Anti-idiotypic single-chain variable fragment antibody partially mimic the functionally spatial structure of Cry2Aa toxin.

The anti-idiotypic antibody is widely used in the field of immunology to simulate structural features or even induce the biological activity of antigens. In this study, we obtained seven anti-idiotypic single-chain variable fragments (scFv) antibodies of Cry2Aa toxin from a phage-displayed mutant library constructed using error-prone PCR technique. A mutant designated 2-12B showed the best binding ability amongst all anti-idiotypic scFv isolates to Plutella xylostella brush border membrane vesicles (BBMVs). 2-12B and Cry2Aa toxin shared a potential receptor of polycalin in P. xylostella BBMVs. Homology modeling and molecular docking demonstrated that 2-12B and Cry2Aa toxin have seven common binding amino acid residues in polycalin. Insect bioassay results suggested that 2-12 had insecticidal efficacy against P. xylostella larvae. These results indicated that the Cry2Aa anti-idiotypic scFv antibody 2-12B partially mimicked the structure and function of Cry2Aa toxin. The anti-idiotypic scFv antibody provides the basic material for the future study of surrogate molecules or new insecticidal materials.

Blocking Allergic Reaction through Targeting Surface-Bound IgE with Low-Affinity Anti-IgE Antibodies.

Allergic disorders have now become a major worldwide public health issue, but the effective treatment options remain limited. We report a novel approach to block allergic reactivity by targeting the surface-bound IgE of the allergic effector cells via low-affinity anti-human IgE Abs with dissociation constants in the 10-6 to 10-8 M range. We demonstrated that these low-affinity anti-IgE mAbs bind to the cell surface-bound IgE without triggering anaphylactic degranulation even at high concentration, albeit they would weakly upregulate CD203c expression on basophils. This is in contrast to the high-affinity anti-IgE mAbs that trigger anaphylactic degranulation at low concentration. Instead, the low-affinity anti-IgE mAbs profoundly block human peanut- and cat-allergic IgE-mediated basophil CD63 induction indicative of anaphylactic degranulation; suppress peanut-, cat-, and dansyl-specific IgE-mediated passive cutaneous anaphylaxis; and attenuate dansyl IgE-mediated systemic anaphylaxis in human FcεRIα transgenic mouse model. Mechanistic studies reveal that the ability of allergic reaction blockade by the low-affinity anti-IgE mAbs was correlated with their capacity to downregulate the surface IgE and FcεRI level on human basophils and the human FcεRIα transgenic mouse bone marrow-derived mast cells via driving internalization of the IgE/FcεRI complex. Our studies demonstrate that targeting surface-bound IgE with low-affinity anti-IgE Abs is capable of suppressing allergic reactivity while displaying an excellent safety profile, indicating that use of low-affinity anti-IgE mAbs holds promise as a novel therapeutic approach for IgE-mediated allergic diseases.

Preliminary assessment of anti-α-Gal IgG and IgM levels in patients with patent Plasmodium vivax infection.

Anti-α-Gal responses may exert a protective effect in falciparum malaria. However, the biological role of such antibodies is still unknown during Plasmodium vivax infections. We investigated IgG and IgM responses to α-Gal in individuals with vivax malaria. Anti-α-Gal IgG and IgM levels were higher in these patients than in controls, but no significant correlation was found between parasitaemia and anti-α-Gal response, nor between this response and ABO blood group status. This is the first study to investigate anti-α-Gal antibodies in P. vivax-infected patients; a larger survey is necessary to achieve a better understanding of host immune response during vivax malaria.

Antigen modulation as a potential mechanism of anti-KEL immunoprophylaxis in mice.

