Significance tests for analyzing gene expression data with small sample sizes.

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.

Estimation for zero-inflated beta-binomial regression model with missing response data.

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.

Specific amyloid ß clearance by a catalytic antibody construct.

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 ß (Aß)-clearing effect of a catabody construct engineered from innate immunity principles. The catabody recognized the Aß C terminus noncovalently and hydrolyzed Aß rapidly, with no reactivity to the Aß precursor protein, transthyretin amyloid aggregates, or irrelevant proteins containing the catabody-sensitive Aß dipeptide unit. The catabody dissolved preformed Aß aggregates and inhibited Aß aggregation more potently than an Aß-binding IgG. Intravenous catabody treatment reduced brain Aß deposits in a mouse Alzheimer disease model without inducing microgliosis or microhemorrhages. Specific Aß hydrolysis appears to be an innate immune function that could be applied for therapeutic Aß removal.

Estimation for zero-inflated over-dispersed count data model with missing response.

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.

Physiological IgM class catalytic antibodies selective for transthyretin amyloid.

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 ß-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 tetrameric TTR (phyTTR). IgM class B cell 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 ß 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.

Small sample GEE estimation of regression parameters for longitudinal data.

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 (N ≤50), either of the bias-corrected methods GEEBc and GEEBr can be used. However, the method GEEBc should be preferred over GEEBr, as the former is computationally easier. For large sample sizes, the GEE method can be used.

Antibodies to a superantigenic glycoprotein 120 epitope as the basis for developing an HIV vaccine.

Failure to induce synthesis of neutralizing Abs to the CD4 binding determinant (CD4BD) of gp120, a central objective in HIV vaccine research, has been alternately ascribed to insufficient immunogen binding to Abs in their germline V region configuration expressed as BCRs, insufficient adaptive mutations in Ab V regions, and conformational instability of gp120. We employed peptide analogs of gp120 residues 421-433 within the CD4BD (CD4BD(core)) to identify Abs produced without prior exposure to HIV (constitutive Abs). The CD4BD(core) peptide was recognized by single-chain Fv fragments from noninfected humans with lupus that neutralized genetically diverse strains belonging to various HIV subtypes. Replacing the framework region (FR) of a V(H)4-family single-chain Fv with the corresponding V(H)3-family FRs from single-chain Fv JL427 improved the CD4BD(core) peptide-binding activity, suggesting a CD4BD(core) binding site outside the pocket formed by the CDRs. Replacement mutations in the FR site vicinity suggested the potential for adaptive improvement. A very small subset of serum CD4BD(core)-specific serum IgAs from noninfected humans without autoimmune disease isolated by epitope-specific chromatography neutralized the virus potently. A CD4BD(core)-specific, HIV neutralizing murine IgM with H and L chain V regions (V(H) and V(L) regions) free of immunogen-driven somatic mutations was induced by immunization with a CD4BD(core) peptide analog containing an electrophilic group that binds B cells covalently. The studies indicate broad and potent HIV neutralization by constitutive Abs as an innate, germline-encoded activity directed to the superantigenic CD4BD(core) epitope that is available for amplification for vaccination against HIV.

Constant domain-regulated antibody catalysis.

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.

Constitutive production of catalytic antibodies to a Staphylococcus aureus virulence factor and effect of infection.

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.

Modified Gaussian estimation for correlated binary data.

In this paper, we develop a Gaussian estimation (GE) procedure to estimate the parameters of a regression model for correlated (longitudinal) binary response data using a working correlation matrix. A two-step iterative procedure is proposed for estimating the regression parameters by the GE method and the correlation parameters by the method of moments. Consistency properties of the estimators are discussed. A simulation study was conducted to compare 11 estimators of the regression parameters, namely, four versions of the GE, five versions of the generalized estimating equations (GEEs), and two versions of the weighted GEE. Simulations show that (i) the Gaussian estimates have the smallest mean square error and best coverage probability if the working correlation structure is correctly specified and (ii) when the working correlation structure is correctly specified, the GE and the GEE with exchangeable correlation structure perform best as opposed to when the correlation structure is misspecified.

