter/enhancer elements and liver-specific microRNA-122 target sites restricted reporter gene expression to adipose tissue. As proof of efficacy, the leptin gene was incorporated into the adipose-targeted expression vector, package into AAV2/8 and administered intravenously to 9- to 10-week-old ob/ob mice. Phenotypic changes were measured over an 8-week period. Leptin mRNA and protein were expressed in adipose and leptin protein was secreted into plasma. Mice responded with reversal of weight gain, decreased hyperinsulinemia and improved glucose tolerance. AAV2/8-mediated systemic delivery of an adipose-targeted expression vector can replace a gene lacking in adipose tissue and correct a mouse model of human disease, demonstrating experimental application and therapeutic potential in disorders of adipose.

Dietary isoflavone intake is associated with evoked responses to inflammatory cardiometabolic stimuli and improved glucose homeostasis in healthy volunteers.

BACKGROUND AND AIMS: Consumption of foods that modulate inflammatory stress in genetically-prone individuals may influence development of cardiometabolic diseases. Isoflavones in soy-derived foods function as phytoestrogens, have antioxidant and anti-inflammatory activity, inhibit protein-tyrosine kinase activity, and may be atheroprotective. We examined the relationship between soy food consumption and inflammatory responses to endotoxemia, postprandial responses to oral lipid tolerance test (OLTT), and insulin sensitivity from frequently sampled intravenous tolerance tests (FSIGTT). METHODS AND RESULTS: We administered low-dose endotoxin (LPS 1 ng/kg) to induce transient endotoxemia in young, healthy volunteers (N = 215) of African (AA), and European (EA) ancestry as part of the GENE Study. We further supported these findings in two independent samples: the MECHE Study and NHANES. Soy food consumption was a significant predictor of peak cytokine response following LPS. Individuals with moderate-high (>1.48 mg/day, N = 65) vs. low-no (<1.48 mg/day, N = 150) isoflavone consumption had significantly higher tumor necrosis factor alpha (TNFα) post-LPS (AUC, P = 0.009). Further, high-isoflavone consumers were protected against inflammation-induced decline in insulin sensitivity (SI) in GENE. We observed significant differences by soy consumption in the interferon gamma (IFNγ) response to OLTT, and the insulin response to OGTT in MECHE, as well as significantly lower fasting insulin, and 2-hour glucose post-OGTT in EA NHANES subjects. CONCLUSION: We demonstrate that soy consumption may influence inflammatory and metabolic responses. In research of nutritional exposures, measuring evoked phenotypes may be more informative than describing resting characteristics. The GENE Study was registered under NCT00953667 and the MECHE Study under NCT01172951, both at clinicaltrials.gov.

Psoriasis is associated with decreased plasma adiponectin levels independently of cardiometabolic risk factors.

BACKGROUND: Psoriasis is an inflammatory skin disease that may be associated with an adverse cardiometabolic profile including modulated plasma adiponectin and leptin levels. Whether these levels are independent of cardiometabolic risk factors, which are also prevalent in psoriasis, is not known. METHODS: A consecutive sample of 122 participants with varying degrees of psoriasis severity, and a random sample of 134 participants without psoriasis, were recruited for this case-control study. Cardiometabolic risk factors including traditional cardiovascular risk factors, waist circumference, insulin resistance, and total plasma adiponectin and leptin were measured. Total plasma adiponectin and leptin levels were compared in unadjusted and adjusted analyses by psoriasis status. RESULTS: Participants with psoriasis had mostly mild disease and were mainly on topical therapies, but still had a more adverse cardiometabolic profile compared with those without psoriasis. Furthermore, plasma adiponectin levels were significantly lower in participants with psoriasis than those without {7.13 µg/mL [interquartile range (IQR) 4.9-11.3) vs. 14.5 µg/mL (IQR 8.4-24.1); P < 0.001]}. Plasma leptin (ng/mL) levels were higher in the psoriasis group but this did not reach statistical significance [11.3 (IQR 6.4-21.8) vs. 9.8 (IQR 4.9-20.5); P = 0.07]. In multivariable modelling, plasma adiponectin levels were still negatively associated with psoriasis status after adjusting for waist size (% difference = -41.2%, P < 0.001), insulin resistance (% difference = -39.5%, P < 0.001), and both waist size and insulin resistance (% difference = -38.5%, P < 0.001). CONCLUSIONS: Plasma levels of adiponectin were lower in psoriasis, and this relationship persisted after adjusting for cardiometabolic risk factors known to decrease adiponectin levels. These findings suggest that inflammation present in psoriasis may be associated with adipose tissue dysfunction; however, direct studies of adipose tissue are needed to confirm this.

