STrengthening the Reporting of OBservational studies in Epidemiology: Molecular Epidemiology STROBE-ME. An extension of the STROBE statement

Advances in laboratory techniques have led to a rapidly increasing use of biomarkers in epidemiological studies. Biomarkers of internal dose, early biological change, susceptibility, and clinical outcomes are used as proxies for investigating the interactions between external and/or endogenous agents and the body components or processes. The need for improved reporting of scientific research led to influential statements of recommendations such as STrengthening Reporting of Observational studies in Epidemiology (STROBE) statement. The STROBE initiative established in 2004 aimed to provide guidance on how to report observational research. Its guidelines provide a user-friendly checklist of 22 items to be reported in epidemiological studies, with items specific to the three main study designs: cohort studies, case-control studies and cross-sectional studies. The present STrengthening the Reporting of OBservational studies in Epidemiology - Molecular Epidemiology (STROBE-ME) initiative builds on the STROBE Statement implementing 9 existing items of STROBE and providing 17 additional items to the 22 items of STROBE checklist. The additions relate to the use of biomarkers in epidemiological studies, concerning collection, handling and storage of biological samples; laboratory methods, validity and reliability of biomarkers; specificities of study design; and ethical considerations. The STROBE-ME recommendations are intended to complement the STROBE recommendations.

The habitat, double life, citizenship, and forgetfulness of IgA

Immunoglobulin A (IgA) is the main secretory immunoglobulin of mucous membranes and is powerfully induced by the presence of commensal microbes in the intestine. B cells undergo class switch recombination to IgA in the mucosa-associated lymphoid tissues, particularly mesenteric lymph nodes (MLNs) and Peyer's patches, through both T-dependent and T-independent pathways. IgA B cells primed in the mucosa traffic from the intestinal lymphoid structures, initially through the lymphatics and then join the bloodstream, to home back to the intestinal mucosa as IgA-secreting plasma cells. Once induced, anti-bacterial IgA can be extremely long-lived but is replaced if there is induction of additional IgA specificities by other microbes. The mucosal immune system is anatomically separated from the systemic immune system by the MLNs, which act as a firewall to prevent penetration of live intestinal bacteria to systemic sites. Dendritic cells sample intestinal bacteria and induce B cells to switch to IgA. In contrast, intestinal macrophages are adept at killing extracellular bacteria and are able to clear bacteria that have crossed the mucus and epithelial barriers. There is both a continuum between innate and adaptive immune mechanisms and compartmentalization of the mucosal immune system from systemic immunity that function to preserve host microbial mutualism.

Monotone Constrained Tensor-product B-spline with application to screening studies

When different markers are responsive to different aspects of a disease, combination of multiple markers could provide a better screening test for early detection. It is also resonable to assume that the risk of disease changes smoothly as the biomarker values change and the change in risk is monotone with respect to each biomarker. In this paper, we propose a boundary constrained tensor-product B-spline method to estimate the risk of disease by maximizing a penalized likelihood. To choose the optimal amount of smoothing, two scores are proposed which are extensions of the GCV score (O'Sullivan et al. (1986)) and the GACV score (Ziang and Wahba (1996)) to incorporate linear constraints. Simulation studies are carried out to investigate the performance of the proposed estimator and the selection scores. In addidtion, sensitivities and specificities based ona pproximate leave-one-out estimates are proposed to generate more realisitc ROC curves. Data from a pancreatic cancer study is used for illustration.

RANDOM EFFECTS MODELS IN A META-ANALYSIS OF THE ACCURACY OF DIAGNOSTIC TESTS WITHIN A GOLD STANDARD IN THE PRESENCE OF MISSING DATA

In evaluating the accuracy of diagnosis tests, it is common to apply two imperfect tests jointly or sequentially to a study population. In a recent meta-analysis of the accuracy of microsatellite instability testing (MSI) and traditional mutation analysis (MUT) in predicting germline mutations of the mismatch repair (MMR) genes, a Bayesian approach (Chen, Watson, and Parmigiani 2005) was proposed to handle missing data resulting from partial testing and the lack of a gold standard. In this paper, we demonstrate an improved estimation of the sensitivities and specificities of MSI and MUT by using a nonlinear mixed model and a Bayesian hierarchical model, both of which account for the heterogeneity across studies through study-specific random effects. The methods can be used to estimate the accuracy of two imperfect diagnostic tests in other meta-analyses when the prevalence of disease, the sensitivities and/or the specificities of diagnostic tests are heterogeneous among studies. Furthermore, simulation studies have demonstrated the importance of carefully selecting appropriate random effects on the estimation of diagnostic accuracy measurements in this scenario.

