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The problem of model selection in regression analysis through the use of forward selection, backward elimination, and stepwise selection has been well explored in the literature. The main assumption in this, of course, is that the data are normally distributed and the main tool used here is either a t test or an F test. However, the properties of these model selection procedures are not well-known. The purpose of this paper is to study the properties of these procedures within generalized linear regression models, considering the normal linear regression model as a special case. The main tool that is being used is the score test. However, the F test and other large sample tests, such as the likelihood ratio and the Wald test, the AIC, and the BIC, are included for the comparison. A systematic study, through simulations, of the properties of this procedure was conducted, in terms of level and power, for symmetric and asymmetric distributions, such as normal, Poisson, and binomial regression models. Extensions for skewed distributions, over-dispersed Poisson (the negative binomial), and over-dispersed binomial (the beta-binomial) regression models, are also given and evaluated. The methods are applied to analyze two health datasets. [ABSTRACT FROM AUTHOR]

The generalized Gamma distribution is widely used in survival analysis, engineering and economics, and it includes the Gamma distribution as a special case. In real life applications, the Generalized Gamma distribution may be an unnecessarily complicated distribution to fit when one of the simpler models may be sufficient to analyze the data. The aim of this paper is to develop tests of goodness of fit of the two parameter gamma distribution against its three parameter counterpart. Specifically, we develop Wald, C (α) , score, likelihood ratio, and gradient tests. These tests are then compared in terms of empirical size and power using a simulation study. [ABSTRACT FROM AUTHOR]

Many problems in biomedical and other sciences are subject to biased estimates (maximum likelihood or of similar types). In two seminal papers Cox and Snell (1968) and Firth (1993) deal with first order bias of maximum likelihood estimates. Cox and Snell obtain a correction term that corrects, approximately, first order bias and Firth uses an adjustment to the score function; the solution of the estimating equation obtained by solving the adjusted score function to zero, removes the first order bias of the maximum likelihood estimates approximately. In many applications authors use one of these two procedures for bias correction without being aware that the other exists or whether these two procedures are equivalent. In this paper we investigate the equivalence issue of the two methods through theoretical analysis, simulation study and data analysis. We show that the two methods yield either exactly the same estimates or that the preventive method has some edge over the other. [ABSTRACT FROM AUTHOR]

Motivation Under two biologically different conditions, we are often interested in identifying differentially expressed genes. It is usually the case that the assumption of equal variances on the two groups is violated for many genes where a large number of them are required to be filtered or ranked. In these cases, exact tests are unavailable and the Welch's approximate test is most reliable one. The Welch's test involves two layers of approximations: approximating the distribution of the statistic by a t -distribution, which in turn depends on approximate degrees of freedom. This study attempts to improve upon Welch's approximate test by avoiding one layer of approximation. Results We introduce a new distribution that generalizes the t -distribution and propose a Monte Carlo based test that uses only one layer of approximation for statistical inferences. Experimental results based on extensive simulation studies show that the Monte Carol based tests enhance the statistical power and performs better than Welch's t -approximation, especially when the equal variance assumption is not met and the sample size of the sample with a larger variance is smaller. We analyzed two gene-expression datasets, namely the childhood acute lymphoblastic leukemia gene-expression dataset with 22 283 genes and Golden Spike dataset produced by a controlled experiment with 13 966 genes. The new test identified additional genes of interest in both datasets. Some of these genes have been proven to play important roles in medical literature. Availability and implementation R scripts and the R package mcBFtest is available in CRAN and to reproduce all reported results are available at the GitHub repository, https://github.com/iullah1980/MCTcodes. Supplementary information Supplementary data is available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Discrete data in the form of proportions with overdispersion and zero inflation can arise in toxicology and other similar fields. In regression analysis of such data, another problem that also may arise in practice is that some responses may be missing. In this paper, we develop estimation procedure for the parameters of a zero-inflated overdispersed binomial model in the presence of missing responses under three different missing data mechanisms. A weighted expectation maximization algorithm is used for the maximum likelihood estimation of the parameters involved. Extensive simulations are conducted to study the properties of the estimates in terms of average of estimates, relative bias, variance, mean squared error, and coverage probability of estimates. Simulations show much superior properties of the estimates obtained using the weighted expectation maximization algorithm. Some illustrative examples and a discussion are given. [ABSTRACT FROM AUTHOR]

We develop tests of goodness of fit of the exponential model against some over/under dispersion family of distributions. In particular, we develop 3 score test statistics and 3 likelihood ratio statistics. These are (S1, L1), (S2, L2), and (S3, L3) based on a general over-dispersed family of distributions, two specific over/under dispersed exponential models, namely, the gamma and the Weibull distributions, respectively. A simulation study shows that the statistics S3 and L3 have best overall performance, in terms of both, level and power. However, the statistic L3 can be liberal in some instances and it needs the maximum likelihood estimates of the parameters of theWeibull distribution as opposed to the statistic S3 which is very simple to use. So, our recommendation is to use the statistic S3 to test the fit of an exponential distribution over any over/under-dispersed exponential distribution. [ABSTRACT FROM AUTHOR]

Data in the form of proportions with extra-dispersion (over/under) arise in many biomedical, epidemiological, and toxicological applications. In some situations, two samples of data in the form of proportions with extra-dispersion arise in which the problem is to test the equality of the proportions in the two groups with unspecified and possibly unequal extra-dispersion parameters. This problem is analogous to the traditional Behrens-Fisher problem in which two normal population means with possibly unequal variances are compared. To deal with this problemwe develop eight tests and compare them in terms of empirical size and power, using a simulation study. Simulations show that a C(α) test based on extended quasi-likelihood estimates of the nuisance parameters holds nominal level most effectively (close to the nominal level) and it is at least as powerful as any other statistic that is not liberal. It has the simplest formula, is based on estimates of the nuisance parameters only under the null hypothesis, and is easiest to calculate. Also, it is robust in the sense that no distributional assumption is required to develop this statistic. [ABSTRACT FROM AUTHOR]