Red blood cell (RBC) alloimmunization is a serious complication of transfusion or pregnancy. Despite the widespread use of Rh immune globulin to prevent pregnancy associated anti-D alloimmunization, its mechanism of action remains elusive. We have previously described a murine model in which immunoprophylaxis with polyclonal anti-KEL sera prevents alloimmunization in wild-type recipients transfused with transgenic murine RBCs expressing the human KEL glycoprotein. To investigate the mechanism of action, we have now evaluated the outcome of immunoprophylaxis treatment in mice lacking Fcγ receptors (FcγRs), complement (C3), both, or none. Whereas polyclonal anti-KEL sera completely prevented alloimmunization in wild-type and single-knockout (KO) mice lacking FcγRs or C3, double-KO mice lacking both FcγRs and C3 became alloimmunized despite immunoprophylaxis. Rapid clearance of essentially all transfused RBCs with detectable KEL glycoprotein antigen occurred within 24 hours in wild-type and single-KO recipients treated with immunoprophylaxis, with the transfused RBCs remaining in circulation having minimal KEL glycoprotein antigen detectable by flow cytometry or western blot. In contrast, transfused RBCs with the KEL glycoprotein antigen fully intact continued to circulate for days in double-KO mice despite treatment with immunoprophylaxis. Further, in vitro phagocytosis assays showed no consumption of opsonized murine RBCs by double-KO splenocytes. Taken in combination, our data suggest that modulation of the KEL antigen (and potentially RBC clearance) by redundant recipient pathways involving both FcγRs and C3 may be critical to the mechanism of action of polyclonal anti-KEL immunoprophylaxis. These findings could have implications for the development of immunoprophylaxis programs in humans.

Surface Modification with a Catechol-Bearing Polypeptide and Sensing Applications.

A novel catechol-bearing polypeptide (CtP) was synthesized and used as a component of electrochemical biosensor involving both enzymatic activity and affinity-based sensing systems. Glucose oxidase (GOx) and anti-immunoglobulin G (Anti-IgG) were selected as model biorecognition elements for the selective analysis of glucose and IgG. Step-by-step surface modifications were followed using various techniques such as cyclic voltammetry (CV) and electrochemical impedance spectrometry (EIS) as well as X-ray photoelectron spectroscopy (XPS). Additionally, contact angles were measured in order to observe surface properties. Amperometric measurements using the GOx biosensor were performed at -0.7 V by following the oxygen consumption due to the enzymatic reaction in different glucose concentrations. Affinity-based interactions via IgG sensor were monitored using the differential pulse voltammetry (DPV) technique. As the "surface design with CtP" approach employed herein is generally applicable and easily adaptable to obtain functional matrices for biomolecule immobilization, CtP-coated surfaces can be promising platforms for the fabrication of various biobased sensing systems.

Structural optimization of an aptamer generated from Ligand-Guided Selection (LIGS) resulted in high affinity variant toward mIgM expressed on Burkitt's lymphoma cell lines.

Aptamers are synthetic, short nucleic acid molecules capable of specific target recognition. Aptamers are selected using a screening method termed Systematic Evolution of Ligands by Exponential enrichment (SELEX). We recently have introduced a variant of SELEX called "Ligand-Guided-Selection" (LIGS) that allows the identification of specific aptamers against known cell-surface proteins. Utilizing LIGS, we introduced three specific aptamers against membrane-bound IgM (mIgM), which is the hallmark of B cells. Out of the three aptamers selected against mIgM, an aptamer termed R1, in particular, was found to be interesting due to its ability to recognize mIgM on target cells and then block anti-IgM antibodies binding their antigen. We systematically truncated parent aptamer R1 to design shorter variants with enhanced affinity. Importantly, herein we show that the specificity of the most optimized variant of R1 aptamer is similar to that of anti-IgM antibody, indicating that the specificity of the ligand utilized in selective elution of the aptamer determines the specificity of the LIGS-generated aptamer. Furthermore, we report that truncated variants of R1 are able to recognize mIgM-positive human B lymphoma BJAB cells at physiological temperature, demonstrating that LIGS-generated aptamers could be re-optimized into higher affinity variants. Collectively, these findings show the significance of LIGS in generating highly specific aptamers with potential applications in biomedicine.