Nature and nurture of catalytic antibodies.

Immunoglobulins (antibodies) frequently express constitutive functions. Two such functions are nucleophilic catalysis and the reversible binding to B-cell superantigens. Constitutive or "naturally-occurring" antibodies are produced spontaneously from germline genetic information. The antibody structural elements mediating the constitutive functions have originated over millions of years of phylogenic evolution, contrasting with antigen-driven, somatic sequence diversification of the complementarity determining regions (CDR) that underlies the better-known high affinity antigen binding function of antibodies. Often, the framework regions (FRs) play a dominant role in antibody constitutive functions. Catalytic antibody subsets with promiscuous, autoantigen-directed and microbe-directed specificities have been identified. Mucosal antibodies may be specialized to express high-level catalytic activity against microbes transmitted by the mucosal route, exemplified by constitutive production of IgA class antibodies in mucosal secretions that catalyze the cleavage of HIV gp120. Catalytic specificity can be gained by constitutive noncovalent superantigen binding at the FRs and by adaptive development of noncovalent classical antigen or superantigen binding, respectively, at the CDRs and FRs. Growing evidence suggests important functional roles for catalytic antibodies in homeostasis, autoimmune disease and protection against infection. Adaptive antibody responses to microbial superantigens are proscribed underphysiological circumstances. Covalent electrophilic immunogen binding to constitutively expressed nucleophilic sites in B-cell receptors bypasses the restriction on adaptive antibody production, and simultaneous occupancy of the CDR binding site by a stimulatory antigenic epitope can also overcome the downregulatory effect of superantigen binding at the FRs. These concepts may be useful for developing novel vaccines that capitalize and improve on constitutive antibody functions for protection against microbes.

Toward effective HIV vaccination: induction of binary epitope reactive antibodies with broad HIV neutralizing activity.

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 (V(H)) domain framework (FR) residues. Substitution of the FR cavity V(H) Lys-19 residue by an Ala residue resulted in attenuated binding of the 421-433 region peptide probe. The CDRs and V(H) 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 V(H)1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp120. 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.

Immunological origin and functional properties of catalytic autoantibodies to amyloid beta peptide.

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 beta peptide (Abeta) 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 Abeta 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 Abeta overexpression, which suggests a beneficial function of the catalytic activity. The catalytic autoantibodies impeded Abeta aggregation, dissolved preformed Abeta aggregates, and inhibited Abeta cytotoxicity in tissue culture. Recombinant catalytic antibodies from a human library have been identified, validating the phenomenon of antibody-catalyzed Abeta cleavage. As a single catalyst molecule inactivates multiple Abeta molecules, catalytic antibodies may clear Abeta efficiently. IVIG did not cleave Abeta, indicating the importance of purification procedures that maintain catalytic site integrity. Traditional Abeta-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 Abeta-specific catalytic subsets present in IgM and IgG from human blood.

Catalytic antibody (catabody) platform for age-associated amyloid disease: From Heisenberg's uncertainty principle to the verge of medical interventions.

Quantum mechanics-based design of useful catalytic antibodies (catabodies) failed because of the uncertain structure of the dynamic catalyst-substrate complex. The Catabody Platform emerged from discovery of beneficial germline gene catabodies that hydrolyzed self-proteins by transient covalent pairing of the strong catabody nucleophile with a weak target protein electrophile. Catabodies have evolved by Darwinian natural selection for protection against misfolded self-proteins that threatened survival by causing amyloid disease. Ancient antibody scaffolds upregulate the catalytic activity of the antibody variable (V) domains. Healthy humans universally produce beneficial catabodies specific for at least 3 misfolded self-proteins, transthyretin, amyloid ß peptide and tau protein. Catabody are superior to ordinary antibodies because of catalyst reuse for thousands of target destruction cycles with little or no risk of causing inflammation, a must for non-toxic removal of abundant targets such as amyloids. Library mining with electrophilic target analogs (ETAs) isolates therapy-grade catabodies (fast, specific). Ex vivo- and in vivo-verified catabodies specific for the misfolded protein are available to dissolve brain, cardiac and vertebral amyloids. Immunization with ETAs overcomes important ordinary vaccine limitations (no catabody induction, poor immunogenicity of key target epitopes). We conceive electrophilic longevity vaccines that can induce catabody synthesis for long-lasting protection against amyloid disease.