In this paper, we develop estimation procedure for the parameters of a zero-inflated over-dispersed/under-dispersed count model in the presence of missing responses. In particular, we deal with a zero-inflated extended negative binomial model in the presence of missing responses. A weighted expectation maximization algorithm is used for the maximum likelihood estimation of the parameters involved. Some simulations are conducted to study the properties of the estimators. Robustness of the procedure is shown when count data follow other over-dispersed models, such as the log-normal mixture of the Poisson distribution or even from a zero-inflated Poisson model. An illustrative example and a discussion leading to some conclusions are given. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Two samples count data with unequal over-dispersions arise in many applied statistics problems (biostatistics, epidemiology, etc.). In this situation, it is of interest to test the equality of the means. The traditional Behrens–Fisher problem is to test the equality of the means μ1and μ2of two normal populations where the variancesandare unknown. The purpose of this paper is to deal with the corresponding problem for over-dispersed count data. We develop six test procedures, namely, a likelihood ratio test LR, a likelihood ratio test based on the bias corrected maximum likelihood estimates of the nuisance parameters LR(bc), a score testT2, a score test based on the bias corrected estimates of the nuisance parametersT2(bc), aC(α) test based on the method of moments estimates of the nuisance parametersT1with Welch's [The significance of the difference between two means when the population variances are unequal. Biometrika. 1937;29:350–362] degree of freedom correction, and a testTNusing the asymptotic normal distribution ofT1. These procedures are then compared in terms of size and power using simulations. Simulations show the best overall performance of the statisticT1in terms of size and power and it is easy to calculate. For large sample sizes, for example, forn1=n2=50, all six statistics do well in terms of level and their power performances are also similar. So, for large sample sizes, the statisticTNshould be used as it is very easy to use in practice. [ABSTRACT FROM PUBLISHER]

Data in the form of survival times arise in many fields of studies such as engineering, manufacturing, aeronautics and bio-medical sciences. A popular model for survival data is the two parameter Weibull distribution. Often lifetime or survival time data that are collected in the form of two independent samples are assumed to have come from two independent Weibull populations with different shape and scale parameters. In such a situation it may be of interest to test the equality of the scale parameters with the shape parameters being unspecified. This is equivalent to testing the equality of the location parameters with the shape parameters being unspecified in two extreme value distributions. Also, this is analogous to the traditional Behrens-Fisher problem of testing the equality of the means µ 1 and µ 2 of two normal populations where the variances σ1² and σ2² are unknown. We develop four test procedures, namely, a likelihood ratio test, a C (α) test based on the maximum likelihood estimates of the nuisance parameters, a C (α) test based on the method of moments estimates of the nuisance parameters by Cran (1988), and a C (α) test based on the method of moments estimates of the nuisance parameters by Teimouri and Gupta (2013). These test statistics are then compared, in terms of empirical size and power, using a simulation study. [ABSTRACT FROM AUTHOR]

Longitudinal or clustered response data arise in many applications such as biostatistics, epidemiology and environmental studies. The repeated responses cannot in general be assumed to be independent. One method of analysing such data is by using the generalized estimating equations (GEE) approach. The current GEE method for estimating regression effects in longitudinal data focuses on the modelling of the working correlation matrix assuming a known variance function. However, correct choice of the correlation structure may not necessarily improve estimation efficiency for the regression parameters if the variance function is misspecified [Wang YG, Lin X. Effects of variance-function misspecification in analysis of longitudinal data. Biometrics. 2005;61:413–421]. In this connection two problems arise: finding a correct variance function and estimating the parameters of the chosen variance function. In this paper, we study the problem of estimating the parameters of the variance function assuming that the form of the variance function is known and then the effect of a misspecified variance function on the estimates of the regression parameters. We propose a GEE approach to estimate the parameters of the variance function. This estimation approach borrows the idea of Davidian and Carroll [Variance function estimation. J Amer Statist Assoc. 1987;82:1079–1091] by solving a nonlinear regression problem where residuals are regarded as the responses and the variance function is regarded as the regression function. A limited simulation study shows that the proposed method performs at least as well as the modified pseudo-likelihood approach developed by Wang and Zhao [A modified pseudolikelihood approach for analysis of longitudinal data. Biometrics. 2007;63:681–689]. Both these methods perform better than the GEE approach. [ABSTRACT FROM AUTHOR]

Longitudinal (clustered) response data arise in many bio-statistical applications which, in general, cannot be assumed to be independent. Generalized estimating equation (GEE) is a widely used method to estimate marginal regression parameters for correlated responses. The advantage of the GEE is that the estimates of the regression parameters are asymptotically unbiased even if the correlation structure is misspecified, although their small sample properties are not known. In this paper, two bias adjusted GEE estimators of the regression parameters in longitudinal data are obtained when the number of subjects is small. One is based on a bias correction, and the other is based on a bias reduction. Simulations show that the performances of both the bias-corrected methods are similar in terms of bias, efficiency, coverage probability, average coverage length, impact of misspecification of correlation structure, and impact of cluster size on bias correction. Both these methods show superior properties over the GEE estimates for small samples. Further, analysis of data involving a small number of subjects also shows improvement in bias, MSE, standard error, and length of the confidence interval of the estimates by the two bias adjusted methods over the GEE estimates. For small to moderate sample sizes ( [ABSTRACT FROM AUTHOR]

Abstract: Clustered (longitudinal) count data arise in many bio-statistical practices in which a number of repeated count responses are observed on a number of individuals. The repeated observations may also represent counts over time from a number of individuals. One important problem that arises in practice is to test homogeneity within clusters (individuals) and between clusters (individuals). As data within clusters are observations of repeated responses, the count data may be correlated and/or over-dispersed. For over-dispersed count data with unknown over-dispersion parameter we derive two score tests by assuming a random intercept model within the framework of (i) the negative binomial mixed effects model and (ii) the double extended quasi-likelihood mixed effects model (). These two statistics are much simpler than a statistic derived by under the framework of the over-dispersed generalized linear model. The first statistic takes the over-dispersion more directly into the model and therefore is expected to do well when the model assumptions are satisfied and the other statistic is expected to be robust. Simulations show superior level property of the statistics derived under the negative binomial and double extended quasi-likelihood model assumptions. A data set is analyzed and a discussion is given. [Copyright &y& Elsevier]

Inference concerning the negative binomial dispersion parameter, denoted by c, is important in many biological and biomedical investigations. Properties of the maximum-likelihood estimator of c and its bias-corrected version have been studied extensively, mainly, in terms of bias and efficiency [W.W. Piegorsch, Maximum likelihood estimation for the negative binomial dispersion parameter, Biometrics 46 (1990), pp. 863–867; S.J. Clark and J.N. Perry, Estimation of the negative binomial parameter κ by maximum quasi-likelihood, Biometrics 45 (1989), pp. 309–316; K.K. Saha and S.R. Paul, Bias corrected maximum likelihood estimator of the negative binomial dispersion parameter, Biometrics 61 (2005), pp. 179–185]. However, not much work has been done on the construction of confidence intervals (C.I.s) for c. The purpose of this paper is to study the behaviour of some C.I. procedures for c. We study, by simulations, three Wald type C.I. procedures based on the asymptotic distribution of the method of moments estimate (mme), the maximum-likelihood estimate (mle) and the bias-corrected mle (bcmle) [K.K. Saha and S.R. Paul, Bias corrected maximum likelihood estimator of the negative binomial dispersion parameter, Biometrics 61 (2005), pp. 179–185] of c. All three methods show serious under-coverage. We further study parametric bootstrap procedures based on these estimates of c, which significantly improve the coverage probabilities. The bootstrap C.I.s based on the mle (Boot-MLE method) and the bcmle (Boot-BCM method) have coverages that are significantly better (empirical coverage close to the nominal coverage) than the corresponding bootstrap C.I. based on the mme, especially for small sample size and highly over-dispersed data. However, simulation results on lengths of the C.I.s show evidence that all three bootstrap procedures have larger average coverage lengths. Therefore, for practical data analysis, the bootstrap C.I. Boot-MLE or Boot-BCM should be used, although Boot-MLE method seems to be preferable over the Boot-BCM method in terms of both coverage and length. Furthermore, Boot-MLE needs less computation than Boot-BCM. [ABSTRACT FROM AUTHOR]