Identification of craniofacial risk factors for obstructive sleep apnoea using three-dimensional MRI.

The alteration of craniofacial structures has been associated with obstructive sleep apnoea (OSA). We hypothesised that: 1) a smaller mandible is a risk factor for OSA; and 2) the previously observed inferiorly positioned hyoid bone in apnoeics is associated with enlarged tongue volume. This is a case-control study using three-dimensional magnetic resonance imaging cephalometry. 55 apneics and 55 controls were matched for age, sex and race. The analysis was stratified by sex and controlled for age, race, height, neck visceral fat, skeletal type and tongue volume. We found that a 1-sd increase in mandibular length and depth were associated with decreased risk of sleep apnoea (OR 0.52, 95% CI 0.28-0.99 and OR 0.46, 95% CI 0.23-0.91, respectively) in males but not in females. Greater hyoid-to-nasion (OR 2.64, 95% CI 1.19-5.89 in males and OR 5.01, 95% CI 2.00-12.52 in females) and supramentale-to-hyoid (OR 2.39, 95% CI 1.12-5.14) in males and OR 3.38, 95% CI 1.49-7.68 in females) distances were associated with increased risk of OSA. The difference for hyoid position between apnoeics and controls was lost after controlling for tongue volume. Enlargement of tongue is likely to be the pathogenic factor for inferior-posterior positioning of hyoid. A small and shallow mandible is an independent risk factor for OSA in males but not in females.

Non-random sampling leads to biased estimates of transcriptome association.

Integration of independent data resources across -omics platforms offers transformative opportunity for novel clinical and biological discoveries. However, application of emerging analytic methods in the context of selection bias represents a noteworthy and pervasive challenge. We hypothesize that combining differentially selected samples for integrated transcriptome analysis will lead to bias in the estimated association between predicted expression and the trait. Our results are based on in silico investigations and a case example focused on body mass index across four well-described cohorts apparently derived from markedly different populations. Our findings suggest that integrative analysis can lead to substantial relative bias in the estimate of association between predicted expression and the trait. The average estimate of association ranged from 51.3% less than to 96.7% greater than the true value for the biased sampling scenarios considered, while the average error was - 2.7% for the unbiased scenario. The corresponding 95% confidence interval coverage rate ranged from 46.4% to 69.5% under biased sampling, and was equal to 75% for the unbiased scenario. Inverse probability weighting with observed and estimated weights is applied as one corrective measure and appears to reduce the bias and improve coverage. These results highlight a critical need to address selection bias in integrative analysis and to use caution in interpreting findings in the presence of different sampling mechanisms between groups.

Human sickle hemoglobin in transgenic mice.

DNA molecules that contain the human alpha- and beta s-globin genes inserted downstream of erythroid-specific, deoxyribonuclease I super-hypersensitive sites were coinjected into fertilized mouse eggs and a transgenic mouse line was established that synthesizes human sickle hemoglobin (Hb S). These animals were bred to beta-thalassemic mice to reduce endogenous mouse globin levels. When erythrocytes from these mice were deoxygenated, greater than 90 percent of the cells displayed the same characteristic sickled shapes as erythrocytes from humans with sickle cell disease. Compared to controls the mice have decreased hematocrits, elevated reticulocyte counts, lower hemoglobin concentrations, and splenomegaly, which are all indications of the anemia associated with human sickle cell disease.

Synthesis of functional human hemoglobin in transgenic mice.