Upregulated expression of substance P in basophils of the patients with chronic spontaneous urticaria: induction of histamine release and basophil accumulation by substance P.

Human basophils have been implicated in the pathogenesis of chronic spontaneous urticaria (CSU), and substance P (SP) is a possible candidate as histamine-releasing factor in some patients with CSU. However, little is known of relationship between basophils and SP in CSU. In the present study, we investigated expression of SP and NK1R on basophils from patients with CSU, and influence of SP on basophil functions by using flow cytometry analysis, basophil challenge, and mouse sensitization model techniques. The results showed that plasma SP level and basophil numbers in CSU patients were higher than that in HC subject. The percentages of SP+ and NK1R+ basophils were markedly elevated in CSU blood in comparison with HC blood. Once added, SP induced up to 41.2 % net histamine release from basophils of CSU patients, which was comparable with that provoked by anti-IgE, and fMLP. It appeared that SP induced dramatic increase in blood basophil numbers of mice following peritoneal injection. Ovalbumin (OVA)-sensitized mice had much more SP+ and NK1R+ basophils in blood than non-sensitized mice. In conclusion, the elevated plasma concentration of SP, upregulated expression of SP and NK1R on basophils, and the ability of SP in induction of basophil degranulation and accumulation indicate strongly that SP is most likely a potent proinflammatory mediator, which contributes greatly to the pathogenesis of CSU through basophils. Inhibitors of SP and blockers of NK1R are likely useful agents for treatment of CSU.

Functionalized electrospun poly(vinyl alcohol) nanofibers for on-chip concentration of E. coli cells.

Positively and negatively charged electrospun poly(vinyl alcohol) (PVA) nanofibers were incorporated into poly(methyl methacrylate) (PMMA) microchannels in order to facilitate on-chip concentration of Escherichia coli K12 cells. The effects of fiber distribution and fiber mat height on analyte retention were investigated. The 3D morphology of the mats was optimized to prevent size-related retention of the E. coli cells while also providing a large enough surface area for analyte concentration. Positively charged nanofibers produced an 87% retention and over 80-fold concentration of the bacterial cells by mere electrostatic interaction, while negatively charged nanofibers reduced nonspecific analyte retention when compared to an empty microfluidic channel. In order to take advantage of this reduction in nonspecific retention, these negatively charged nanofibers were then modified with anti-E. coli antibodies. These proof-of-principle experiments showed that antibody-functionalized negatively charged nanofiber mats were capable of the specific capture of 72% of the E. coli cells while also significantly reducing nonspecific analyte retention within the channel as expected. The ease of fabrication and immense surface area of the functionalized electrospun nanofibers make them a promising alternative for on-chip concentration of analytes. The pore size and fiber mat morphology, as well as surface functionality of the fibers, can be tailored to allow for specific capture and concentration of a wide range of analytes.

Development of an ELISA using anti-idiotypic antibody for diagnosis of opisthorchiasis.

Monoclonal antibody specific for an epitope of cretory-secretory antigen protein of Opisthorchis felineus (Rivolta, 1884) (Trematoda: Opisthorchiidae) with a molecular weight of 28 kDa was used in a sandwich enzyme-linked immunosorbent assay (ELISA) for immobilisation of liver fluke specific antigen to the solid phase. Examination of human sera by this ELISA compared with commercial assays demonstrated that the monoclonal antibody epitope is located within this significant parasite protein. Anti-idiotypic antibody specific for the paratope of this monoclonal antibody was obtained by a hybridoma technique. Mimicking an epitope of excretory-secretory antigen of O. felineus, it had the capacity to bind specific antibody and elicit an antibody response. The value of anti-idiotypic antibody as a substitute for the liver fluke antigen was tested by ELISA using serum samples of infected dogs. Anti-idiotypic antibody proved to be of value in both an indirect-ELISA and a competitive-ELISA for diagnosis of opisthorchiasis. Mature trematodes were isolated from all infected animals. The faecal egg counts were negative in dogs with a relatively small number of parasites, despite finding antibodies in serum by ELISA. Substitution of parasite antigen with anti-idiotype avoids the use of experimental animals and also reduces time-consuming steps of antigen preparation.

