A 600 kb deletion syndrome at 16p11.2 leads to energy imbalance and neuropsychiatric disorders

BACKGROUND: The recurrent ~600 kb 16p11.2 BP4-BP5 deletion is among the most frequent known genetic aetiologies of autism spectrum disorder (ASD) and related neurodevelopmental disorders. OBJECTIVE: To define the medical, neuropsychological, and behavioural phenotypes in carriers of this deletion. METHODS: We collected clinical data on 285 deletion carriers and performed detailed evaluations on 72 carriers and 68 intrafamilial non-carrier controls. RESULTS: When compared to intrafamilial controls, full scale intelligence quotient (FSIQ) is two standard deviations lower in carriers, and there is no difference between carriers referred for neurodevelopmental disorders and carriers identified through cascade family testing. Verbal IQ (mean 74) is lower than non-verbal IQ (mean 83) and a majority of carriers require speech therapy. Over 80% of individuals exhibit psychiatric disorders including ASD, which is present in 15% of the paediatric carriers. Increase in head circumference (HC) during infancy is similar to the HC and brain growth patterns observed in idiopathic ASD. Obesity, a major comorbidity present in 50% of the carriers by the age of 7 years, does not correlate with FSIQ or any behavioural trait. Seizures are present in 24% of carriers and occur independently of other symptoms. Malformations are infrequently found, confirming only a few of the previously reported associations. CONCLUSIONS: The 16p11.2 deletion impacts in a quantitative and independent manner FSIQ, behaviour and body mass index, possibly through direct influences on neural circuitry. Although non-specific, these features are clinically significant and reproducible. Lastly, this study demonstrates the necessity of studying large patient cohorts ascertained through multiple methods to characterise the clinical consequences of rare variants involved in common diseases.

Cholesteryl ester accumulation and accelerated cholesterol absorption in intestine-specific hormone sensitive lipase-null mice

Hormone sensitive lipase (HSL) regulates the hydrolysis of acylglycerols and cholesteryl esters (CE) in various cells and organs, including enterocytes of the small intestine. The physiological role of this enzyme in enterocytes, however, stayed elusive. In the present study we generated mice lacking HSL exclusively in the small intestine (HSLiKO) to investigate the impact of HSL deficiency on intestinal lipid metabolism and the consequences on whole body lipid homeostasis. Chow diet-fed HSLiKO mice showed unchanged plasma lipid concentrations. In addition, feeding with high fat/high cholesterol (HF/HC) diet led to unaltered triglyceride but increased plasma cholesterol concentrations and CE accumulation in the small intestine. The same effect was observed after an acute cholesterol load. Gavaging of radioactively labeled cholesterol resulted in increased abundance of radioactivity in plasma, liver and small intestine of HSLiKO mice 4h post-gavaging. However, cholesterol absorption determined by the fecal dual-isotope ratio method revealed no significant difference, suggesting that HSLiKO mice take up the same amount of cholesterol but in an accelerated manner. mRNA expression levels of genes involved in intestinal cholesterol transport and esterification were unchanged but we observed downregulation of HMG-CoA reductase and synthase and consequently less intestinal cholesterol biosynthesis. Taken together our study demonstrates that the lack of intestinal HSL leads to CE accumulation in the small intestine, accelerated cholesterol absorption and decreased cholesterol biosynthesis, indicating that HSL plays an important role in intestinal cholesterol homeostasis.

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Elevated transaminases in asymptomatic patients can be detected in more than 5 % of the investigations. If there are no obvious reasons, the finding should be confirmed within the next 3 months. Frequent causes are non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcohol, hepatitis B or C, hemochromatosis and drugs or toxins. Rarer causes are autoimmune hepatitis, M. Wilson and α1-antitrypsine deficiency. There are also non-hepatic causes such as celiac disease or hemolysis and myopathies in the case of an exclusive increase of ASAT. I recommend a two-step investigational procedure; the more frequent causes are examined first before the rare causes are studied. The value of the proposed investigations is discussed.