Human alpha- and beta-globin genes were separately fused downstream of two erythroid-specific deoxyribonuclease (DNase) I super-hypersensitive sites that are normally located 50 kilobases upstream of the human beta-globin gene. These two constructs were coinjected into fertilized mouse eggs, and expression was analyzed in transgenic animals that developed. Mice that had intact copies of the transgenes expressed high levels of correctly initiated human alpha- and beta-globin messenger RNA specifically in erythroid tissue. An authentic human hemoglobin was formed in adult erythrocytes that when purified had an oxygen equilibrium curve identical to the curve of native human hemoglobin A (Hb A). Thus, functional human hemoglobin can be synthesized in transgenic mice. This provides a foundation for production of mouse models of human hemoglobinopathies such as sickle cell disease.

Prothrombotic factors enhance heparin-induced thrombocytopenia and thrombosis in vivo in a mouse model.

BACKGROUND: Heparin-induced thrombocytopenia/thrombosis (HIT/T) is a common cause of life- and limb-threatening thrombosis. The development of antibodies that react with complexes of heparin and platelet factor 4 (PF4) is fundamental to the development of the disease. However, anti-PF4/heparin antibodies are far more common than is HIT/T and there is less understanding of the factors that contribute to thrombosis in only a subset of patients. OBJECTIVES: Both qualitative and quantitative differences in multiple factors (e.g. antibodies, heparin and platelets) may influence the clinical course of patients who develop anti-PF4/heparin antibodies. We examined the hypothesis that host-specific factors, such as comorbid prothrombotic conditions, would exacerbate the pathologic effects of anti-PF4/heparin antibodies. METHODS AND RESULTS: A mouse model transgenic for human Fcgamma RIIa and PF4 and null for mouse PF4 was used to study the influence of prothrombotic conditions on the effects of anti-PF4/heparin antibodies in vivo. To simulate a prothrombotic milieu, mice were fed a hypercholesterolemic diet (HD). HD-fed mice had elevated plasma cholesterol, increased platelet reactivity and increased endothelial activation relative to mice fed a standard diet (SD). Age- and sex-matched mice from each diet group were treated with an anti-PF4/heparin antibody and heparin. HD-fed mice developed more severe thrombocytopenia than similarly treated SD-fed mice. Mice with moderate to severe thrombocytopenia had elevated plasma levels of thrombin-antithrombin complexes, indicative of increased thrombin generation in vivo. Platelet-fibrin thrombi were observed in multiple organs of HD-fed mice that developed severe thrombocytopenia. CONCLUSIONS: Host-specific factors, such as prothrombotic changes in platelet reactivity and/or endothelial activation, may influence the development of thrombosis in a subset of patients who develop anti-PF4/heparin antibodies.

Relationship between tetramer-dimer assembly and the stability of Hb Malmö (alpha 2 beta 2 97Gln).

The effects of the mutation of the alpha 1 beta 2 contact in Hb Malmö (alpha 2 beta 2 97(FG4)His----Gln) on oxygen-binding properties, ease of dissociation into dimeric hemoglobin and stability were studied. The P50 value of Hb Malmö in the absence of organic phosphates was 1.9 mmHg, in contrast to 8.8 mmHg of Hb A. The n-value of Hb Malmö was 1.6. The overall free energy of interaction of oxygen with Hb Malmö was about 25% that of Hb A. The Adair constant, K1, of Hb Malmö was about 10-times larger than that of Hb A, but the K4 of Hb Malmö was similar to that of Hb A. The liganded form of Hb Malmö was found to dissociate into dimers more readily than Hb A by gel filtration on Sephadex G-100. Dissociation into dimeric hemoglobin was enhanced in dilute solutions. Increased instability during mechanical agitation of diluted samples was greater for Hb Malmö than for Hb A. The denaturation rate constants of tetramers of the oxyform of Hb A and Hb Malmö were about 20-times greater than those of dimers of these hemoglobins. The instability of Hb Malmö depends on a greater alpha 1 beta 2 dissociation constant compared with that of Hb A. These findings allow an examination of the role of the intersubunit contact in determining the functional properties and the stability of the hemoglobin molecule.