Efficacy of immunotherapy with TG4040, peg-interferon, and ribavirin in a Phase 2 study of patients with chronic HCV infection.

BACKGROUND & AIMS: TG4040 is a modified vaccinia Ankara (MVA) virus that expresses the hepatitis C virus (HCV) proteins NS3, NS4, and NS5B. We performed a phase II open-label study to determine the efficacy, safety, and immunotherapeutic properties of TG4040 in combination with pegylated interferon α-2a and ribavirin (PEG-IFNα/RBV) in patients with chronic HCV infection. METHODS: Treatment-naive patients with HCV genotype 1 infection were assigned randomly to 1 of the following groups: PEG-IFNα/RBV for 48 weeks (group A, n = 31), PEG-IFNα/RBV for 4 weeks followed by PEG-IFNα/RBV for 44 weeks with 6 injections of TG4040 (group B, n = 63), or TG4040 for 12 weeks (7 injections) followed by PEG-IFNα/RBV for 48 weeks with 6 injections of TG4040 (group C, n = 59). The primary end point was complete early virologic response (cEVR), defined as HCV-RNA level less than 10 IU/mL after 12 weeks of PEG-IFNα/RBV treatment. RESULTS: In group C, 64.2% of evaluable patients achieved cEVR, compared with 30.0% in group A and 45.9% in group B (P = .0003 for group C vs A). A higher percentage of patients achieved a sustained virologic response 24 weeks after therapy ended in group C (58.2%) than in groups A (48.4%) or B (50.8%). HCV- and MVA-specific T-cell responses were observed predominantly in group C. As expected, most patients given injections of TG4040 developed anti-MVA antibodies. The combination of TG4040 and PEG-IFNα/RBV was reasonably well tolerated. However, PEG-IFNα-associated thrombocytopenia developed in 3 patients who carried the class II HLA allele DRB01*04. CONCLUSIONS: A higher percentage of patients with chronic HCV infection who received immunotherapy with TG4040 followed by TG4040 and PEG-IFNα/RBV achieved a cEVR compared with patients who received only PEG-IFNα/RBV therapy. These findings show that immunotherapies that activate T cells are effective in patients with chronic HCV infection. ClinicalTrials.gov number, NCT01055821.

IgM rheumatoid factor amplifies the inflammatory response of macrophages induced by the rheumatoid arthritis-specific immune complexes containing anticitrullinated protein antibodies.

OBJECTIVES: Anticitrullinated protein antibodies (ACPA) are specifically associated with rheumatoid arthritis (RA) and produced in inflamed synovial membranes where citrullinated fibrin, their antigenic target, is abundant. We showed that immune complexes containing IgG ACPA (ACPA-IC) induce FcγR-mediated tumour necrosis factor (TNF)-α secretion in macrophages. Since IgM rheumatoid factor (RF), an autoantibody directed to the Fc fragment of IgG, is also produced and concentrated in the rheumatoid synovial tissue, we evaluated its influence on macrophage stimulation by ACPA-IC. METHODS: With monocyte-derived macrophages from more than 40 healthy individuals and different human IgM cryoglobulins with RF activity, using a previously developed human in vitro model, we evaluated the effect of the incorporation of IgM RF into ACPA-IC. RESULTS: IgM RF induced an important amplification of the TNF-α secretion. This effect was not observed in monocytes and depended on an increase in the number of IgG-engaged FcγR. It extended to the secretion of interleukin (IL)-1ß and IL-6, was paralleled by IL-8 secretion and was not associated with overwhelming secretion of IL-10 or IL-1Ra. Moreover, the RF-induced increased proinflammatory bioactivity of the cytokine response to ACPA-IC was confirmed by an enhanced, not entirely TNF-dependent, capacity of the secreted cytokine cocktail to prompt IL-6 secretion by RA synoviocytes. CONCLUSIONS: By showing that it can greatly enhance the proinflammatory cytokine response induced in macrophages by the RA-specific ACPA-IC, these results highlight a previously undescribed, FcγR-dependent strong proinflammatory potential of IgM RF. They clarify the pathophysiological link between the presence of ACPA and IgM RF, and RA severity.

