From the Cover: Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire

Antibody repertoire diversity, potentially as high as 10¹¹ unique molecules in a single individual, confounds characterization by conventional sequence analyses. In this study, we present a general method for assessing human antibody sequence diversity displayed on phage using massively parallel pyrosequencing, a novel application of Kabat column-labeled profile Hidden Markov Models, and translated complementarity determining region (CDR) capture-recapture analysis. Pyrosequencing of domain amplicon and RCA PCR products generated 1.5 × 10⁶ reads, including more than 1.9 × 10⁵ high quality, full-length sequences of antibody variable fragment (Fv) variable domains. Novel methods for germline and CDR classification and fine characterization of sequence diversity in the 6 CDRs are presented. Diverse germline contributions to the repertoire with random heavy and light chain pairing are observed. All germline families were found to be represented in 1.7 × 10⁴ sequences obtained from repeated panning of the library. While the most variable CDR (CDR-H3) presents significant length and sequence variability, we find a substantial contribution to total diversity from somatically mutated germline encoded CDRs 1 and 2. Using a capture-recapture method, the total diversity of the antibody library obtained from a human donor Immunoglobulin M (IgM) pool was determined to be at least 3.5 × 10¹⁰. The results provide insights into the role of IgM diversification, display library construction, and productive germline usages in antibody libraries and the humoral repertoire.     

Evidence for genetic transmission of thyroid peroxidase autoantibody epitopic "fingerprints"

Autoimmune thyroid disease is characterized by the tendency to cluster in families and by IgG class autoantibodies to antigens such as thyroid peroxidase (TPO). The epitopes recognized by polyclonal serum autoantibodies can be quantitatively fingerprinted using four recombinant human TPO autoantibodies (expressed as Fab) that define A and B domain epitopes in an immunodominant region. To determine whether these fingerprints are genetically transmitted, we analyzed fingerprints of 63 members of 7 multiplex Old Order Amish families and 17 individuals from 4 Hashimoto thyroiditis families. Inhibition of serum autoantibody binding to [125I]TPO by the recombinant Fab was used to assess recognition of the TPO immunodominant region (4 Fab combined) and recognition of domain A or B (individual Fab). Complex segregation analysis was performed using a unified model (POINTER). For the 4 Fab combined inhibition phenotype, the no transmission model was rejected (chi2(4) = 20.67; P < 0.0032), and the most parsimonious model includes a major gene effect. More importantly, evidence for genetic transmission was obtained for the phenotype defined by the ratio of inhibition by subdomain Fab B1:B2. Thus, for this ratio (reflecting recognition of the B domain), the no transmission model was rejected chi2(4) = 63.59; P < 0.000008). Moreover, the polygenic hypothesis could be rejected, but not the major locus hypothesis, suggesting that major genes might be involved in familial transmission of this trait. In conclusion, our findings suggest that autoantibody recognition of the TPO immunodominant region and the TPO B domain is genetically transmitted. These data may open the way to the identification by candidate analysis or positional cloning of at least one gene responsible for the development of Hashimoto's thyroiditis.     

Clinical events after transjugular intrahepatic portosystemic shunt: Correlation with hemodynamic findings

Background & Aims: Transjugular intrahepatic portosystemic shunt (TIPS) procedures are increasingly being used, but the relationship between the hemodynamic effects of TIPS and the clinical events on follow-up remains undefined. Hence, we have investigated the hemodynamic correlations of portal hypertension–related events after a TIPS procedure. Methods: Prospective follow-up of 122 cirrhotic patients who had a TIPS procedure performed because of variceal hemorrhage was conducted. Results: The portacaval pressure gradient (PPG) significantly decreased after the TIPS procedure (from 19.7 ± 4.6 to 8.6 ± 2.7 mm Hg; P > 0.001), but increased thereafter and at rebleeding (n = 25) was >12 mm Hg in all patients (18.4 ± 4.6 mm Hg). Twenty-six patients developed ascites; the PPG (measured in 19) was always >12 mm Hg. Increasing the PPG to >12 mm Hg occurred very frequently (83% at 1 year). Within 1 year, 77% of patients underwent balloon angioplasty or restenting. However, 80% had again a PPG of >12 mm Hg 1 year after reintervention. Hepatic encephalopathy developed in 31% of patients at 1 year; 21 of 23 patients had a PPG of <12 mm Hg. Conclusions: Total protection from the risk of recurrent complications of portal hypertension after a TIPS procedure requires that the PPG be decreased and maintained <12 mm Hg. However, reintervention will be required in most patients within 1 year and again the second year. On the other hand, such portal decompression is associated with an increased risk of hepatic encephalopathy.GASTROENTEROLOGY 1998;114:1296-1303     

State of the art: new developments in cardiac imaging

Cardiac imaging continues to reveal new anatomical and functional insights into heart disease. In echocardiography, both transesophageal and transthoracic three-dimensional imaging have been fully developed and optimized, and the value of the techniques that have increased our understanding of cardiac mechanics and ventricular function is well established. At the same time, the healthcare industry has released new devices onto the market which, although they are easier to use, have limitations that restrict their use for routine assessment. Tomography's diagnostic and prognostic value in coronary artery disease continues to increase while radiation exposure becomes progressively lower. With cardiac magnetic resonance imaging, myocardial injury and recovery in ischemic heart disease and following acute coronary syndrome can be monitored in exquisite detail. The emergence of new combined tomographic and gamma camera techniques, exclusively developed for nuclear cardiology, have improved the quality of investigations and reduced radiation exposure. The hybrid or fusion images produced by combining different techniques, such as nuclear cardiology techniques and tomography, promise an exciting future.     