Novel pressure-to-cornea index in glaucoma

BACKGROUND: Several conversion tables and formulas have been suggested to correct applanation intraocular pressure (IOP) for central corneal thickness (CCT). CCT is also thought to represent an independent glaucoma risk factor. In an attempt to integrate IOP and CCT into a unified risk factor and avoid uncertain correction for tonometric inaccuracy, a new pressure-to-cornea index (PCI) is proposed. METHODS: PCI (IOP/CCT(3)) was defined as the ratio between untreated IOP and CCT(3) in mm (ultrasound pachymetry). PCI distribution in 220 normal controls, 53 patients with normal-tension glaucoma (NTG), 76 with ocular hypertension (OHT), and 89 with primary open-angle glaucoma (POAG) was investigated. PCI's ability to discriminate between glaucoma (NTG+POAG) and non-glaucoma (controls+OHT) was compared with that of three published formulae for correcting IOP for CCT. Receiver operating characteristic (ROC) curves were built. RESULTS: Mean PCI values were: Controls 92.0 (SD 24.8), NTG 129.1 (SD 25.8), OHT 134.0 (SD 26.5), POAG 173.6 (SD 40.9). To minimise IOP bias, eyes within the same 2 mm Hg range between 16 and 29 mm Hg (16-17, 18-19, etc) were separately compared: control and NTG eyes as well as OHT and POAG eyes differed significantly. PCI demonstrated a larger area under the ROC curve (AUC) and significantly higher sensitivity at fixed 80% and 90% specificities compared with each of the correction formulas; optimum PCI cut-off value 133.8. CONCLUSIONS: A PCI range of 120-140 is proposed as the upper limit of "normality", 120 being the cut-off value for eyes with untreated pressures or=22 mm Hg. PCI may reflect individual susceptibility to a given IOP level, and thus represent a glaucoma risk factor. Longitudinal studies are needed to prove its prognostic value.

Self-reactivity in the dimeric intravenous immunoglobulin fraction

Therapeutic intravenous immunoglobulin (IVIg) preparations contain antibodies reflecting the cumulative antigen experience of the donor population. IVIg contains variable amounts of monomeric and dimeric IgG, but there is little information available on their comparative antibody specificities. We have isolated highly purified fractions of monomeric and dimeric IgG by size-exclusion chromatography. Following treatment of all fractions at pH4, analyses by immunodot and immunocytology on human cell lines showed a preferential recognition of autoantigens in the dimeric IgG fraction. Investigation of the HEp-2 cytoplasmic proteome by 2D-PAGE, Western blot, and subsequent identification of IVIg reactive spots by mass spectrometry (LC-MS/MS) showed that IVIg recognized only a restricted set of the total proteins. Similar experiments showed that more antigens were recognized by the dimeric IgG fraction, especially when the dissociated dimer fraction was used, as compared to its monomeric counterpart. These observations are consistent with idiotype-anti-idiotype masking of auto-specific Abs in the dimeric fraction of IVIg.

Clinical performance of a new laser fluorescence device for detection of occlusal caries lesions in permanent molars

