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We have previously described an autoantibody-catalyzed hydrolysis of vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide with airway smooth muscle relaxant and anti-inflammatory properties.[1] Catalytic antibodies can be expected, by [...]

HIV is vulnerable to antibodies that recognize a linear CD4 binding site epitope of gp120 (CLIN), but inducing CLIN-directed antibody synthesis by traditional vaccine principles is difficult. We wished to understand the basis for deficient CLIN-directed antibody synthesis and validate correction of the deficiency by an electrophilic gp120 analog (E-gp120) immunogen that binds B-cell receptors covalently.

This research study investigates the factors influencing the appearance of an exterior coated automobile finish as measured by the BYK wave-scan DOI instrument. Sets of 10 to 16 painted panels of three different colours; silver, white and blue were visually evaluated by 60 or 30 panellists of various demographic characteristics, who ranked the panels individually from best to worst. The median rank for each panel was correlated to a linear combination of the measured wave-scan values for that panel. The medium ranks of silver and white painted panels exhibited strong correlations (R 2 = 82% and 90%, respectively) with wave-scan parameters. Even though blue was the most preferred colour among the study group, the resulting model exhibited a weak correlation (53%) between rank and the wave-scan parameters. Further studies need to be conducted to establish a scientific method to obtain an acceptable value for BYK parameters.

For the analysis of Data in the form of paired (pre-treatment, post-treatment) counts we propose a zero-inflated bivariate Poisson regression (ZIBPR) model. We develop EM algorithm to obtain maximum likelihood estimates of the parameters of the ZIBPR model. Further, we obtain exact Fisher information matrix of the maximum likelihood estimates of the parameters of the ZIBPR model which can be used in a procedure for testing treatment effects. The Procedure to detect treatment effects based on the ZIBPR model is compared, in terms of size, by simulations, with an earlier procedure using a zero-inflated Poisson regression (ZIPR) model of the post- treatment count with the pre-treatment count treated as a covariate. The procedure based on the ZIBPR model holds level most effectively. A further simulation study indicates good power property of the procedure based on the ZIBPR model. We then analyze the DMFT index data based on the ZIBPR model and compare with the analysis using the ZIPR model.

In this paper we derive explicit expressions for the elements of the exact Fisher information matrix of the Dirichlet-multinomial distribution. We show that exact calculation is based on the beta-binomial probability function rather than that of the Dirichlet-multinomial and this makes the exact calculation quite easy. The exact results are expected to be useful for the calculation of standard errors of the maximum likelihood estimates of the beta-binomial parameters and those of the Dirichlet-multinomial parameters for data that arise in practice in toxicology and other similar fields. Standard errors of the maximum likelihood estimates of the beta-binomial parameters and those of the Dirichlet-multinomial parameters, based on the exact and the asymptotic Fisher information matrix based on the Dirichlet distribution, are obtained for a set of data from Haseman and Soares (1976), a dataset from Mosimann (1962) and a more recent dataset from Chen, Kodell, Howe and Gaylor (1991). There is substantial difference between the standard errors of the estimates based on the exact Fisher information matrix and those based on the asymptotic Fisher information matrix. (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The immunoregulatory neuropeptide vasoactive intestinal peptide (VIP) was cleaved by purified IgG from Fas‐defective C3H/gldmice, lupus patients, and autoimmune thyroiditis patients. No VIPase activity was detected in IgG from control mice and humans. Kinetic analyses of VIPase IgG preparations suggested low‐affinity recognition of VIP. Yet the VIPase activity was VIP selective, judged by lack of correlation with other protease activities expressed by the IgG and by noninterference of unrelated peptides in the activity. Recombinant Fv constructs selected from a human lupus phage show library displayed VIPase activity, confirming that the active site is located in the V domains. Inhibition of the VIPase activity by di‐isopropylfluorophosphate suggested a serine protease‐like mechanism of catalysis. Irreversible binding of a biotinyated phosphonate diester by the IgG and Fv preparations was observed, consistent with the presence of activated nucleophiles similar to those in enzymes capable of covalent catalysis. These observations show that VIP is a target for specific catalytic autoantibodies in autoimmune disease.