Metabolic syndrome in Spain: prevalence and coronary risk associated with harmonized definition and WHO proposal. DARIOS study

Introduction and objectives

To update the prevalence of metabolic syndrome and associated coronary risk in Spain, using the harmonized definition and the new World Health Organization proposal (metabolic premorbid syndrome), which excludes diabetes mellitus and cardiovascular disease.

Methods

Individual data pooled analysis study of 24 670 individuals from 10 autonomous communities aged 35 to 74 years. Coronary risk was estimated using the REGICOR function.

Results

Prevalence of metabolic syndrome was 31% (women 29% [95% confidence interval, 25%-33%], men 32% [95% confidence interval, 29%-35%]). High blood glucose (P=.019) and triglycerides (P<.001) were more frequent in men with metabolic syndrome, but abdominal obesity (P<.001) and low high-density lipoprotein cholesterol (P=.001) predominated in women. Individuals with metabolic syndrome showed moderate coronary risk (8% men, 5% women), although values were higher (P<.001) than in the population without the syndrome (4% men, 2% women). Women and men with metabolic syndrome had 2.5 and 2 times higher levels of coronary risk, respectively (P<.001). Prevalence of metabolic premorbid syndrome was 24% and the increase in coronary risk was also proportionately larger in women than in men (2 vs 1.5, respectively; P<.001).

Conclusions

Prevalence of metabolic syndrome is 31%; metabolic premorbid syndrome lowers this prevalence to 24% and delimits the population for primary prevention. The increase in coronary risk is proportionally larger in women, in both metabolic syndrome and metabolic premorbid syndrome.Full English text available from:www.revespcardiol.org     

ST segment elevation at 72 hours in patients with a first anterior myocardial infarction best correlates with pre-discharge and 1-year regional contractility and ventricular dilatation.

AIMS: To analyse the relationship between the in-hospital course of ST segment elevation (STE) and negative T wave (NTW) with ejection fraction, regional contractility and left ventricular end-diastolic volume at pre-discharge and at 1 year in patients with a first anterior STE acute myocardial infarction (AMI). METHODS AND RESULTS: ECG changes were measured during hospitalization and at 1 year whereas ejection fraction, regional contractility score and end-diastolic volume index were measured by isotopic ventriculography at pre-discharge and at 1 year. At 72h but not earlier patients with SigmaSTE >0.6mV (group A, n: 35) had a lower ejection fraction (P<0.001), a higher regional contractility score (P<0.001) and a larger end-diastolic volume index (P<0.001) at discharge than those with <0.6mV (group B, n: 26). Negative T wave did not provide additional information. At 1 year, group A continued to show a more impaired ejection fraction and regional contractility than group B and a larger end-diastolic volume. CONCLUSION: Although reportedly changes in STE within the first hours correlate with coronary reperfusion our findings indicate that additional assessment of STE as early as at 72h correlates with wall motion, ejection fraction and ventricular dilatation at discharge and at 1 year.     

Comparison of clinical and angiographic features and longterm follow-up events between patients with variant angina and patients with ST elevation myocardial infarction

We investigated to what extent patients with variant angina and significant coronary stenosis (>or=70%) present a clinical and angiographic profile similar to patients with ST elevation myocardial infarction. Thus, the clinical and angiographic features as well as follow-up events of 200 patients were prospectively analyzed and were compared with those of 422 patients with a first ST elevation myocardial infarction survivors of the early phase (3 days) and those of 70 patients with variant angina and non significant stenosis. Age and incidence of smoking, systemic hypertension, diabetes and maximum ST elevation were similar in the 2 groups. Furthermore, among patients with significant coronary stenosis, stenosis severity and the proportion of eccentric lesions were also comparable. Incidence of recent-within 30 days prior to admission-angina at rest was higher in variant angina patients with significant stenosis (67% vs. 27%, p<0.001) than in those with myocardial infarction but long standing angina at rest (>30 days) was low and comparable in these 2 groups (15% vs. 11%, ns). Also, in a 5-year follow-up most patients from these 2 groups were free from angina at rest (86% vs. 84%) which in variant angina patients was largely attributable to a high revascularization rate (72%). Moreover, the rate of myocardial infarction/cardiac death (20% vs. 19%) was also similar. Patients with variant angina and non-significant stenosis, however, had longer antecedent angina, more frequent follow-up angina and a lower incidence of cardiac events than the other 2 groups. Thus, these findings suggest that patients with variant angina and significant coronary stenosis generally behave as an acute coronary syndrome-likely associated with an acutely complicated plaque-rather than as recurrent vasospastic angina, and should be managed accordingly.     