Pitfalls when examining gap junction hemichannels: interference from volume-regulated anion channels

Human HeLa cells transfected with mouse connexin45 were used to explore the experimental conditions suitable to measure currents carried by gap junction hemichannels. Experiments were performed with a voltage-clamp technique and whole-cell recording. Lowering [Ca(2+)](o) from 2 mM to 20 nM evoked an extra current, I (m), putatively carried by Cx45 hemichannels. However, the variability of I (m) (size, voltage sensitivity, kinetics) suggested the involvement of other channels. The finding that growth medium in the incubator increased the osmolarity with time implied that volume-regulated anion channels (VRAC) may participate. This assumption was reinforced by the following observations. On the one hand, keeping [Ca(2+)](o) normal while the osmolarity of the extracellular solution was reduced from 310 to 290 mOsm yielded a current characteristic of VRAC; I (VRAC) activated/deactivated at negative/positive voltage, giving rise to the conductance functions g (VRAC,inst)=f(V (m)) (inst: instantaneous; V (m): membrane potential) and g (VRAC,ss)=f(V (m)) (ss: steady state). Moreover, it was reversibly inhibited by mibefradil, a Cl(-)channel blocker (binding constant K (d)=38 microM, Hill coefficient n=12), but not by the gap junction channel blocker 18alpha-glycyrrhetinic acid. On the other hand, minimizing the osmotic imbalance while [Ca(2+)](o) was reduced led to a current typical for Cx45 hemichannels; I (hc) activated/deactivated at positive/negative voltage. Furthermore, it was reversibly inhibited by 18alpha-glycyrrhetinic acid or palmitoleic acid, but not by mibefradil. Computations based on g (VRAC,ss)=f(V (m)) and g (hc,ss)=f(V (m)) indicated that the concomitant operation of both currents results in a bell-shaped conductance-voltage relationship. The functional implications of the data presented are discussed. Conceivably, VRAC and hemichannels are involved in a common signaling pathway.

Prevalence of risk factors for cardiovascular disease in HIV-infected patients over time: the Swiss HIV Cohort Study

OBJECTIVE: Metabolic changes caused by antiretroviral therapy (ART) may increase the risk of coronary heart disease (CHD). We evaluated changes in the prevalence of cardiovascular risk factors (CVRFs) and 10-year risk of CHD in a large cohort of HIV-infected individuals. METHODS: All individuals from the Swiss HIV Cohort Study (SHCS) who completed at least one CVRF questionnaire and for whom laboratory data were available for the period February 2000 to February 2006 were included in the analysis. The presence of a risk factor was determined using cut-offs based on the guidelines of the National Cholesterol Education Program (NCEP ATP III), the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC7), the American Diabetes Association, and the Swiss Society for Cardiology. RESULTS: Overall, 8,033 individuals completed at least one CVRF questionnaire. The most common CVRFs in the first completed questionnaire were smoking (57.0%), low high-density lipoprotein (HDL) cholesterol (37.2%), high triglycerides (35.7%), and high blood pressure (26.1%). In total, 2.7 and 13.8% of patients were categorized as being at high (>20%) and moderate (10-20%) 10-year risk for CHD, respectively. Over 6 years the percentage of smokers decreased from 61.4 to 47.6% and the percentage of individuals with total cholesterol >6.2 mmol/L decreased from 21.1 to 12.3%. The prevalence of CVRFs and CHD risk was higher in patients currently on ART than in either pretreated or ART-naive patients. CONCLUSION: During the 6-year observation period, the prevalence of CVRFs remains high in the SHCS. Time trends indicate a decrease in the percentage of smokers and individuals with high cholesterol.

Intramyocellular lipid stores increase markedly in athletes after 1.5 days lipid supplementation and are utilized during exercise in proportion to their content

Intramyocellular lipids (IMCL) and muscle glycogen provide local energy during exercise (EX). The objective of this study was to clarify the role of high versus low IMCL levels at equal initial muscle glycogen on fuel selection during EX. After 3 h of depleting exercise, 11 endurance-trained males consumed in a crossover design a high-carbohydrate (7 g kg(-1) day(-1)) low-fat (0.5 g kg(-1) day(-1)) diet (HC) for 2.5 days or the same diet with 3 g kg(-1) day(-1) more fat provided during the last 1.5 days of diet (four meals; HCF). Respiratory exchange, thigh muscle substrate breakdown by magnetic resonance spectroscopy, and plasma FFA oxidation ([1-(13)C]palmitate) were measured during EX (3 h, 50% W (max)). Pre-EX IMCL concentrations were 55% higher after HCF. IMCL utilization during EX in HCF was threefold greater compared with HC (P < 0.001) and was correlated with aerobic power and highly correlated (P < 0.001) with initial content. Glycogen values and decrements during EX were similar. Whole-body fat oxidation (Fat(ox)) was similar overall and plasma FFA oxidation smaller (P < 0.05) during the first EX hour after HCF. Myocellular fuels contributed 8% more to whole-body energy demands after HCF (P < 0.05) due to IMCL breakdown (27% Fat(ox)). After EX, when both IMCL and glycogen concentrations were again similar across trials, a simulated 20-km time-trial showed no difference in performance between diets. In conclusion, IMCL concentrations can be increased during a glycogen loading diet by consuming additional fat for the last 1.5 days. During subsequent exercise, IMCL decrease in proportion to their initial content, partly in exchange for peripheral fatty acids.