Autoantibodies to the recombinant extracellular domain of epidermal growth factor receptor (exEGFR) were detected by ELISA in the serum of Fas‐defective old MRL/MpJ/lpr and C3H/HeJ/gld mice, but not young mice from these strains, or nonautoimmune young and old BALB/c, MRL/MpJ/++, and C3H/HeJ/MMTV mice. Compared with control human subjects without autoimmune disease, the frequency of exEGFR‐binding autoantibodies was increased in scleroderma (systemic sclerosis) patients and to a lesser extent in lupus patients. Phage autoantibodies (Fv fragments) isolated from a lupus library by selection on a linear epitope of EGFR (residues 294–310) displayed the ability to bind exEGFR. Treatment of EGFR‐expressing A431 cells with autoantibodies purified by affinity chromatography on immobilized exEGFR resulted in specific staining of the cells. Shortlived but strong inhibition of cellular DNA synthesis was observed in the presence of the autoantibodies. We concluded that autoantibody responses to EGFR hold the potential of fulfilling a pathogenic role in autoimmune disease.—Planque, S., Zhou, Y.‐X., Nishiyama, Y., Sinha, M., O'Connor‐McCourt, M., Arnett, F.C., Paul, S. Autoantibodies to the epidermal growth factor receptor in systemic sclerosis, lupus, and autoimmune mice. FASEB J. 17, 136–143 (2003)

Monoclonal antibodies are suitable for therapeutic applications by virtue of their excellent target binding characteristics (specificity, affinity) and long half-life in vivo. Catalytic antibodies (CAbs) potentially represent a new generation of therapeutics with enhanced antigen inactivation capability. Here, we describe prospects for development of therapeutic CAbs to the envelope protein gp120 of HIV. The strategy consists of exploiting the natural tendency of the immune system to synthesize germline-encoded, serine protease-like CAbs. Lupus patients were found to develop antibodies to a conserved component of the CD4 binding site of gp120, potentially offering a means to obtain human antibodies expressing broad reactivity with various HIV strains. Covalently reactive antigen analogs (CRAs) capable of selective recognition of nucleophilic Abs were synthesized and applied to isolate Fv and L chain catalysts from lupus phage repertoires. CRA binding by the recombinant Ab fragments was statistically correlated with catalytic cleavage of model peptide substrates. A peptidyl CRA composed of residues 421-431 with a phosphonate diester moiety at its C terminus was validated as a reagent that combines noncovalent and covalent binding interactions in recognition of a gp120ase L chain. A general challenge in the field is the apparent instability of the catalytic conformation of the Abs. In reference to therapy of HIV infection, assurance is required that the Abs recognize the native conformation of gp120 expressed as a trimer on the virus surface.

The autoimmune repertoire is well known from previous studies to be capable of producing catalytic antibodies directed to self-antigens. In the present study, we explored the ability of 26 monoclonal light chains (Lchains) from multiple myeloma patients to cleave radiolabeled gp 120, a foreign protein. One L chain with this activity was identified. 125I-gp120 and unlabeled gp 120 were cleaved at several sites by the L chain, as shown by SDS-polyacrylamide gel electrophoresis, autoradiography, and immunoblotting, respectively. The apparent dissociation constant of the L chain was 130–145 nM, indicating high-affinity gp 120 recognition. 125I-albumin was not cleaved by the L chain, and various proteins and peptides did not inhibit gp 120 cleavage by the L chain, suggesting that the activity is not a nonspecific phenomenon. The substrate recognition determinants may be conserved in different HIV-1 strains, because gp 120 isolated from strains SF2, MN, and IIIB was found to be cleaved by the L chain. Micromolar concentrations of a synthetic peptide corresponding to residues 23–30 of gp 120 inhibited the cleavage of 125I-gp 120, suggesting that these residues are components of the epitope recognized by the L chain. The toxic effect of gp120 in neuronal cultures was reduced by about 100-fold by pretreatment of the protein with the L chain. These observations open the possibility of utilizing gp120-cleaving antibodies in the treatment of AIDS.

Proteolytic antibodies appear to utilizecatalytic mechanisms akin to nonantibody serine proteases, assessed from mutagenesis and protease-inhibitor studies. The catalytic efficiency derives substantially from the ability to recognize the ground state with high affinity. Because the proteolytic activity is germline-encoded, catalysts with specificity for virtually any target polypeptide could potentially be developed by applying appropriate immunogens and selection strategies. Analysis of transition-state stabilizing interactions suggests that chemical reactivity ofactive-site serine residues is an important contributor to catalysis. A prototype antigen analog capable of reacting covalently with nucleophilic serine residues permitted enrichment of the catalysts from a phage-displayed lupus light-chain library. Further mechanistic developments in understanding proteolytic antibodies may lead to the isolation of catalysts suitable for passive immunotherapy of major diseases, and elicitation of catalytic immunity as a component of prophylactic vaccination.