Proprotein convertase substilisin/kexin type 9 antagonism reduces low-density lipoprotein cholesterol in statin-treated hypercholesterolemic nonhuman primates

Proprotein convertase substilisin/kexin type 9 (PCSK9) promotes the degradation of low-density lipoprotein (LDL) receptor (LDLR) and thereby increases serum LDL-cholesterol (LDL-C). We have developed a humanized monoclonal antibody that recognizes the LDLR binding domain of PCSK9. This antibody, J16, and its precursor mouse antibody, J10, potently inhibit PCSK9 binding to the LDLR extracellular domain and PCSK9-mediated down-regulation of LDLR in vitro. In vivo, J10 effectively reduces serum cholesterol in C57BL/6 mice fed normal chow. J16 reduces LDL-C in healthy and diet-induced hypercholesterolemic cynomologous monkeys, but does not significantly affect high-density lipoprotein-cholesterol. Furthermore, J16 greatly lowered LDL-C in hypercholesterolemic monkeys treated with the HMG-CoA reductase inhibitor simvastatin. Our data demonstrate that anti-PCSK9 antibody is a promising LDL-C-lowering agent that is both efficacious and potentially additive to current therapies.     

Large‐conductance calcium‐activated potassium channels modulate vascular tone in experimental cirrhosis

Background: Large‐conductance calcium‐activated potassium (BK_Ca) channels regulate vascular tone in different vascular systems. Moreover, activated hepatic stellate cells (HSC) contain BK_Ca channels. The aim of this study was to evaluate the role of BK_Ca channels in the regulation of vascular tone in control (CT) and carbon tetrachloride‐cirrhotic (CH) rat livers. Methods: Changes in intrahepatic vascular resistance were assessed by evaluating the portal perfusion pressure (PP) response to methoxamine (Mtx) in the presence of Iberiotoxin (Ibtx; a BK_Ca channel blocker), NS1619 (a BK_Ca channel opener), Ibtx plus the nitric oxide (NO) synthase inhibitor, N^G‐nitro‐l ‐arginine (l ‐NNA) or l ‐NNA alone. In addition, in CH livers, PP dose–response curves to the NO donor, S‐nitroso‐N‐acetyl‐d,l ‐penicillamine (SNAP), were performed after pre‐incubation with Ibtx or its vehicle. BK_Ca mRNA expression was assessed in liver homogenates, and BK_Ca protein expression in HSC isolated from CT and CH livers. Results: In CH livers, Ibtx significantly increased baseline PP and exacerbated the PP response to Mtx. Conversely, NS1619 induced a mild nonsignificant decrease of baseline PP and attenuated the hyperresponse to Mtx. CH livers exhibited an upregulation of both mRNA and protein of the α‐subunit of BK_Ca. Conclusion: Large‐conductance calcium‐activated potassium channels are overexpressed in CH livers and might represent a compensatory mechanism modulating the increased hepatic vascular tone of cirrhosis.     

Emergency care of patients with upper gastrointestinal bleeding in regional hospitals in Catalonia

Objective

To evaluate the resources available in Catalan regional hospitals for the emergency care of upper gastrointestinal hemorrhage.

Methods

We analyzed a survey sent to 32 hospitals on the availability, composition and resources of a duty endoscopy service for the year 2009.

Results

Responses were obtained from 24 centers, covering 3,954,000 inhabitants. Duty endoscopists were available in 12 hospitals. A total of 1,483,000 inhabitants were unable to access a duty endoscopist in the referral center. Centers with duty endoscopists had more beds and had a larger catchment area. Duty services were composed of 4.5 endoscopists (range 2-11), covering 82.1 (33.2-182.5) duty shifts/year. Seventeen centers reported 1,571 episodes (51%, range: 3-280, 39.68/100,000 inhabitants). Centers with a duty service reported a greater number of cases (76 vs. 43, p = 0.047). Centers without this service referred a greater number of patients (147 vs. 17, p = 0.001). Patients in the emergency department were under the care of the internal medicine department in four centers, the surgery department in 14 centers and under the care of both departments in six. Admitted patients were under the care of the gastroenterology department in only six hospitals. The most widely used procedures were ligation of varicose bleeding and injection therapies in non-varicose bleeding. Twenty-one percent of centers did not perform combined treatment.

Conclusions

A significant proportion of the population does not have access to a duty endoscopist in referral centers. Duty shifts represent significant workload in regional hospitals. Coordination among health professionals and centers would allow the efficient application of therapeutic resources and a duty endoscopy service to be established in centers lacking this resource.