Catalytic antibodies to HIV: physiological role and potential clinical utility.

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.

Catalytic antibodies to amyloid beta peptide in defense against Alzheimer disease.

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

Antibodies to the superantigenic site of HIV-1 gp120: hydrolytic and binding activities of the light chain subunit.

Antibodies (Abs) to the superantigenic determinant of HIV gp120 (gp120(SAg)) 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 gp120(SAg) independent of the heavy chain. Unlike frequent gp120(SAg) recognition by intact Abs attributable to V(H) domain structural elements, the isolated light chains expressed this activity rarely. Four light chains capable of gp120(SAg) recognition were identified by fractionating phage displayed light chains using peptide probes containing gp120 residues 421-433, a gp120(SAg) component. Three light chains expressed non-covalent gp120(SAg) binding and one expressed gp120(SAg) 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 gp120(SAg) 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 gp120(SAg) 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 gp120(SAg)-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 gp120(SAg) without the participation of the heavy chain. The pairing of such light chains with heavy chains capable of gp120(SAg) recognition represents a potential mechanism for generating protective Abs with enhanced HIV binding strength and anti-viral proteolytic activity.

Characterization of gp120 hydrolysis by IgA antibodies from humans without HIV infection.

Antibody hydrolysis of the superantigenic gp120 site and HIV-1 neutralization was studied as a potential anti-HIV mechanism in uninfected humans. gp120 hydrolysis by purified serum and salivary antibodies was determined by electrophoresis and peptide sequencing, the proteolytic mechanism was analyzed using electrophilic peptide analogs, and viral neutralization was studied using peripheral blood mononuclear cells as hosts. Polyclonal and monoclonal IgA but not IgG preparations selectively catalyzed the cleavage of HIV gp120 at rates sufficient to predict biologically relevant protection against the virus. The IgA hydrolytic reaction proceeded by noncovalent recognition of gp120 residues 421-433, a component of the superantigenic site of gp120, coordinated with peptide bond cleavage via a serine protease-like mechanism. The Lys-432-Ala-433 bond was one of the cleavage sites. Infection of peripheral blood mononuclear cells by a primary isolate of HIV was neutralized by the IgA but not IgG fractions. The neutralizing activity was specifically inhibited by an electrophilic inhibitor of the catalytic activity. The existence of catalytic IgAs to gp120 in uninfected humans suggests their role in resistance to HIV.

Naturally occurring catalytic antibodies: evidence for preferred development of the catalytic function in IgA class antibodies.

IgG class antibodies express catalytic activities rarely and at very low levels. Here, we studied polyclonal IgA and IgG preparations from healthy human sera and saliva for the ability to hydrolyze model peptidyl-aminomethylcoumarin (peptide-AMC) substrates. These substrates permit objective evaluation of the catalytic potential of the antibody classes with minimal effects of noncovalent interactions occurring at sites remote from the reaction center. The IgA preparations hydrolyzed Glu-Ala-Arg-AMC at rates 3-orders of magnitude greater than IgG preparations from the same individuals. The cleavage occurred preferentially on the C terminal side of a basic residue. The activity was confirmed using monoclonal IgAs isolated from patients with multiple myeloma. Active site-directed inhibitors of serine proteases inhibited the catalytic activity and were bound irreversibly by the IgA, suggesting the involvement of a serine protease-like mechanism similar to that utilized by previously described IgM antibodies. These observations suggest that mechanisms underlying B cell clonal selection favor the retention and improvement of catalytic activity in the IgA, but not the IgG compartment of the immune response.