Autoantibody catalysis is now a well-established phenomenon, but the initial finding of autoantibody-catalyzed VIP cleavage was suspected to be an artefact by certain practitioners of designer antibody catalysis, mainly because of conceptual complexities not foreseen in our training about the immune system and about the mechanisms of biological catalysis. Confirmation that antibodies can acquire proteolytic activity by entirely natural means has emerged, ironically, in part from the field of designer catalytic antibodies. Recent studies have provided insight into the molecular strategies whereby antibodies can combine antigen binding with chemical catalysis, and the contributions of innate and adaptive immune mechanisms in the proteolytic activity. The history of this field illustrates the dangers of assuming that novel observations must fit into the simple confines of established theories. Scientific theories are changeable entities, dependent on empirical data and interpretations of the data, and their growth is better served by keeping an open mind.

Abstract: The light (L) chain of a model antibody (Ab) was deduced to contain a serine protease-like catalytic site capable of cleaving peptide bonds. The catalytic site is encoded by a germline VL gene. The catalytic activity can potentially be improved by somatic sequence diversification and pairing of the L chain with the appropriate heavy chain. Autoimmune disease is associated with increased synthesis of antigen (Ag)-specific Abs, but the reasons for this phenomenon are not known. Only recently has attention turned to the functional role of the catalytic function. Preliminary studies confirm that the catalytic cleavage of peptide bonds is a more potent means to achieve Ag neutralization, compared to reversible Ag binding. Administration of a monoclonal Ab to VIP in experimental animals induces an inflammatory response in the airways, suggesting that catalytic autoantibodies to this peptide found in airway disease and lupus are capable of causing airway dysfunction. The phenomenon of autoantibody catalysis can potentially be applied to isolate efficient catalysts directed against tumor or microbial Ags by exposing the autoimmune repertoire to such Ags or their analogs capable of recruiting the germline VL gene encoding the catalytic site.

Catalytic activities are known to arise naturally in antibodies. Several naturally‐occurring peptides, synthetic protease substrates, DNA, and esters are known to be cleaved by antibodies. There is increased production of antigen‐specific catalytic antibodies in autoimmune disease. Polyreactive catalytic antibodies are present in unimmunized donors. Antibody light chains isolated from multiple myeloma patients frequently express proteolytic activity. Immunization protocols using as immunogens the ground state of a naturally‐occurring polypeptide, anti‐enzyme antibodies, or the enzyme itself are known to provoke catalytic antibody synthesis. Active site residues in the light chain subunit serve as the catalytic residues in a model antibody with peptide bond cleaving activity. A split‐site model in which distinct amino acids serve as the essential catalytic residues and substrate ground‐state recognition residues has been developed from mutagenesis studies. Engineering of the available antibodies could potentially generate improved catalysts. The possible mechanisms underlying proteolysis by natural antibodies and evolution of the catalytic activity are reviewed.

Abstract: Human ϰ -light chains (L chains) were amplified by the reverse transcriptase-polymerase chain reaction (PCR) and cloned into a phagemid vector. Phage particles displaying L chains were fractionated on immobilized vasoactive intestinal peptide (VIP). The resultant phage preparation displayed saturable binding of (tyr10_125I)VIP. One of the L-chain clones (hk13) was deduced to be related to subgroup I of ϰ-light chains based on its nucleotide sequence. The VIP binding activity of the soluble and phage-displayed form of this L chain was confirmed by radioimmunoassay and ELISA, respectively. These observations demonstrate the potential of selecting antigen-specific L chains from phage-display libraries.

Abstract: Catalytic antibodies may be produced over the natural course of antibody-affinity maturation by placement of chemically reactive residues in antibody-active sites by somatic hypermutation or V-D-J-gene rearrangement. This hypothesis has received support from recent observations on the chemical reactivity of antibodies to vasoactive intestinal peptide (VIP), DNA, and steroid-and dinitrophenyl-esters. Recent studies reveal that monoclonal antibodies raised against the ground state of VIP can accelerate the cleavage of peptide bonds. The light-chain (L-chain) subunit of human autoantibodies display increased hydrolytic rate and diminished VIP-binding affinity compared to the parent antibody, consistent with increased turnover owing to weaker binding of the substrate ground state. These observations reveal an essential limitation of catalytic antibodies, i.e., large turnover rates may be associated with diminished substrate specificity. The hydrolysis of VIP by IgG purified by affinity chromatography from asthma patients and nonasthmatic controls was compared. IgG from the majority of asthma patients displayed VIP-hydrolyzing activity. Vmax values for IgG from asthmatics tended to be higher than those from the nonasthmatic group. In principle, catalysis by antibodies may be an important mediator of immunological defense, regulation, and autoimmune dysfunction. The verification of these possibilities will require studies that utilize efficient assays of antibody catalysis during experimental immunization and autoimmune disease, as well as mechanistic investigation of catalysis by antibodies and their subunits.