Red blood cell membrane and density changes under ambient and hypoxic conditions in transgenic mice producing human sickle hemoglobin.

Red blood cells (RBC) from patients with sickle cell disease (SCD) are characterized by membrane lesions caused by cell sickling and oxidative damage due to denatured hemichromes. We have developed three lines of transgenic human sickle hemoglobin (Hb S) mice, which produce 30, 50, and 80% human beta sickle globin (h beta S), by crossing transgenic progeny with nonthalassemic, heterozygous, or homozygous beta-thalassemic mice, respectively. Transgenic mice that produce Hb A, developed in a similar fashion, were used as controls. RBC from each transgenic line were examined for pathologic changes. RBC from 50 and 80% h beta S mice sickle upon deoxygenation in vitro while RBC from 30% h beta S mice and all Hb A mice do not. Density gradients of RBC from each Hb S line, including those from 30% h beta S that do not sickle, show broad distributions with increased dense fractions, similar to those of patients with SCD. RBC from Hb S lines exhibit elevated membrane-associated denatured hemoglobin (MADH) levels (0.250 +/- 0.080%) when compared to RBC from nontransgenic (0.073 +/- 0.021%) and transgenic Hb A (0.062 +/- 0.033%) mice. Elevated MADH levels in RBC from the 30% h beta S line suggest that membrane changes occur even though these cells do not sickle. These Hb S-dependent pathologic changes suggest that transgenic Hb S lines may be useful for the study of not only RBC sickling in vivo but also membrane oxidative damage and other chronic changes attributed to abnormal properties of both oxygenated and deoxygenated Hb S.

Frequency, risk factors, and clinical outcomes of left ventricular assist device-associated ventricular thrombus.

A retrospective, transesophageal study of 51 consecutive patients receiving a left ventricular (LV) assist device (AD) over a 2-year period showed that LVAD-associated LV thrombosis (16%) was predicted by acute myocardial infarction, atrial cannulation, and postimplantation bleeding, and was associated with a fourfold increased risk of stroke compared with patients without thrombosis. LV cannulation, when using short-term LVADs, may decrease the incidence of LV thrombosis, and early transition to Heartmate-LVAD support may improve outcome.

Cerebrovascular emboli related to pulmonary venous thrombosis after lung transplantation.

We describe a case of cerebral emboli related to pulmonary venous thrombosis after bilateral lung transplantation in a young man with cystic fibrosis. The diagnosis was made by transesophageal echocardiography, leading to aggressive anticoagulation within 24 hours of surgery. Hemodynamic deterioration in the following hours was of concern for the development of obstructive thrombus but was found to be due to pericardial tamponade, which, remarkably, resolved during a repeat transesophageal echocardiography.

Impulsivity and voucher versus money preference in polydrug-dependent participants enrolled in a contingency-management-based substance abuse treatment program.

Thirty-four polydrug-dependent participants enrolled in a voucher-based substance abuse treatment program were given choices between hypothetical amounts of money and hypothetical amounts of vouchers, which are traded for goods and services, to determine their preferences for the two payment modalities. It was hypothesized that the majority of participants would prefer money to voucher because under the circumstances of the treatment program, the delay associated with money exchange is shorter than the delay associated with voucher exchange. It was further hypothesized that those participants who selected money over voucher also would have greater levels of impulsivity as assessed by the Barratt Impulsiveness Rating Scale (BIS) (Barratt, 1965). The results show large individual differences in money/voucher preference with approximately half of the participants preferring money to voucher when the two amounts are equivalent. In addition, as the magnitude of the money/voucher comparisons increased from 0.50 dollars to 32.00 dollars, the percentage of participants that preferred money increased. No correlations were found between money/voucher preference and impulsivity scores.

No thanks, I'm good. Any more and I'll be sick: comment on Lynch and Carroll (2001).

W. J. Lynch and M. E. Carroll's (2001) excellent analyses of drug intake from a regulation perspective are formalized in terms of control systems. Satiation corresponds to the set point, deviations below which are called hunger or craving, deviations above which are called surfeit. Although simple, the model provides a unifying framework for many of the phenomena Lynch and Carroll describe.