The importance of the normal distribution for fitting continuous data is well known. However, in many practical situations data distribution departs from normality. For example, the sample skewness and the sample kurtosis are far away from 0 and 3, respectively, which are nice properties of normal distributions. So, it is important to have formal tests of normality against any alternative. D'Agostino et al. [A suggestion for using powerful and informative tests of normality, Am. Statist. 44 (1990), pp. 316-321] review four procedures Z2(g1), Z2(g2), D and K2 for testing departure from normality. The first two of these procedures are tests of normality against departure due to skewness and kurtosis, respectively. The other two tests are omnibus tests. An alternative to the normal distribution is a class of skew-normal distributions (see [A. Azzalini, A class of distributions which includes the normal ones, Scand. J. Statist. 12 (1985), pp. 171-178]). In this paper, we obtain a score test (W) and a likelihood ratio test (LR) of goodness of fit of the normal regression model against the skew-normal family of regression models. It turns out that the score test is based on the sample skewness and is of very simple form. The performance of these six procedures, in terms of size and power, are compared using simulations. The level properties of the three statistics LR, W and Z2(g1) are similar and close to the nominal level for moderate to large sample sizes. Also, their power properties are similar for small departure from normality due to skewness (γ1≤0.4). Of these, the score test statistic has a very simple form and computationally much simpler than the other two statistics. The LR statistic, in general, has highest power, although it is computationally much complex as it requires estimates of the parameters under the normal model as well as those under the skew-normal model. So, the score test may be used to test for normality against small departure from normality due to skewness. Otherwise, the likelihood ratio statistic LR should be used as it detects general departure from normality (due to both skewness and kurtosis) with, in general, largest power. [ABSTRACT FROM AUTHOR]

Objectives: Objectives The objectives of this study are to (1) evaluate the ability of the immune system to synthesize specific antibodies that catalyze the degradation of amyloid β peptide (Aβ) and to (2) evaluate the prospect of developing a catalytic IVIG (CIVIG) formulation for therapy of Alzheimer's disease (AD). Conclusions: Polyclonal autoantibodies from humans without dementia hydrolyzed Aβ specifically. The catalytic activity improved as a function of age. Patients with AD produced catalytic antibodies at increased levels. IgM-class antibodies expressed the activity at levels superior to IgGs. Production of catalytic autoantibodies appears to be an innate immunity function with adaptive improvements occurring upon Aβ overexpression, which suggests a beneficial function of the catalytic activity. The catalytic autoantibodies impeded Aβ aggregation, dissolved preformed Aβ aggregates, and inhibited Aβ cytotoxicity in tissue culture. Recombinant catalytic antibodies from a human library have been identified, validating the phenomenon of antibody-catalyzed Aβ cleavage. As a single catalyst molecule inactivates multiple Aβ molecules, catalytic antibodies may clear Aβ efficiently. IVIG did not cleave Aβ, indicating the importance of purification procedures that maintain catalytic site integrity. Traditional Aβ-binding antibodies form immune complexes that can induce inflammatory reaction and vascular dysfunction. Catalysts do not form stable immune complexes, minimizing these risks. Criteria appropriate for developing a CIVIG formulation with potential therapeutic utility are discussed, including isolation of the Aβ-specific catalytic subsets present in IgM and IgG from human blood. [ABSTRACT FROM AUTHOR]

Risk difference (RD) is an important measure in epidemiological studies where the probability of developing a disease for individuals in an exposed group, for example, is compared with that in a control group. There are varying cluster sizes in each group and the binary responses within each cluster cannot be assumed independent. Under the cluster sampling scenario, Lui ( Statistical Estimation of Epidemiological Risk. Wiley: CA, 2004; 7-27) discusses four methods for the construction of a confidence interval for the RD. In this paper we introduce two very simple methods. One method is based on an estimator of the variance of a ratio estimator ( Sampling Techniques (3rd edn). Wiley: New York, 1977; 30-67) and the other method is based on a sandwich estimator of the variance of the regression estimator using the generalized estimating equations approach of Zeger and Liang ( Biometrics 1986; 42:121-130). These two methods are then compared, by simulation, in terms of maintaining nominal coverage probability and average coverage length, with the four methods discussed by Lui ( Statistical Estimation of Epidemiological Risk. Wiley: CA, 2004; 7-27). Simulations show at least as good properties of these two methods as those of the others. The method based on an estimate of the variance of a ratio estimator performs best overall. It involves a very simple variance expression and can be implemented with a very few computer codes. Therefore, it can be considered as an easily implementable alternative. Copyright © 2008 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: Antibodies (Abs) with proteolytic and other catalytic activities have been characterized in the blood and mucosal secretions of humans and experimental animals. The catalytic activity can be traced to nucleophilic sites of innate origin located in Ab germline variable regions. Discoveries of the natural chemical reactivity of Abs were initially met with bewilderment, as the notion had taken hold that catalytic activities can be introduced into Abs by artificial means, but somatically operative selection pressures are designed only to adapt non-covalent Ab binding to antigen ground states. Unsurprisingly, initial efforts to engineer Abs with catalytic activity were oriented towards improving the non-covalent binding at the atoms immediately within the transition state reaction center. Slowly, however, dogmatic approaches to Ab catalysis have given way to the realization that efficient and specific catalytic Abs can be prepared by improving the natural nucleophilic reactivity combined with non-covalent recognition of epitope regions remote from the reaction center. The field remains beset, however, with controversy. This article attempts to provide a rational basis for natural Ab catalysis, in the hope that understanding this phenomenon will stimulate medical and basic science advances in the field. [Copyright &y& Elsevier]