OBJECTIVES: To determine the clinical performance of a laser fluorescence device (DIAGNOdent pen, KaVo) to discriminate between different occlusal caries depths (D(0)-D(1-4); D(0-2)-D(3,4)) in permanent molars. METHODS: In this prospective, randomized two-centre-study 120 sound/uncavitated carious sites in 120 patients were measured after visual and radiographic caries assessment. In cases of operative intervention (n=86), the lesion depths after caries removal were recorded (reference). In cases of preventive intervention (n=34), the sites were reassessed visually/radiographically after 12 months to verify the status assessed before (reference). The discrimination performance was determined statistically (Mann-Whitney test, Spearman's rho coefficient, and areas under the receiver operating characteristic curves (AUCs)). Sensitivities (SE) and specificities (SP) were plotted as a function of the measured values and cut-off values for the mentioned thresholds suggested. RESULTS: Sound sites (n=13) had significantly minor fluorescence values than carious sites (n=107) (P<0.0001) as had sites with no/enamel caries (n=63) compared to dentinal caries (n=57). The AUCs for the same discriminations were 0.92 and 0.78 (P<0.001). For the D(0)-D(1-4) threshold, a cut-off at a value of 12 (SE: 0.88, SP: 0.85) and for the D(0-2)-D(3,4) threshold at 25 (SE: 0.67, SP: 0.79) can be suggested. A moderate positive correlation between the measurements and the caries depths was calculated (rho=+0.57, P=0.01). CONCLUSION: Within this study, the device's discrimination performance for different caries depths was moderate to very good and it may be recommended as adjunct tool in the diagnosis of occlusal caries.

Performance of fluorescence methods, radiographic examination and ICDAS II on occlusal surfaces in vitro

This study compared the performance of fluorescence-based methods, radiographic examination, and International Caries Detection and Assessment System (ICDAS) II on occlusal surfaces. One hundred and nineteen permanent human molars were assessed twice by 2 experienced dentists using the laser fluorescence (LF and LFpen) and fluorescence camera (FC) devices, ICDAS II and bitewing radiographs (BW). After measuring, the teeth were histologically prepared and assessed for caries extension. The sensitivities for dentine caries detection were 0.86 (FC), 0.78 (LFpen), 0.73 (ICDAS II), 0.51 (LF) and 0.34 (BW). The specificities were 0.97 (BW), 0.89 (LF), 0.65 (ICDAS II), 0.63 (FC) and 0.56 (LFpen). BW presented the highest values of likelihood ratio (LR)+ (12.47) and LR- (0.68). Rank correlations with histology were 0.53 (LF), 0.52 (LFpen), 0.41 (FC), 0.59 (ICDAS II) and 0.57 (BW). The area under the ROC curve varied from 0.72 to 0.83. Inter- and intraexaminer intraclass correlation values were respectively 0.90 and 0.85 (LF), 0.93 and 0.87 (LFpen) and 0.85 and 0.76 (FC). The ICDAS II kappa values were 0.51 (interexaminer) and 0.61 (intraexaminer). The BW kappa values were 0.50 (interexaminer) and 0.62 (intraexaminer). The Bland and Altman limits of agreement were 46.0 and 38.2 (LF), 55.6 and 40.0 (LFpen) and 1.12 and 0.80 (FC), for intra- and interexaminer reproducibilities. The posttest probability for dentine caries detection was high for BW and LF. In conclusion, LFpen, FC and ICDAS II presented better sensitivity and LF and BW better specificity. ICDAS II combined with BW showed the best performance and is the best combination for detecting caries on occlusal surfaces.

Synergy-inducing chemokines enhance CCR2 ligand activities on monocytes

The migration of monocytes to sites of inflammation is largely determined by their response to chemokines. Although the chemokine specificities and expression patterns of chemokine receptors are well defined, it is still a matter of debate how cells integrate the messages provided by different chemokines that are concomitantly produced in physiological or pathological situations in vivo. We present evidence for one regulatory mechanism of human monocyte trafficking. Monocytes can integrate stimuli provided by inflammatory chemokines in the presence of homeostatic chemokines. In particular, migration and cell responses could occur at much lower concentrations of the CCR2 agonists, in the presence of chemokines (CCL19 and CCL21) that per se do not act on monocytes. Binding studies on CCR2(+) cells showed that CCL19 and CCL21 do not compete with the CCR2 agonist CCL2. Furthermore, the presence of CCL19 or CCL21 could influence the degradation of CCL2 and CCL7 on cells expressing the decoy receptor D6. These findings disclose a new scenario to further comprehend the complexity of chemokine-based monocyte trafficking in a vast variety of human inflammatory disorders.