Evidence that FcRn mediates the transplacental passage of maternal IgE in the form of IgG anti-IgE/IgE immune complexes.

BACKGROUND: The mechanism(s) responsible for acquisition of maternal antibody isotypes other than IgG are not fully understood. This uncertainty is a major reason underlying the continued controversy regarding whether cord blood (CB) IgE originates in the mother or fetus. OBJECTIVE: To investigate the capacity of maternal IgE to be transported across the placenta in the form of IgG anti-IgE/IgE immune complexes (ICs) and to determine the role of the neonatal Fc receptor (FcRn) in mediating this process. METHODS: Maternal and CB serum concentrations of IgE, IgG anti-IgE, and IgG anti-IgE/IgE ICs were determined in a cohort of allergic and non-allergic mother/infant dyads. Madin-Darby canine kidney (MDCK) cells stably transfected with human FcRn were used to study the binding and transcytosis of IgE in the form of IgG anti-IgE/IgE ICs. RESULTS: Maternal and CB serum concentrations of IgG anti-IgE/IgE ICs were highly correlated, regardless of maternal allergic status. IgG anti-IgE/IgE ICs generated in vitro bound strongly to FcRn-expressing MDCK cells and were transcytosed in an FcRn-dependent manner. Conversely, monomeric IgE did not bind to FcRn and was not transcytosed. IgE was detected in solutions of transcytosed IgG anti-IgE/IgE ICs, even though essentially all the IgE remained in complex form. Similarly, the majority of IgE in CB sera was found to be complexed to IgG. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that human FcRn facilitates the transepithelial transport of IgE in the form of IgG anti-IgE/IgE ICs. They also strongly suggest that the majority of IgE in CB sera is the result of FcRn-mediated transcytosis of maternal-derived IgG anti-IgE/IgE ICs. These findings challenge the widespread perception that maternal IgE does not cross the placenta. Measuring maternal or CB levels of IgG anti-IgE/IgE ICs may be a more accurate predictor of allergic risk.

Autoimmunity to the alpha 3 chain of type IV collagen in glomerulonephritis is triggered by 'autoantigen complementarity'.

'Autoantigen complementarity' is a theory proposing that the initiator of an autoimmune response is not necessarily the autoantigen or its molecular mimic, but may instead be a peptide that is 'antisense/complementary' to the autoantigen. We investigated whether such complementary proteins play a role in the immunopathogenesis of autoimmune glomerulonephritis. Experimental autoimmune glomerulonephritis, a model of anti-glomerular basement membrane (GBM) disease, can be induced in Wistar Kyoto (WKY) rats by immunization with the α3 chain of type IV collagen. In this study, WKY rats were immunized with a complementary α3 peptide (c-α3-Gly) comprised of amino acids that 'complement' the well characterized epitope on α3(IV)NC1, pCol(24-38). Within 8 weeks post-immunization, these animals developed cresentic glomerulonephritis, similar to pCol(24-38)-immunized rats, while animals immunized with scrambled peptide were normal. Anti-idiotypic antibodies to epitopes from c-α3-Gly-immunized animals were shown to be specific for α3 protein, binding in a region containing sense pCol(24-38) sequence. Interestingly, anti-complementary α3 antibodies were identified in sera from patients with anti-GBM disease, suggesting a role for 'autoantigen complementarity' in immunopathogenesis of the human disease. This work supports the idea that autoimmune glomerulonephritis can be initiated through an immune response against a peptide that is anti-sense or complementary to the autoantigen. The implications of this discovery may be far reaching, and other autoimmune diseases could be due to responses to these once unsuspected 'complementary' antigens.

Activation of TLR9 by incorporated pDNA within PEG-coated lipoplex enhances anti-PEG IgM production.