The influence of exchange delays on cigarette versus money choice: a laboratory analog of voucher-based reinforcement therapy.

Contingency-management interventions that provide reinforcement in the form of exchangeable vouchers, contingent on drug abstinence, are among the most effective substance abuse treatment strategies available. Factors known to contribute to the efficacy of these interventions include voucher magnitude and the schedule with which vouchers are made available. Another potential factor may be the delay between earning a voucher and exchanging it for a desired good or service. The authors adapted a laboratory analog of a voucher program to examine the effects of immediacy of reinforcement and its interaction with reinforcer magnitude. Abstinent cigarette smokers made repeated choices between puffs on a cigarette and points worth a variety of monetary values (10 cents-2 dollars). The time at which these points could be exchanged for money varied from the end of the session to 1 or 3 weeks. Results indicated that longer exchange delays and tower magnitude reinforcers increased the number of choices for drug.

Effect of an allosteric modifier of hemoglobin, RSR-4, on oxygen affinity and oxygen saturation of hemoglobin in rabbits.

Theoretically, if the arterial partial oxygen pressure (PaO2) does not change, a right shift in the oxygen equilibrium curve (OEC) of hemoglobin should reduce arterial oxygen saturation. In this study we investigate whether a right shift in the OEC of hemoglobin decreases transcutaneous oxygen saturation (Tc-SO2) following the administration of an allosteric effector, 2-[4-(((3, 5-dichloroanilino)-carbonyl) methyl) phenoxy]-2-methylpropionic acid (RSR-4). The effect of RSR-4 on hemoglobin oxygen affinity was studied in four New Zealand white male rabbits. Following intraperitoneal administration of RSR-4, Tc-SO2 decreased in a dose-dependent manner. P50 (partial oxygen pressure at 50% hemoglobin oxygen saturation) in whole blood increased as the concentration of RSR-4 increased. Tc-SO2 decreased as whole-blood affinity (1/P50) decreased. There was no positive correlation between Tc-SO2 and PaO2. We concluded that a decrease in hemoglobin oxygen affinity following RSR-4 administration reduced arterial oxygen saturation. This decrease in the presence of an allosteric effector such as RSR-4 in vivo can be detected and monitored as a reduction in Tc-SO2.

Mechanisms underlying the effects of unsignaled delayed reinforcement on key pecking of pigeons under variable-interval schedules.

Three experiments were conducted to test an interpretation of the response-rate-reducing effects of unsignaled nonresetting delays to reinforcement in pigeons. According to this interpretation, rates of key pecking decrease under these conditions because key pecks alternate with hopper-observing behavior. In Experiment 1, 4 pigeons pecked a food key that raised the hopper provided that pecks on a different variable-interval-schedule key met the requirements of a variable-interval 60-s schedule. The stimuli associated with the availability of the hopper (i.e., houselight and keylight off, food key illuminated, feedback following food-key pecks) were gradually removed across phases while the dependent relation between hopper availability and variable-interval-schedule key pecks was maintained. Rates of pecking the variable-interval-schedule key decreased to low levels and rates of food-key pecks increased when variable-interval-schedule key pecks did not produce hopper-correlated stimuli. In Experiment 2, pigeons initially pecked a single key under a variable-interval 60-s schedule. Then the dependent relation between hopper presentation and key pecks was eliminated by arranging a variable-time 60-s schedule. When rates of pecking had decreased to low levels, conditions were changed so that pecks during the final 5 s of each interval changed the keylight color from green to amber. When pecking produced these hopper-correlated stimuli, pecking occurred at high rates, despite the absence of a peck-food dependency. When peck-produced changes in keylight color were uncorrelated with food, rates of pecking fell to low levels. In Experiment 3, details (obtained delays, interresponse-time distributions, eating times) of the transition from high to low response rates produced by the introduction of a 3-s unsignaled delay were tracked from session to session in 3 pigeons that had been initially trained to peck under a conventional variable-interval 60-s schedule. Decreases in response rates soon after the transition to delayed reinforcement were accompanied by decreases in eating times and alterations in interresponse-time distributions. As response rates decreased and became stable, eating times increased and their variability decreased. These findings support an interpretation of the effects of delayed reinforcement that emphasizes the importance of hopper-observing behavior.