Human intestinal maltase-glucoamylase: crystal structure of the N-terminal catalytic subunit and basis of inhibition and substrate specificity

Human maltase-glucoamylase (MGAM) is one of the two enzymes responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM is anchored to the small-intestinal brush-border epithelial cells and contains two homologous glycosyl hydrolase family 31 catalytic subunits: an N-terminal subunit (NtMGAM) found near the membrane-bound end and a C-terminal luminal subunit (CtMGAM). In this study, we report the crystal structure of the human NtMGAM subunit in its apo form (to 2.0 A) and in complex with acarbose (to 1.9 A). Structural analysis of the NtMGAM-acarbose complex reveals that acarbose is bound to the NtMGAM active site primarily through side-chain interactions with its acarvosine unit, and almost no interactions are made with its glycone rings. These observations, along with results from kinetic studies, suggest that the NtMGAM active site contains two primary sugar subsites and that NtMGAM and CtMGAM differ in their substrate specificities despite their structural relationship. Additional sequence analysis of the CtMGAM subunit suggests several features that could explain the higher affinity of the CtMGAM subunit for longer maltose oligosaccharides. The results provide a structural basis for the complementary roles of these glycosyl hydrolase family 31 subunits in the bioprocessing of complex starch structures into glucose.

First-trimester serum levels of soluble endoglin and soluble fms-like tyrosine kinase-1 as first-trimester markers for late-onset preeclampsia

OBJECTIVE: The aim of this investigation was to assess soluble endoglin (sEng) and soluble fms-like tyrosine kinase-1 (sFlt1) as first-trimester serum markers to predict preeclampsia. STUDY DESIGN: First-trimester sera were obtained from 46 women with subsequent late-onset preeclampsia and from 92 controls. sEng and sFlt1 concentrations were determined immunoanalytically. Correlation analysis with inhibin A and placental growth factor levels was performed. RESULTS: sEng and sFlt1 serum concentrations were higher in women with subsequent preeclampsia than in controls (mean +/- SD, sEng: 5.57 +/- 1.18 ng/mL vs 5.02 +/- 1.01 ng/mL, P = .009; sFlt1: 1764 +/- 757 pg/mL vs 1537 +/- 812 pg/mL, P = .036). Sensitivities and specificities for predicting preeclampsia were 63% and 57% for sEng and 64% and 56% for sFlt1, respectively. When sEng and inhibin A were combined, the sensitivity increased to 68%, whereas the specificity was 61%. CONCLUSION: sEng and sFlt1 are increased in the first trimester in women with subsequent late-onset preeclampsia and might therefore prove useful to predict preeclampsia.

Intravenous immunoglobulin contains a broad repertoire of anticarbohydrate antibodies that is not restricted to the IgG2 subclass

BACKGROUND: Specificities for carbohydrate IgG antibodies, thought to be predominantly of the IgG2 subclass, have never been broadly examined in healthy human subjects. OBJECTIVE: To examine commercial intravenous immunoglobulin (IVIG) preparations for their ability to recognize a wide range of glycans and to determine the contribution of IgG2 to the binding pattern observed. METHODS: We used a glycan microarray to evaluate IVIG preparations and a control mix of similar proportions of human myeloma IgG1 and IgG2 for binding to 377 glycans, courtesy of the Consortium for Functional Glycomics Core H. Glycans recognized were categorized using public databases for their likely cellular sources. IgG2 was depleted from IVIG by using immunoaffinity chromatography, and depletion was confirmed by using nephelometry and surface plasmon resonance. RESULTS: Nearly half of the glycans bound IgG. Some of the glycans with the greatest antibody binding can be found in structures of human pathogenic bacteria (eg, Streptococcus pneumoniae, Mycobacterium tuberculosis, Vibrio cholera) and nonpathogenic bacteria, including LPS and lipoteichoic acid, capsular polysaccharides, and exopolysaccharides. Surprisingly, depletion of IgG2 had only a modest effect on anticarbohydrate recognition patterns compared with the starting IVIG preparation. Little to no binding activity was detected to human endogenous glycans, including tumor-associated antigens. CONCLUSIONS: This novel, comprehensive analysis provides evidence that IVIG contains a much wider range than previously appreciated of anticarbohydrate IgG antibodies, including those recognizing both pathogenic and non-pathogen-associated prokaryotic glycans.