Cationic liposome represents a promising alternative to viral vectors for the delivery of therapeutic genes. For in vivo use, surface modification of the liposome with polyethylene glycol (PEG) is frequently applied to achieve gene-expression in the targeted tissue. However, we have reported that PEG-coated liposomes have induced anti-PEG IgM, which has caused subsequent doses of PEG-coated liposome to be rapidly cleared from blood circulation, and the complexation of pDNA electrostatically associated with liposome surface has enhanced this antibody response. In this study, we investigated how a Toll-like receptor (TLR) might enhance anti-PEG IgM production. PEG-coated pDNA-lipoplex (PDCL) was injected into either wild type, MyD88 (all TLR adaptor protein, independent of TLR3) knock out (KO) or TLR9 KO mice, and the anti-PEG IgM production levels were detected. Attenuated anti-PEG IgM production following the injection of PDCL was observed in both MyD88 and TLR9 KO mice compared to wild type mice, probably due to the abolished induction of cytokines in both MyD88 and TLR9 KO mice. Our results suggest that TLR, exclusively TLR9, signaling plays a potential role in the enhanced anti-PEG IgM production following the injection of PDCL. This result may have important implications for the design and development of an efficient PEG-coated non-viral gene vector.

Low anti-RhD IgG-Fc-fucosylation in pregnancy: a new variable predicting severity in haemolytic disease of the fetus and newborn.

Haemolytic disease of the fetus and newborn (HDFN) may occur when maternal IgG antibodies against red blood cells (RBCs), often anti-RhD (anti-D) antibodies, cross the placenta and mediate the destruction of RBCs via phagocytic IgG-Fc-receptors (FcγR). Clinical severity is not strictly related to titre and is more accurately predicted by the diagnostically-applied monocyte-based antibody-dependent cellular cytotoxicity (ADCC), a sensitive test with relatively low specificity. This suggests that other factors are involved in the pathogenesis of HDFN. Binding of IgG to FcγR requires the N-linked glycan at position 297 in the IgG-Fc-region, consisting of several different glycoforms. We therefore systematically analysed IgG-derived glycopeptides by mass spectrometry from 70 anti-D IgG1 antibodies purified from the plasma of alloimmunized pregnant women. This revealed a variable decrease in Fc-fucosylation in the majority of anti-D IgG1 (even down to 12%), whereas the total IgG of these patients remained highly fucosylated, like in healthy individuals (>90%). The degree of anti-D fucosylation correlated significantly with CD16 (FcγRIIIa)-mediated ADCC, in agreement with increased affinity of defucosylated IgG to human FcγRIIIa. Additionally, low anti-D fucosylation correlated significantly with low fetal-neonatal haemoglobin levels, thus with increased haemolysis, suggesting IgG-fucosylation to be an important pathological feature in HDFN with diagnostic potential.

Increased T-helper 2 cytokines in bile from patients with IgG4-related cholangitis disrupt the tight junction-associated biliary epithelial cell barrier.

BACKGROUND & AIMS: IgG4-related cholangitis is a chronic inflammatory biliary disease that involves different parts of the pancreatobiliary system, but little is known about its mechanisms of pathogenesis. A T-helper (Th) 2 cell cytokine profile predominates in liver tissues from these patients. We investigated whether Th2 cytokines disrupt the barrier function of biliary epithelial cells (BECs) in patients with IgG4-related cholangitis. METHODS: We assessed the Th2 cytokine profile in bile samples and brush cytology samples from 16 patients with IgG4-related cholangitis and respective controls, and evaluated transcription of tight junction (TJ)-associated proteins in primary BECs from these patients. The effect of Th2 cytokines on TJ-mediated BEC barrier function and wound closure was examined by immunoblot, transepithelial resistance, charge-selective Na(+)/Cl(-) permeability, and 4-kDa dextran flux analyses. RESULTS: Bile samples from patients with IgG4-related cholangitis had significant increases in levels of Th2 cytokines, interleukin (IL)-4, and IL-5. IL-13 was not detected in bile samples, but polymerase chain reaction analysis of whole-brush cytology samples from patients with IgG4-related cholangitis revealed increased levels of IL-13 mRNA, compared with controls. BECs isolated from the brush cytology samples revealed decreased levels of claudin-1 and increased levels of claudin-2 mRNAs. In vitro, IL-4 and IL-13 significantly reduced TJ-associated BEC barrier function by activating claudin-2-mediated paracellular pore pathways. Th2 cytokines also impaired wound closure in BEC monolayers. CONCLUSIONS: Th2 cytokines predominate in bile samples from patients with IgG4-related cholangitis and disrupt the TJ-mediated BEC barrier in vitro. Subsequent increases in biliary leaks might contribute to the pathogenesis of chronic biliary inflammation in these patients.