Discrimination of methadone and cocaine by pigeons without explicit discrimination training.

Pigeons were trained to peck a key on a variable-interval 2-min schedule of food reinforcement. Prior to each session, either 2.0 mg/kg methadone (n = 3), 3.0 mg/kg cocaine (n = 4), or 5.6 mg/kg cocaine (n = 2) was administered. When each pigeon's rate of pecking was stable, a range of doses of the training drug and saline were administered prior to 20-min extinction sessions separated by at least four training sessions. Rate of pecking during these extinction tests was generally an increasing function of dose, with the lowest rates obtained following saline and low doses and the highest rates obtained following doses near the training doses. Dose functions from pigeons trained with 5.6 mg/kg cocaine were steeper than those from pigeons trained with 3.0 mg/kg cocaine. Pigeons trained with methadone or 3.0 mg/kg cocaine were then given discrimination training, in which food reinforcement followed drug administration and 20-min extinction sessions followed saline administration. Rates of pecking under these conditions quickly diverged until near-zero rates were obtained following saline and high rates were obtained following drug. Discrimination training steepened dose functions for the training drugs, and the effects of several other substituted drugs depended on the pharmacology of the training drug. The pigeons trained with 5.6 mg/kg cocaine were tested with d-amphetamine, methadone, and morphine prior to discrimination training. d-Amphetamine increased rates dose dependently, and methadone and morphine did not. The results suggest that discriminative control by methadone and cocaine was established without explicit discrimination training.

Polymorphism of feline beta-globins studied by reversed-phase high-performance liquid chromatography.

OBJECTIVE: To characterize the feline beta-globin system with respect to the number and genetics of adult beta-globin chains. ANIMALS: 84 domestic and purebred cats (9 breeds), 14 cats were mildly to severely anemic. For genetic analyses, 12 litters with 1 to 5 offspring for a total of 29 kittens. PROCEDURE: Feline globins from erythrocyte lysates were separated by reversed-phase high-performance liquid chromatography and analyzed on the basis of retention time and area of each beta-globin peak. Mixing studies with half the amount of hemoglobin (Hb) from 2 cats were performed to assign the beta-globin peaks in each cat. The Hb from 60 cats were also separated by standard isoelectric focusing methods. RESULTS: Heme and alpha- and beta-globins were effectively separated by high-performance liquid chromatography. Although there was only 1 alpha-globin, a total of 6 beta-globins (I through VI) were identified, with each cat having 1 to 4 beta-globin chains. From analysis of the number of beta-globins, their relative amounts, and Hb mixing studies, 17 beta-globin patterns were observed. The beta-globin pattern in healthy and anemic cats was identical. Family studies documented an autosomal codominant mode of inheritance of the adult beta-globins and a linkage of 2 beta-chains forming haplotypes. CONCLUSIONS: Biochemical and genetic evidence for the greatest polymorphism of adult beta-globins thus far described in any species is provided. A feline beta-globin gene region with 2 linked beta-globin gene loci and 2 and 5 alleles is proposed. CLINICAL RELEVANCE: Better understanding of feline Hb will likely be helpful for diagnosis of hemoglobinopathies in anemic cats.

The renaissance of the experimental analysis of human behavior.

Ten years ago, a number of authors commented on the dismal state of the basic research area known as the experimental analysis of human behavior (EAHB). At that time, data on the number of research articles using human subjects published in the Journal of the Experimental Analysis of Behavior (JEAB) indicated little progress since the early 1960s. However, updated publication data through 1991 reveal that EAHB research has accelerated in the last decade, reaching a peak of nearly half of all research articles published in JEAB, with an increasing trend evident. The increase in this percentage is not due solely to a long-term declining trend in the total number of experimental articles in JEAB using either human or nonhuman subjects, a trend that appears to have slowed or stabilized in the last 6 years. These data indicate that the EAHB has made dramatic progress in a decade and is healthy and growing.