Divalent metal-ion transporter DMT1 mediates both H+ -coupled Fe2+ transport and uncoupled fluxes

The H(+) -coupled divalent metal-ion transporter DMT1 serves as both the primary entry point for iron into the body (intestinal brush-border uptake) and the route by which transferrin-associated iron is mobilized from endosomes to cytosol in erythroid precursors and other cells. Elucidating the molecular mechanisms of DMT1 will therefore increase our understanding of iron metabolism and the etiology of iron overload disorders. We expressed wild type and mutant DMT1 in Xenopus oocytes and monitored metal-ion uptake, currents and intracellular pH. DMT1 was activated in the presence of an inwardly directed H(+) electrochemical gradient. At low extracellular pH (pH(o)), H(+) binding preceded binding of Fe(2+) and its simultaneous translocation. However, DMT1 did not behave like a typical ion-coupled transporter at higher pH(o), and at pH(o) 7.4 we observed Fe(2+) transport that was not associated with H(+) influx. His(272) --> Ala substitution uncoupled the Fe(2+) and H(+) fluxes. At low pH(o), H272A mediated H(+) uniport that was inhibited by Fe(2+). Meanwhile H272A-mediated Fe(2+) transport was independent of pH(o). Our data indicate (i) that H(+) coupling in DMT1 serves to increase affinity for Fe(2+) and provide a thermodynamic driving force for Fe(2+) transport and (ii) that His-272 is critical in transducing the effects of H(+) coupling. Notably, our data also indicate that DMT1 can mediate facilitative Fe(2+) transport in the absence of a H(+) gradient. Since plasma membrane expression of DMT1 is upregulated in liver of hemochromatosis patients, this H(+) -uncoupled facilitative Fe(2+) transport via DMT1 can account for the uptake of nontransferrin-bound plasma iron characteristic of iron overload disorders.

Significance of primary stability for osseointegration of dental implants

AIM: To investigate the significance of the initial stability of dental implants for the establishment of osseointegration in an experimental capsule model for bone augmentation. MATERIAL AND METHODS: Sixteen male rats were used in the study. In each rat, muscle-periosteal flaps were elevated on the lateral aspect of the mandibular ramus on both sides, resulting in exposure of the bone surface. Small perforations were then produced in the ramus. A rigid, hemispherical Teflon capsule with a diameter of 6 mm and a height of 4 mm and with a hole in its middle portion, prepared to fit the circumference of an ITI HC titanium implant of 2.8 mm in diameter, was fixed to the ramus using 4 mini-screws. On one side of the jaw, the implant was placed through the hole in such a way that its apex did not make contact with the mandibular ramus (test). This placement of the implant did not ensure primary stability. On the other side of the jaw, a similar implant was placed through the hole of the capsule in such a way that contact was made between the implant and the surface of the ramus (control). This provided primary stability of the implant. After placement of the implants, the soft tissues were repositioned over the capsules and sutured. After 1, 3, 6 and 9 months, four animals were sacrificed and subjected to histometric analysis. RESULTS: The mean height of direct bone-to-implant contact of implants with primary stability was 38.8%, 52.9%, 64.6% and 81.3% of the implant length at 1, 3, 6 and 9 months, respectively. Of the bone adjacent to the implant surface, 28.1%, 28.9%, 52.6% and 69.6%, respectively, consisted of mineralized bone. At the test implants, no bone-to-implant contact was observed at any observation time or in any of these non-stabilized specimens. CONCLUSION: The findings of the present study indicate that primary implant stability is a prerequisite for successful osseointegration, and that implant instability results in fibrous encapsulation, thus confirming previously made clinical observations.

HAART and the heart: changes in coronary risk factors and implications for coronary risk in men starting antiretroviral therapy

OBJECTIVES: To estimate changes in coronary risk factors and their implications for coronary heart disease (CHD) rates in men starting highly active antiretroviral therapy (HAART). METHODS: Men participating in the Swiss HIV Cohort Study with measurements of coronary risk factors both before and up to 3 years after starting HAART were identified. Fractional polynomial regression was used to graph associations between risk factors and time on HAART. Mean risk factor changes associated with starting HAART were estimated using multilevel models. A prognostic model was used to predict corresponding CHD rate ratios. RESULTS: Of 556 eligible men, 259 (47%) started a nonnucleoside reverse transcriptase inhibitor (NNRTI) and 297 a protease inhibitor (PI) based regimen. Levels of most risk factors increased sharply during the first 3 months on HAART, then more slowly. Increases were greater with PI- than NNRTI-based HAART for total cholesterol (1.18 vs. 0.98 mmol L(-1)), systolic blood pressure (3.6 vs. 0 mmHg) and BMI (1.04 vs. 0.55 kg m(2)) but not HDL cholesterol (0.24 vs. 0.32 mmol L(-1)) or glucose (1.02 vs. 1.03 mmol L(-1)). Predicted CHD rate ratios were 1.40 (95% CI 1.13-1.75) and 1.17 (0.95-1.47) for PI- and NNRTI-based HAART respectively. CONCLUSIONS: Coronary heart disease rates will increase in a majority of patients starting HAART: however the increases corresponding to typical changes in risk factors are relatively modest and could be offset by lifestyle changes.