A popular model to analyse over/under-dispersed proportions is to assume the extended beta-binomial model with dispersion (intraclass correlation) parameter ϕ and then to estimate this parameter by maximum likelihood. However, it is well known that maximum likelihood estimate (MLE) may be biased when the sample size n or the total Fisher information is small. In this paper we obtain a bias-corrected maximum likelihood (BCML) estimator of the intraclass correlation parameter and compare it, by simulation, in terms of bias and efficiency, with the MLE, an estimator Q2 based on optimal quadratic estimating equations of Crowder and recommended by Paul et al. and a double extended quasi-likelihood (DEQL) estimator proposed by Lee. The BCML estimator has superior bias and efficiency properties in most instances. Analyses of a set of toxicological data from Paul and a set of medical data pertaining to chromosomal abnormalities among survivors of the atomic bomb in Hiroshima from Otake and Prentice show, in general, much improvement in standard errors of the BCML estimates over the other three estimates. Copyright © 2005 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Antibodies (Abs) and enzymes are structural and functional relatives. Abs with promiscuous peptidase activity are ubiquitous in healthy humans, evidently derived from germline variable domain immunoglobulin genes encoding the serine protease-like nucleophilic function. Exogenous and endogenous electrophilic antigens can bind the nucleophilic sites covalently, and recent evidence suggests that immunization with such antigens can induce proteolytic antibodies. Previously, Ab catalytic activities have been linked to pathogenic autoimmune reactions, but recent studies indicate that proteolytic Abs may also serve beneficial functions. An example is the rapid and selective cleavage of the HIV-1 coat protein gp120 by IgMs found in uninfected humans. The selectivity of this reaction appears to derive from recognition of gp120 as a superantigen. A second example is the cleavage of amyloid ß-peptide by IgM and IgG from aged humans, a phenomenon that may represent a specific proteolytic response to a neurotoxic endogenous peptide implicated in the pathogenesis of Alzheimer’s disease. [ABSTRACT FROM AUTHOR]

We derive a first-order bias-corrected maximum likelihood estimator for the negative binomial dispersion parameter. This estimator is compared, in terms of bias and efficiency, with the maximum likelihood estimator investigated by(1990,Biometrics46,863–867), the moment and the maximum extended quasi-likelihood estimators investigated by(1989,Biometrics45,309–316), and a double-extended quasi-likelihood estimator. The bias-corrected maximum likelihood estimator has superior bias and efficiency properties in most instances. For ease of comparison we give results for the two-parameter negative binomial model. However, an example involving negative binomial regression is given. [ABSTRACT FROM AUTHOR]

Tests d'homogénéité de plusieurs populations à deux paramètres: Les auteurs étendent la méthode proposée par Fisher pour combiner deux statistiques indépendantes servant à tester l'homogénéité de plusieurs populations à deux paramètres. Ils explorent deux procédures combinant des statistiques asymptotiquement indépendantes: la première regroupe deux statistiques du rapport des vraisemblances et la seconde. deux statistiques de type score. Ils s'int6ressent plus spécifiquement au problème de tester l'homogénéité de plusieurs populations normales, binomiales négatives ou bêta-binomiales. Leurs simulations montrent que dam ce contexte. la méthode de Fisher donne d'assez bons résultats, même lorsque les statistiques ne sont indépendantes qu'asymptotiquement. Ils sont amenés à recommander le test de Fisher basé sur les statistiques scores, puisque ces dernères sont simples, faciles à calculer et maintiennent leur seuil uniformément bien. [ABSTRACT FROM AUTHOR]

We report the selective catalytic cleavage of the HIV coat protein gp120, a B cell superantigen, by IgM antibodies (Abs) from uninfected humans and mice that had not been previously exposed to gp120. The rate of IgM-catalyzed gp120 cleavage was greater than of other polypeptide substrates, including the bacterial superantigen protein A. The kinetic parameters of gp120 cleavage varied over a broad range depending on the source of the IgMs, and turnover numbers as great as 2.1/min were observed, suggesting that different Abs possess distinct gp120 recognition properties. IgG Abs failed to cleave gp120 detectably. The Fab fragment of a monoclonal IgM cleaved gp120, suggesting that the catalytic activity belongs to the antibody combining site. The electrophoretic profile of gp120 incubated with a monoclonal human IgM suggested hydrolysis at several sites. One of the cleavage sites was identified as the Lys432-Ala433 peptide bond, located within the region thought to be the Ab-recognizable superantigenic determinant. A covalently reactive peptide analog (CRA) corresponding to gp120 residues 421–431 with a C-terminal amidino phosphonate diester mimetic of the Lys432-Ala433 bond was employed to probe IgM nucleophilic reactivity. The peptidyl CRA inhibited the IgM-catalyzed cleavage of gp120 and formed covalent IgM adducts at levels exceeding a control hapten CRA devoid of the peptide sequence. These observations suggest that IgMs can selectively cleave gp120 by a nucleophilic mechanism and raise the possibility of their role as defense enzymes. [ABSTRACT FROM AUTHOR]

A procedure for testing for treatment effect in data similar to the data on premature ventricular contractions (PVC) is presented. We consider a zero-inflated beta-binomial model. Based on this model, we develop score tests to test for treatment effect in the data in which observations in the form of counts are recorded before and after applying a therapy. Results of a small simulation experiment, to study small sample behaviour of a score test and a likelihood ratio test, are reported and the PVC data are analysed. Both the score and the likelihood ratio tests show good level properties. Either the score tests or the likelihood ratio tests can be used for testing the presence of treatment effect. The score tests, however, may be preferable because they use estimates of the parameters only under the null hypothesis and in the important range π<0.5 power of the score test statistic S1 is slightly better than the likelihood ratio statistic LR1. Copyright © 2004 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

This paper is concerned with properties (bias, standard deviation, mean square error and efficiency) of twenty six estimators of the intraclass correlation in the analysis of binary data. Our main interest is to study these properties when data are generated from different distributions. For data generation we considered three over-dispersed binomial distributions, namely, the beta-binomial distribution, the probit normal binomial distribution and a mixture of two binomial distributions. The findings regarding bias, standard deviation and mean squared error of all these estimators, are that (a) in general, the distributions of biases of most of the estimators are negatively skewed. The biases are smallest when data are generated from the beta-binomial distribution and largest when data are generated from the mixture distribution; (b) the standard deviations are smallest when data are generated from the beta-binomial distribution; and (c) the mean squared errors are smallest when data are generated from the beta-binomial distribution and largest when data are generated from the mixture distribution. Of the 26, nine estimators including the maximum likelihood estimator, an estimator based on the optimal quadratic estimating equations of Crowder (1987), and an analysis of variance type estimator is found to have least amount of bias, standard deviation and mean squared error. Also, the distributions of the bias, standard deviation and mean squared error for each of these estimators are, in general, more symmetric than those of the other estimators. Our findings regarding efficiency are that the estimator based on the optimal quadratic estimating equations has consistently high efficiency and least variability in the efficiency results. In the important range in which the intraclass correlation is small (≤0 5), on the average, this estimator shows best efficiency performance. The analysis of variance type estimator seems to do well for larger values of the... [ABSTRACT FROM AUTHOR]

Examines the natural nucleophilic activity of antibodies for the purpose of specific cleavage of the human immunodeficiency virus-1 coat protein gp120. Enzyme-linked immunoabsorbent assay; Nucleophilic reactivity; Proteolysis.