The recombinant light chain (L chain) of an antibody raised by immunization with vasoactive intestinal polypeptide (VIP) cleaved this peptide on the C-terminal side of basic residues. The major sites of cleavage in VIP were two adjacent peptide bonds, Lys20Lys21 and Lys21Tyr22. Lower levels of cleavage were evident at Arg14Lys15 and Lys15Gln16. Hydrolysis of radiolabeled VIP by the L chain was inhibited by two serine protease inhibitors, diisopropylfluorophosphate and aprotinin, but not by soybean or lima bean trypsin inhibitors or inhibitors of other classes of proteases. To probe the role of the VHdomain, single chain Fvconstructs composed of the VLdomain of the anti-VIP L chain linked viaa 14-residue peptide to its natural VHdomain partner or an irrelevant anti-lysozyme VHdomain (hybrid Fv) were prepared. The anti-VIP Fvhydrolyzed VIP with Ks21.4-fold lower than the L chain and 250-fold lower than the hybrid Fv, suggesting increased affinity for the substrate ground state due to the anti-VIP VHdomain. The kinetic efficiency (kcat/Ks) of the anti-VIP Fvwas 6.6-fold greater compared to the L chain and 29.4-fold greater compared to the hybrid Fv. Peptide-MCA substrates unrelated in sequence to VIP were hydrolyzed by the anti-VIP Fvand L chain at equivalent rates. These observations lead to a model of catalysis by the anti-VIP Fvin which the essential catalytic residues are located in the VLdomain and additional residues from the VHdomain are involved in high affinity binding of the substrate.

Amino acid residues in a proteolytic antibody light chain selected by molecular modeling were substituted with Ala by site-directed mutagene sis. Hydrolysis of vasoactive intestinal polypeptide (VIP), the immunogen employed to elicit the antibody light chain, was reduced by>95% by replacement of Ser27a or His93 by Ala residues. Similar reductions in the activity were observed using synthetic protease substrates containing Arg-methylcoumarinamide (MCA) and Lys-MCA bonds. Turnover of the Ser27a and His93 mutants was lower than that of wild-type protein by about two orders of magnitude. The activity of the wild-type protein was inhibited selectively by diisopropylfluorophosphate (DFP), a serine protease inhibitor, but the residual activity of the Ser26 mutant was refractory to DFP. The affinity of the wild-type light chain for the substrate ground state was nearly unaffected by mutations at Ser27a and His93. In contrast, a Ser26 single mutant and aHis27d/Asp28 double mutant displayed increasedK_m(by about tenfold) and increased turnover (by about tenfold) using VIP as substrate. The kinetic constants for these mutants and the wild type protein were essentially identical with Boc-Glu-Ala-Arg as substrate. Thus, two types of residues participating in catalysis by the light chain have been identified. Ser27a and His93 are essential for catalysis but not for initial high affinity complexation and substrate. Ser26 and His27d or Asp28 participate in VIP binding and limit turnover indirectly.

Monoclonal human light chains, i.e.Bence Jones proteins, and their recombinant variable fragments (VL) were screened for proteolytic activity using peptide-methylcoumarinamide (peptide-MCA) conjugates and vasoactive intestinal polypeptide (VIP) as substrates. Sixteen of 21 Bence Jones proteins and one of three VLfragments were capable of detectable cleavage of one or more substrates. The magnitude and kinetic characteristics of the activity varied with different substrates. Among the peptide-MCA substrates, the presence of tripeptide or tetrapeptide moieties with a basic residue at the scissile bond generally favored expression of the activity. The influence of N-terminal flanking residue recognition was evident from differing values of Kmand kcat(turnover number) observed using different Arg-containing peptide-MCA substrates. Different light chains displayed different kinetic parameters for the same substrate, suggesting unique catalytic sites. Hydrolysis of VIP was characterized by nanomolar Michaelis-Menten constants (Km), suggesting comparatively high affinity recognition of this peptide. The 25-kDa monomer and the 50-kDa dimer forms of one light chain preparation were resolved by gel filtration in 6 M guanidine hydrochloride. Following renaturation, the monomer displayed 51-fold greater peptide-MCA-hydrolyzing activity than the dimer. A renatured VLdomain prepared by gel filtration in 6 M guanidine hydrochloride displayed VIP-hydrolyzing activity in the 12.5-kDa peak fractions. These results provide evidence for the proteolytic activity of certain human light chains and imply that this phenomenon may have a pathophysiological significance.