Regulation of endothelial monocyte-activating polypeptide II release by apoptosis

Endothelial monocyte-activating polypeptide II (EMAP II) is a proinflammatory cytokine and a chemoattractant for monocytes. We show here that, in the mouse embryo, EMAP II mRNA was most abundant at sites of tissue remodeling where many apoptotic cells could be detected by terminal deoxynucleotidyltransferase-mediated dUTP end labeling. Removal of dead cells is known to require macrophages, and these were found to colocalize with areas of EMAP II mRNA expression and programmed cell death. In cultured cells, post-translational processing of pro-EMAP II protein to the mature released EMAP II form (23 kDa) occurred coincidentally with apoptosis. Cleavage of pro-EMAP II could be abrogated in cultured cells by using a peptide-based inhibitor, which competes with the ASTD cleavage site of pro-EMAP II. Our results suggest that the coordinate program of cell death includes activation of a caspase-like activity that initiates the processing of a cytokine responsible for macrophage attraction to the sites of apoptosis.

Fritz: a secreted frizzled-related protein that inhibits Wnt activity

Signaling molecules of the Wnt gene family are involved in the regulation of dorso-ventral, segmental and tissue polarity in Xenopus and Drosophila embryos. Members of the frizzled gene family, such as Drosophila frizzled-2 and rat frizzled-1, have been shown encode Wnt binding activity and to engage intracellular signal transduction molecules known to be part of the Wnt signaling pathway. Here we describe the cloning and characterization of Fritz, a mouse (mfiz) and human (hfiz) gene which codes for a secreted protein that is structurally related to the extracellular portion of the frizzled genes from Drosophila and vertebrates. The Fritz protein antagonizes Wnt function when both proteins are ectopically expressed in Xenopus embryos. In early gastrulation, mouse fiz mRNA is expressed in all three germ layers. Later in embryogenesis fiz mRNA is found in the central and peripheral nervous systems, nephrogenic mesenchyme and several other tissues, all of which are sites where Wnt proteins have been implicated in tissue patterning. We propose a model in which Fritz can interfere with the activity of Wnt proteins via their cognate frizzled receptors and thereby modulate the biological responses to Wnt activity in a multitude of tissue sites.

Alternative vaccination against equine botulism (BoNT/C)

REASON FOR PERFORMING STUDY: In Europe the incidence of botulism in horses has increased in the last decade due to the growing popularity of haylage feeding. Recombinant vaccines are safer and less expensive to produce and are generally better tolerated than toxoids. OBJECTIVES: To investigate whether the recombinant C-terminal half of the heavy chain of the botulinum neurotoxin C (Hc BoNT/C) in combination with an immunstimulatory adjuvant is an appropriate vaccine candidate for horses by testing its efficacy to induce neutralising antibodies and by comparing its immunogenic properties and adverse reactions to a commercial toxoid vaccine. Formation of oedema and local pain reactions were assessed. ELISA and Western blot assay against Hc BoNT/C and testing of neutralising antibody induction in a mouse protection assay were used to evaluate the immune response. RESULTS: With the recombinant vaccine, only minor local swelling with full recovery after 5 days was noted after brisket injections. The toxoid vaccine produced local, painful reactions with longer recovery periods of up to 2 weeks. Horses vaccinated with either vaccine induced neutralising antibodies after the second booster vaccination, while seroconversion on ELISA and Western blot to Hc BoNT/C was apparent after the first recombinant vaccination, and at various time points in the vaccination schedule in horses that received commercial toxoid vaccine. CONCLUSION: The recombinant vaccine showed fewer adverse reactions compared to the only commercially available vaccine but induced similar concentrations of neutralising antibodies. There was no correlation between the serological response to Hc BoNT/C and the neutralising capacity of serum. POTENTIAL RELEVANCE: Recombinant Hc BoNT/C is an appropriate vaccine candidate to stimulate production of neutralising antibodies against botulinum neurotoxin C in horses and creates only minor local reactions at the injection site.