Les auteurs développent des procédures scores permettant de tester l'adéquation de modèles linéaires généralisés discrets lorsque la valeur zéro est en surnombre dans l'échantillon. Les modéles binomial et de Poisson font l'objet d'une attention particuliére et, dans ce dernier cas, le test obtenu se ramène à celui de Broek (1995). Des simulations et un exemple sont également présentés. [ABSTRACT FROM AUTHOR]

Presents information on a study that determines whether a monoclonal anti-vasoactive intestinal peptide (VIP) antibody attenuates VIP vasorelaxation in vivo. Effects of the insertion of VIP on the surface of sterically stabilized liposomes to this response; Methodology used in the study; What the results suggest.

Determines whether a monoclonal anti-vasoactive intestinal peptide (VIP) antibody, which binds VIP with high affinity and specificity and catalyzes cleavage of the peptide in vitro, attenuates VIP vasorelaxation in vivo. Attenuation of VIP-induced vasodilation; Absence of significant effects of the anti-VIP antibody on vasodilation.

The intraclass correlation coefficient ρ is used as a measure of the degree of resemblance among family members with respect to some biological or psychological attribute, such as blood pressure level or IQ. Many studies have been done in the area. However, a significant gap in the literature is that few statistics are available to test the significance ( H 0 : ρ = 0 against H A : ρ ≠ 0 ) of this important biological measure and no study is available as to the performance of these statistics with respect to level and power. In this paper we develop three large sample test statistics and compare these, in terms of level and power, using extensive simulation studies of these, the Wald test developed in this paper is found to have a very simple form and highest power while maintaining level well. [ABSTRACT FROM AUTHOR]

Catalytic antibodies (CAbs) occur naturally in healthy individuals where they may form part of the innate immune system, but are preferentially found in those with autoimmune disease. CAbs can also be artificially engineered or elicited by immunizations. Their mechanisms of action include nucleophilic catalysis, induction of conformational strain, coordination with metal ions, and stabilization of transition states. Recent applications of CAbs with clinical significance include the conversion of cocaine to a non-psychoactive form, the degradation of nicotine, activation of prodrugs for targeted chemotherapy, protection from ultraviolet radiation, inhibition of HIV infectivity, and the destruction of aggregates of β-amyloid implicated in Alzheimer''s disease. Artificial CAbs are likely to find increasing applications in research, clinical medicine, diagnostics and manufacturing. [Copyright &y& Elsevier]

Classical immunization methods do not generate catalytic antibodies (catabodies), but recent findings suggest that the innate antibody repertoire is a rich catabody source. We describe the specificity and amyloid β (Aβ)-clearing effect of a catabody construct engineered from innate immunity principles. The catabody recognized the AβC terminus noncovalently and hydrolyzed Aβ rapidly, with no reactivity to the Aβ precursor protein, transthyretin amyloid aggregates, or irrelevant proteins containing the catabody-sensitive Aβ dipeptide unit. The catabody dissolved preformed Aβ aggregates and inhibited Aβ aggregation more potently than an Aβ-binding IgG. Intravenous catabody treatment reduced brain Aβ deposits in amouse Alzheimer disease model without inducing microgliosis or microhemorrhages. Specific Aβ hydrolysis appears to be an innate immune function that could be applied for therapeutic Aβ removal. [ABSTRACT FROM AUTHOR]

Highlights: [•] Chelation of bound metal inhibited Aβ hydrolysis by a catalytic antibody fragment. [•] The metal accelerated a reaction step after initial antibody nucleophilic attack. [•] The hydrolytic but not chelator-inactivated antibody dissolved Aβ aggregates. [•] Zn-induced conformational transitions were associated with restored catalysis. [•] The antibody requires bound metal for efficient Aβ hydrolysis and dissolution. [ABSTRACT FROM AUTHOR]

Peptide bond-hydrolyzing catalytic antibodies (catabodies) could degrade toxic proteins, but acquired immunity principles have not provided evidence for beneficial catabodies. Transthyretin (TTR) forms misfolded β-sheet aggregates responsible for age-associated amyloidosis. We describe nucleophilic catabodies from healthy humans without amyloidosis that degraded misfolded TTR (misTTR) without reactivity to the physiological tetramericTTR(phyTTR). IgM classBcell receptors specifically recognized the electrophilic analog of misTTR but not phyTTR. IgM but not IgG class antibodies hydrolyzed the particulate and soluble misTTR species. No misTTR-IgM binding was detected. The IgMs accounted for essentially all of the misTTR hydrolytic activity of unfractionated human serum. The IgMs did not degrade non-amyloidogenic, non-superantigenic proteins. Individual monoclonal IgMs (mIgMs) expressed variable misTTR hydrolytic rates and differing oligoreactivity directed to amyloid β peptide and microbial superantigen proteins. A subset of the mIgMs was monoreactive for misTTR. Excess misTTR was dissolved by a hydrolytic mIgM. The studies reveal a novel antibody property, the innate ability of IgMs to selectively degrade and dissolve toxic misTTR species as a first line immune function. [ABSTRACT FROM AUTHOR]

Amyloidosis involves the extracellular deposition of proteinaceous amyloid fibrils and accessory molecules in organ(s) and/or tissue(s), and is associated with a host of human diseases, including Alzheimer disease, diabetes, and heart disease. Unfortunately, the amyloidoses are currently incurable, and there is an urgent need for less invasive diagnostics. To address this, we have generated 22 monoclonal antibodies (mAbs) against aggregates formed by a blood transport protein, transthyretin (TTR), which primarily forms amyloid fibrils in a patient's heart and/or peripheral nerves. Four of the mAbs, 2T5C9, 2G9C, T1F11, and TB2H7, demonstrated diagnostic potential in enzyme-linked immunosorbent assays (ELISA) by their low to sub-nanomolar cross-reactivity with recombinant wild-type (WT) and mutant TTR aggregates and lack of binding to native TTR or amyloid fibrils formed by other peptides or proteins. Notably, in the presence of normal human sera, three of the four mAbs, 2T5C9, 2G9C, and T1F11, retained low nM binding to TTR amyloid fibrils derived from two patients with familial amyloidotic polyneuropathy (FAP). The two most promising mAbs, 2T5C9 and 2G9C, were also shown by immunohistochemistry to have low nM binding to TTR amyloid deposits in cardiac tissue sections from two FAP patients. Taken together, these findings strongly support further investigations on the diagnostic utility of TTR aggregate specific mAbs for patients with TTR amyloidoses. [ABSTRACT FROM AUTHOR]