DARPins as bispecific receptor antagonists analyzed for immunoglobulin E receptor blockage

The concept of multispecific antibodies is of high therapeutic interest but has failed to produce pharmaceutical products due to the poor biophysical properties of such molecules. Here, we propose an alternative and simple way to generate bispecific binding molecules using designed ankyrin repeat proteins (DARPins). For this purpose, monovalent DARPins with different epitope specificities were selected against the alpha chain of the high-affinity receptor for human immunoglobulin E (IgE) (FcepsilonRIalpha). Two of the isolated binders interfering with IgE binding to the receptor were joined to each other or to themselves via a flexible protein linker. The resulting bivalent and bispecific DARPins were tested for their ability to prevent allergen-induced cell degranulation using rat basophilic leukemia cells stably transfected with human FcepsilonRIalpha. The bispecific DARPin construct was the most potent one, efficiently blocking the IgE-FcepsilonRI interaction and preventing the release of proinflammatory mediators. Noteworthy, the multivalent and multispecific DARPin construct did not show any alteration of the beneficial biophysical properties of the monovalent parental DARPins. Hence, bispecific DARPins may be used to generate receptor antagonists simultaneously targeting different epitopes on the same molecule. Moreover, they easily overcome the limiting immunoglobulin binding paradigm (one binding molecule=one epitope) and thereby represent an alternative to monoclonal antibodies in cases where the immunoglobulin scaffold is unsuitable.

Mycobacterium tuberculosis NAD+-dependent DNA ligase is selectively inhibited by glycosylamines compared with human DNA ligase I

DNA ligases are important enzymes which catalyze the joining of nicks between adjacent bases of double-stranded DNA. NAD1-dependent DNA ligases (LigA) are essential in bacteria and are absent in humans. They have therefore been identified as novel, validated and attractive drug targets. Using virtual screening against an in-house database of compounds and our recently determined crystal structure of the NAD1 binding domain of the Mycobacterium tuberculosis LigA, we have identified N1, Nn-bis-(5-deoxy-a-D-xylofuranosylated) diamines as a novel class of inhibitors for this enzyme. Assays involving M.tuberculosis LigA, T4 ligase and human DNA ligase I show that these compounds specifically inhibit LigA from M.tuberculosis. In vitro kinetic and inhibition assays demonstrate that the compounds compete with NAD1 for binding and inhibit enzyme activity with IC50 values in the mM range. Docking studies rationalize the observed specificities and show that among several glycofuranosylated diamines, bis xylofuranosylated diamines with aminoalkyl and 1, 3-phenylene carbamoyl spacers mimic the binding modes of NAD1 with the enzyme. Assays involving LigA-deficient bacterial strains show that in vivo inhibition of ligase by the compounds causes the observed antibacterial activities. They also demonstrate that the compounds exhibit in vivo specificity for LigA over ATPdependent ligase. This class of inhibitors holds out the promise of rational development of new anti-tubercular agents.

QSAR Studies on Thiazolidines: A Biologically Privileged Scaffold

A large number of drugs and biologically relevant molecules contain heterocyclic systems. Often the presence of hetero atoms or groupings imparts preferential specificities in their biological responses. Amongst the heterocyclic systems, thiazolidine is a biologically important scaffold known to be associated with several biological activities. Some of the prominent biological responses attributed to this skeleton are antiviral, antibacterial, antifungal, antihistaminic, hypoglycemic, anti-inflammatory activities. This diversity in the biological response profiles of thiazolidine has attracted the attention of many researchers to explore this skeleton to its multiple potential against several activities. Many of these synthetic and biological explorations have been subsequently analyzed in detailed quantitative structure-activity relationship (QSAR) studies to correlate the respective structural features and physicochemical properties with the activities to identify the important structural components in deciding their activity behavior. In this, drugs or any biologically active molecules may be viewed as structural frames consisting of strategically positioned functional groups that will interact effectively with the complementary groups/sites of the receptor. With this in focus, the present article reviews the QSAR studies of diverse biological activities of the thiazolidines published during the past decade.