A recombinant antibody light chain (L chain) maintained under non‐denaturing conditions displayed preferential cleavage of synthetic peptides conjugated to methylcoumarinamide (MCA) on the C‐terminal side of Arg and Lys residues. The same L chain renatured from a denaturing solvent (guanidine hydrochloride) acquired the capability of cleaving Tyr–MCA and Leu–MCA bonds, and its ability to cleave MCA linked to basic residues was decreased. The altered cleavage preference was accompanied by a conformational transition in the protein, evident from the fluorescence emission spectra. These observations suggest the feasibility of redirecting the cleavage specificity via alterations in the conformation of proteolytic antibody combining sites.

Planque: Covalent Immunology Products: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Escobar:Covalent Immunology Products: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Nishiyama:Covalent Immunology Products: Consultancy, Equity Ownership, Patents & Royalties. Paul:Covalent Immunology Products: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

In epidemiological cohort studies, the probability of developing a disease for individuals in a treatment/intervention group is compared with that of a control group. The groups involve varying cluster sizes, and the binary responses within each cluster cannot be assumed independently. Three major measures of association used to report the efficacy of treatments or effectiveness of public health intervention programs in case of prospective studies are Risk Difference (RD), Risk Ratio (RR) and Relative Risk Difference (RED). The preference of one measure of association over the other in drawing statistical inference depends on design of study. Lui (2004) discusses a number of methods of constructing confidence intervals for each of these measures. Specifically, Lui (2004) discusses four methods for RD, four methods for RR and three methods for RED. For the construction of confidence intervals for RD, Paul and Zaihra (2008) compare the four methods discussed by Lui (2004), using extensive simulations with a method based on an estimator of the variance of a ratio estimator by Cochran (1977) and a method based on a sandwich estimator of the variance of the regression estimator using the generalized estimating equations approach of Zeger and Liang (1986). Paul and Zaihra (2008) conclude that the method based on an estimate of the variance of a ratio estimator performs best overall. In this paper, we extend the two new methodologies introduced in Paul and Zaihra (2008) to confidence interval construction of the risk measures RR and RED. Extensive simulations show that the method based on an estimate of the variance of a ratio estimator performs best overall for constructing confidence interval for the other two risk measures RR and RED as well. This method involves a very simple variance expression which can be implemented with a very few computer codes. Therefore, it can be considered as an easily implementable alternative for all the three measures of association.

High titer antibodies (Abs) to Factor VIII (FVIII) can render FVIII replacement therapy ineffective in severe hemophilia A patients. An antigen capable of specific and covalent binding is predicted to reverse the effects of pathogenic Abs potently compared to conventional noncovalently binding antigens, as dissociation of the former type of antigen from the immune complexes is precluded. Recently, we described the apparently universal presence of enzyme-like nucleophiles in the antigen binding sites of Abs (Planque et al, J Biol Chem, 2003). Here, we report a covalently reactive antigen analog (CRA) of FVIII that inactivates anti-FVIII Abs specifically. Recombinant FVIII was derivitized at Lys side chains with phosphonate diester groups, positioning these electrophiles within the antigenic epitopes expressed by FVIII. Comparable binding of FVIII-CRA and underivitized FVIII by a monoclonal Ab and polyclonal Abs from 5 hemophilia patients was evident, suggesting maintenance of the FVIII epitope structure necessary for noncovalent Ab recognition. FVIII-CRA formed immune complexes with polyclonal anti-FVIII Abs from hemophilia patients that were not dissociated by sodium dodecyl sulfate (2%), a detergent that disrupts noncovalent interactions. Removal of free FVIII-CRA from the reaction mixtures by affinity chromatography on Protein G did not restore the FVIII-binding activity of the Abs, confirming irreversible occupancy of the Ab binding sites. No binding of the Abs by an irrelevant peptidyl CRAs was observed, suggesting the role of noncovalent antigen binding in imparting specificity to the covalent reaction. Ab inhibition of the functional role of FVIII in coagulation was studied by the chromogenic assay. Treatment of the Abs with the FVIII-CRA followed by removal of free FVIII-CRA relieved the inhibitory effect of the Abs on FVIII mediated activation of FIXa/FX. An irrelevant peptidyl CRA did not relieve the Ab inhibitory effect. Compared to FVIII, the FVIII-CRA activated FIXa/FX poorly, suggesting that the integrity of the FIXa binding site had been disrupted. These observations encourage the development of electrophilic CRA analogs of FVIII for treatment of patients with inhibitory Abs to FVIII.