Some antibodies contain variable (V) domain catalytic sites. We report the superior amide and peptide bond-hydrolyzing activity of the same heavy and light chain V domains expressed in the IgM constant domain scaffold compared with the IgG scaffold. The superior catalytic activity of recombinant IgM was evident using two substrates, a small model peptide that is hydrolyzed without involvement of high affinity epitope binding, and HIV gp120, which is recognized specifically by noncovalent means prior to the hydrolytic reaction. The catalytic activity was inhibited by an electrophilic phosphonate diester, consistent with a nucleophilic catalytic mechanism. All 13 monoclonal IgMs tested displayed robust hydrolytic activities varying over a 91-fold range, consistent with expression of the catalytic functions at distinct levels by different V domains. The catalytic activity of polyclonal IgM was superior to polyclonal IgG from the same sera, indicating that on average IgMs express the catalytic function at levels greater than IgGs. The findings indicate a favorable effect of the remote IgM constant domain scaffold on the integrity of the V-domain catalytic site and provide a structural basis for conceiving antibody catalysis as a first line immune function expressed at high levels prior to development of mature IgG class antibodies. [ABSTRACT FROM AUTHOR]

Antibodies that recognize microbial B lymphocyte superantigenic epitopes are produced constitutively with no requirement for adaptive immune maturation. We report cleavage of the Staphylococcus aureus virulence factor extracellular fibrinogen-binding protein (Efb) by catalytic antibodies produced with no exposure to the bacterium and reduction of the catalytic antibody activity following infection. IgG catalytic antibodies that specifically hydrolyzed Efb via a nucleophilic catalytic mechanism were found in the blood of healthy humans and aseptic mice free of S. aureus infection. IgG hydrolyzed peptide bonds on the C-terminal side of basic amino acids, including a bond located within the C3b-binding domain of Efb. Efb digested with the IgG lost its ability to bind C3b and inhibit complement-dependent antibody-mediated red blood cell lysis. In addition to catalysis, the IgG expressed saturable Efb binding activity. IgG from S. aureus-infected mice displayed reduced Efb cleaving activity and increased Efb binding activity compared with uninfected controls, suggesting differing effects of the infection on the antibody subsets responsible for the two activities. IgG from children hospitalized for S. aureus infection also displayed reduced Efb cleavage compared with healthy children. These data suggest a potential defense function for constitutively produced catalytic antibodies to a putative superantigenic site of Efb, but an adaptive catalytic response appears to be proscribed. [ABSTRACT FROM AUTHOR]

Accumulation of amyloid-β protein (Aβ) in the brain is thought to be a causal event in Alzheimer's disease (AD). Immunotherapy targeting Aβ holds great promise for reducing Aβ in the brain. Here, we evaluated the efficacy and safety of anti-Aβ single-chain antibody (scFv59) delivery via recombinant adeno-associated virus (rAAV) on reducing Aβ deposits in an AD mouse model (TgAβPPswe/PS1dE9). First, delivery of scFv59 to the brain was optimized by injecting rAAV serotypes 1, 2, and 5 into the right lateral ventricle. Symmetrical high expression of scFv59 was found throughout the hippocampus and partly in the neocortex in both hemispheres via rAAV1 or rAAV5, while scFv59 expression via rAAV2 was mostly limited to one hemisphere. rAAV1, however, induced apoptosis and microglial activation but rAAV5 did not. Therefore, rAAV5 was selected for therapeutic scFv59 delivery in TgAβPPswe/PS1dE9 mice. rAAV5 was similarly injected into the ventricle of 10-month-old TgAβPPswe/PS1dE9 mice and 5 months later its efficacy and safety were evaluated. Immunoreactive Aβ deposits reduced in the hippocampus. Aβ42 levels in cerebrospinal fluid (CSF) tended to increase and the Aβ40 : 42 ratio decreased in CSF, suggesting that Aβ42 was relocated from the parenchyma to CSF. Hemorrhages associated with a focal increase in blood vessel amyloid were found in the brain. While immunotherapy has great potential for clearing cerebral Aβ, caution for cerebrovascular effects should be exercised when rAAV-mediated anti-Aβ immunotherapy is applied. [ABSTRACT FROM AUTHOR]

Accumulation of amyloid-β protein (Aβ) in the brain is thought to be a causal event in Alzheimer's disease (AD). Immunotherapy targeting Aβ holds great promise for reducing Aβ in the brain. Here, we evaluated the efficacy and safety of anti-Aβ single-chain antibody (scFv59) delivery via recombinant adeno-associated virus (rAAV) on reducing Aβ deposits in an AD mouse model (TgAβPPswe/PS1dE9). First, delivery of scFv59 to the brain was optimized by injecting rAAV serotypes 1, 2, and 5 into the right lateral ventricle. Symmetrical high expression of scFv59 was found throughout the hippocampus and partly in the neocortex in both hemispheres via rAAV1 or rAAV5, while scFv59 expression via rAAV2 was mostly limited to one hemisphere. rAAV1, however, induced apoptosis and microglial activation but rAAV5 did not. Therefore, rAAV5 was selected for therapeutic scFv59 delivery in TgAβPPswe/PS1dE9 mice. rAAV5 was similarly injected into the ventricle of 10-month-old TgAβPPswe/PS1dE9 mice and 5 months later its efficacy and safety were evaluated. Immunoreactive Aβ deposits reduced in the hippocampus. Aβ42 levels in cerebrospinal fluid (CSF) tended to increase and the Aβ40 : 42 ratio decreased in CSF, suggesting that Aβ42 was relocated from the parenchyma to CSF. Hemorrhages associated with a focal increase in blood vessel amyloid were found in the brain. While immunotherapy has great potential for clearing cerebral Aβ, caution for cerebrovascular effects should be exercised when rAAV-mediated anti-Aβ immunotherapy is applied. [ABSTRACT FROM AUTHOR]

We review attempts to treat Alzheimer disease with antibodies that bind amyloid beta peptide (Abeta) and the feasibility of developing catalytic antibodies for this purpose. Naturally occurring immunoglobulin M (IgM) class antibodies that hydrolyze Abeta and inhibit Abeta aggregation were identified. The production of these antibodies increases as a function of age, ostensibly reflecting an attempt by the immune system to protect against the deleterious effect of Abeta accumulation in old age. A search for catalytic antibodies in a library of human immunoglobulins variable (IgV) domains yielded catalysts that hydrolyzed Abeta specifically at exceptionally rapid rates. The catalytic IgVs contained the light-chain variable domains within scaffolds that are structurally reminiscent of phylogenetically ancient antibodies. Inclusion of the heavy-chain variable domain in the IgV constructs resulted in reduced catalysis. We present our view that catalytic antibodies are likely to emerge as more efficacious and safer immunotherapy reagents compared to traditional Abeta-binding antibodies. [ABSTRACT FROM AUTHOR]

We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures. Epitope mapping with synthetic peptides and electrophilic peptide analogs indicated binary recognition of two distinct gp120 regions by anti-E-gp120 mAbs, the 421-433 and 288-306 peptide regions. Univalent Fab and single chain Fv fragments expressed the ability to recognize both peptides. X-ray crystallography of an anti-E-gp120 Fab fragment revealed two neighboring cavities, the typical antigen-binding cavity formed by the complementarity determining regions (CDRs) and another cavity dominated by antibody heavy chain variable (VH) domain framework (FR) residues. Substitution of the FR cavity VH Lys-19 residue by an Ala residue resulted in attenuated binding of the 421-433 region peptide probe. The CDRs and VH FR replacement/silent mutation ratios exceeded the ratio for a random mutation process, suggesting adaptive development of both putative binding sites. All mAbs studied were derived from VH1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp 120. The conserved 421- 433 region of gp120 is essential for HIV binding to host CD4 receptors. This region is recognized weakly by the FR of antibodies produced without exposure to HIV, but it usually fails to induce adaptive synthesis of neutralizing antibodies. We present models accounting for improved CD4-binding site recognition and broad HIV neutralizing activity of the mAbs, long sought goals in HIV vaccine development. [ABSTRACT FROM AUTHOR]

The antigen recognition site of antibodies consists of the heavy and light chain variable domains (VL and VH domains). VL domains catalyze peptide bond hydrolysis independent of VH domains (Mei, S., Mody, B., Ekiund, S. H., and Paul, S. (1991) I. Biol. Chem. 266, 15571-15574). VH domains bind antigens noncovalently independent of VL domains (Ward, E. S., Güssow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989) Nature 341, 544-546). We describe specific hydrolysis of fusion proteins of the hepatitis C virus E2 protein with glutathione S-transferase (GST-E2) or FLAG peptide (FLAG-E2) by antibodies containing the VH domain of an anti-E2 IgG paired with promiscuously catalytic VL domains. The hybrid IgG hydrolyzed the E2 fusion proteins more rapidly than the unpaired light chain. An active site-directed inhibitor of serine proteases inhibited the proteolytic activity of the hybrid IgG, indicating a serine protease mechanism. The hybrid IgG displayed noncovalent E2 binding in enzyme-linked immunosorbent assay tests. Immunoblotting studies suggested hydrolysis of FLAG-E2 at a bond within E2 located ∼11 kDa from the N terminus. GST-E2 was hydrolyzed by the hybrid lgG at bonds in the GST tag. The differing cleavage pattern of FLAG-E2 and GST-E2 can be explained by the split-site model of catalysis, in which conformational differences in the E2 fusion protein substrates position alternate peptide bonds in register with the antibody catalytic subsite despite a common noncovalent binding mechanism. These studies provide proof-of-principle that the catalytic activity of a light chain can be rendered antigen-specific by pairing with a noncovalently binding heavy chain subunit. [ABSTRACT FROM AUTHOR]

Nucleophilic sites in the paired variable domains of the light and heavy chains (V[subL] and V[subH] domains) of Ig can catalyze peptide bond hydrolysis. Amyloid β (Aβ)-binding Igs are under consideration for immunotherapy of Alzheimer disease. We searched for Aβ-hydrolyzing human IgV domains (IgVs) in a library containing a majority of single chain Fv clones mimicking physiological V[subL]-V[subH]-combining sites and minority IgV populations with nonphysiological structures generated by cloning errors. Random screening and covalent selection of phage-displayed IgVs with an electrophilic Aβ analog identified rare IgVs that hydrolyzed Aβ mainly at His[sup14]-Gln[sup15]. Inhibition of IgV catalysis and irreversible binding by an electrophilic hapten suggested a nucleophilic catalytic mechanism. Structural analysis indicated that the catalytic IgVs are nonphysiological structures, a two domain heterodimeric V[subL] (IgV[subL2]-t) and single domain V[subL] clones with aberrant polypeptide tags (IgV[subL]-t'). The IgVs hydrolyzed Aβ at rates superior to naturally occurring Igs by 3-4 orders of magnitude. Forced pairing of the single domain V[subL] with V[subH] or V[subL] domains resulted in reduced Aβ hydrolysis, suggesting catalysis by the unpaired V[subL] domain. Angstrom level amino acid displacements evident in molecular models of the two domain and unpaired V[subL] domain clones explain alterations of catalytic activity. In view of their superior catalytic activity, the V[subL] domain IgVs may help attain clearance of medically important antigens more efficiently than natural Igs. [ABSTRACT FROM AUTHOR]

Abstract: Immunoglobulins (Igs) in uninfected humans recognize residues 421–433 located in the B cell superantigenic site (SAg) of the HIV envelope protein gp120 and catalyze its hydrolysis by a serine protease-like mechanism. The catalytic activity is encoded by germline Ig variable (V) region genes, and is expressed at robust levels by IgMs and IgAs but poorly by IgGs. Mucosal IgAs are highly catalytic and neutralize HIV, suggesting that they constitute a first line of defense against HIV. Lupus patients produce the Igs at enhanced levels. Homology of the 421–433 region with an endogenous retroviral sequence and a bacterial protein may provide clues about the antigen driving anti-SAg synthesis in lupus patients and uninfected subjects. The potency and breadth of HIV neutralization revives hopes of clinical application of catalytic anti-421–433 Igs as immunotherapeutic and topical microbicide reagents. Adaptive improvement of anti-SAg catalytic Igs in HIV infected subjects is not customary. Further study of the properties of the naturally occurring anti-SAg catalytic Igs should provide valuable guidance in designing a prophylactic vaccine that amplifies protective catalytic immunity to HIV. [Copyright &y& Elsevier]

The antigen-binding sites of antibodies (Abs) can express enzyme-like nucleophiles that react covalently with electrophilic compounds. We examined the irreversible and specific inactivation of antibodies (Abs) to Factor VIII (FVIII) responsible for failure of FVIII replacement therapy in hemophilia A (HA) patients. Electrophilic analogs of FVIII (E-FVIII) and its C2 domain (E-C2) were prepared by placing the strongly electrophilic phosphonate groups at surface-exposed Lys side chains of diverse antigenic epitopes. IgG Abs to FVIII from HA patients formed stable immune complexes with E-FVIII and E-C2 that were refractory to dissociation by SDS treatment and boiling, procedures that dissociate noncovalent Ab-antigen complexes. The rate-limiting step in the reaction was formation of the initial noncovalent complexes. Conversion of the initial complexes to the irreversible state occurred rapidly. The antigenic epitopes of E-FVIII were largely intact, and most of the Abs were consumed covalently. E-FVIII expressed poor FVIII cofactor activity in clotting factor assays. Nonspecific interference by E-FVIII in clotting factor function was not evident. Treatment with E-FVIII, and to a lesser extent E-C2, irreversibly relieved the FVIII inhibitory effect of HA IgG in clotting factor assays. Small FVIII peptides did not display useful reactivity, highlighting the diverse epitope specificities of the Abs and the conformational character of FVIII epitopes. E-FVIII is a prototype reagent able to attain irreversible and specific inactivation of pathogenic Abs. [ABSTRACT FROM AUTHOR]

Abstract: Immunoglobulins (Igs) that bind amyloid β peptide (Aβ) are under clinical trials for immunotherapy of Alzheimer disease (AD). We have identified IgMs and recombinant Ig fragments that hydrolyze Aβ. Hydrolysis of peripheral Aβ by the IgMs may induce increased Aβ release from the brain. The catalytic IgMs are increased in AD patients, presumably reflecting a protective autoimmune response. Reduced Aβ aggregation and neurotoxicity attributable to the catalytic function were evident. These findings provide a foundation for development of catalytic Igs for AD immunotherapy. [Copyright &y& Elsevier]

We describe IgM class human autoantibodies that hydrolyze amyloid β peptide 1-40 (Aβ40). A monoclonal IgM from a patient with Waldenström's macroglobulinemia hydrolyzed Aβ340 at the Lys-28-Gly-29 bond and Lys-16-Ala-17 bonds. The catalytic activity was inhibited stoichiometrically by an electrophilic serine protease inhibitor. Treatment with the catalytic IgM blocked the aggregation and toxicity of Aβ40 in neuronal cell cultures. IgMs purified from the sera of patients with Alzheimer disease (AD) hydrolyzed Aβ40 at rates superior to IgMs from age-matched humans without dementia. IgMs from non-elderly humans expressed the least catalytic activity. The reaction rate was sufficient to afford appreciable degradation at physiological Aβ and IgM concentrations found in peripheral circulation. Increased Aβ concentrations in the AD brain are thought to induce neurodegenerative effects. Peripheral administration of Aβ binding antibodies has been suggested as a potential treatment of AD. Our results suggest that catalytic IgM autoantibodies can help clear Aβ, and they open the possibility of using catalytic Abs for AD immunotherapy. [ABSTRACT FROM AUTHOR]

Rare monoclonal antibodies (Abs) can form irreversible complexes with antigens by enzyme-like covalent nucleophile-electrophile pairing. To determine the feasibility of applying irreversible antigen inactivation by Abs as the basis of vaccination against microbes, we studied the polyclonal nucleophilic Ab response induced by the electrophilic analog of a synthetic peptide corresponding to the principal neutralizing determinant (PND) of human immunodeficiency virus type-1 (HIV) gp120 located in the V3 domain. Abs from mice immunized with the PND analog containing electrophilic phosphonates (E-PND) neutralized a homologous HIV strain (MN) ∼50-fold more potently than control Abs from mice immunized with PND. The IgG fractions displayed binding to intact HIV particles. HIV complexes formed by anti-E-PND IgG dissociated noticeably more slowly than the complexes formed by anti-PND IgG. The slower dissociation kinetics are predicted to maintain long-lasting blockade of host cell receptor recognition by gp120. Pre-treatment of the anti-PND IgG with a haptenic electrophilic phosphonate compound resulted in more rapid dissociation of the HIV-IgG complexes, consistent with the hypothesis that enhanced Ab nucleophilic reactivity induced by electrophilic immunization imparts irreversible character to the complexes. These results suggest that electrophilic immunization induces a sufficiently robust nucleophilic Ab response to enhance the anti-microbial efficacy of candidate polypeptide vaccines. [ABSTRACT FROM AUTHOR]

Abstract: Antibodies (Abs) to the superantigenic determinant of HIV gp120 (gp120SAg) are potential protective agents against HIV infection. We report that the light chain subunits of Abs cloned from lupus patients using phage library methods bind and hydrolyze gp120SAg independent of the heavy chain. Unlike frequent gp120SAg recognition by intact Abs attributable to VH domain structural elements, the isolated light chains expressed this activity rarely. Four light chains capable of gp120SAg recognition were identified by fractionating phage displayed light chains using peptide probes containing gp120 residues 421–433, a gp120SAg component. Three light chains expressed non-covalent gp120SAg binding and one expressed gp120SAg hydrolyzing activity. The hydrolytic light chain was isolated by covalent phage fractionation using an electrophilic analog of residues 421–433. This light chain hydrolyzed a reporter gp120SAg substrate and full-length gp120. Other peptide substrates and proteins were hydrolyzed at lower rates or not at all. Consistent with the expected nucleophilic mechanism of hydrolysis, the light chain reacted selectively and covalently with the electrophilic gp120SAg peptide analog. The hydrolytic reaction entailed a fast initial step followed by a slower rate limiting step, suggesting rapid substrate acylation and slow deacylation. All four gp120SAg-recognizing light chains contained sequence diversifications relative to their germline gene counterparts. These observations indicate that in rare instances, the light chain subunit can bind and hydrolyze gp120SAg without the participation of the heavy chain. The pairing of such light chains with heavy chains capable of gp120SAg recognition represents a potential mechanism for generating protective Abs with enhanced HIV binding strength and anti-viral proteolytic activity. [Copyright &y& Elsevier]

Conventional antibodies react with antigens reversibly. We report the formation of unusually stable complexes of HIV gp120 and nucleophilic antibodies raised by immunization with an electrophilic HIV gp120 analog (E-gp120). The stability of the complexes was evident from their very slow dissociation in a nondenaturing solvent (approximate t½ 18.5 days) and their resistance to dissociation by a denaturant commonly employed to disrupt noncovalent protein-protein binding (sodium dodecyl sulfate). Kinetic studies indicated time-dependent and virtually complete progression of the antibody-gp120 complexes from the initial noncovalent state to a poorly dissociable state. The antibodies to E-gp120 displayed improved covalent reactivity with an electrophilic phosphonate probe compared to control antibodies, suggesting their enhanced nucleophilicity. One of the stably binding antibodies neutralized the infectivity of CCR5-dependent primary HIV strains belonging to clades B and C. These findings suggest the feasibility of raising antibodies capable of long-lasting inactivation of antigens by electrophilic immunization. Copyright © 2006 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]