B-cell tolerance to the B-cell receptor variable regions.

An enormous number of B cells with different B-cell receptors (BCRs) are continuously produced in the bone marrow. BCRs are further diversified during the germinal center reaction. Due to extensive recirculation, B cells with mutually binding BCR are likely to meet in lymphoid organs. We have addressed possible outcomes of such an encounter in vitro. B lymphoma cells were transfected with complementary BCR, one transfectant expressing an Idiotype⁺ (Id⁺) BCR and the other an anti-Id BCR. To exclude confounding effects of secreted Ig, the transfected B lymphoma cells only expressed membrane IgD. Coincubation of paired Id⁺/anti-Id lymphoma cells results in conjugate formation, signaling, activation of Caspase 3/7, and apoptosis of at least one of the two cells in the pair. Our data provide suggestive evidence for a mechanism whereby the B-cell compartment is partly purged of B cells with complementary BCRs.

Why specific anti-integrase antibodies from HIV-infected patients can efficiently hydrolyze 21-mer oligopeptide corresponding to antigenic determinant of human myelin basic protein.

Human immunodeficiency virus-infected patients possess anti-integrase (IN) catalytic IgGs and IgMs (abzymes), which, unlike canonical proteases, specifically hydrolyze only intact globular IN. Anti-myelin MBP abzymes from patients with multiple sclerosis and systemic lupus erythematosus efficiently hydrolyze only intact MBP. Anti-MBP and anti-IN abzymes do not hydrolyze several other tested control globular proteins. Here, we show that anti-IN abzymes efficiently hydrolyze a 21-mer oligopeptide (OP21) corresponding to one antigenic determinant (AGD) of MBP, whereas anti-MBP abzymes extremely poorly cleave oligopeptides corresponding to AGDs of IN. All sites of IgG-mediated and IgM-mediated proteolysis of OP21 by anti-IN abzymes were found for the first time by a combination of reverse phase and thin layer chromatography and mass spectrometry. Several clustered sites of OP21 cleavage were revealed and compared with the cleavage sites within the complete IN. Several fragments of OP21 had good homology with many fragments of the IN sequence. The active sites of anti-IN abzymes are known to be located on their light chains, whereas heavy chains are responsible for the affinity for protein substrates. Interactions of intact IN with both light and heavy chains of the abzymes provide high affinity for IN and the specificity of its hydrolysis. Our data suggest that OP21 interacts mainly with the light chains of polyclonal anti-IN abzymes, which possess lower affinity and specificity for substrate. The hydrolysis of the non-cognate OP21 oligopeptide may be also less specific than the hydrolysis of the globular IN because in contrast to previously described serine protease-like abzymes against different proteins, anti-IN abzymes possess serine, thiol, acidic, and metal-dependent protease activities.

Activation of the B cell antigen receptor triggers reactivation of latent Kaposi's sarcoma-associated herpesvirus in B cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus and the cause of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. Latently infected B cells are the main reservoir of this virus in vivo, but the nature of the stimuli that lead to its reactivation in B cells is only partially understood. We established stable BJAB cell lines harboring latent KSHV by cell-free infection with recombinant virus carrying a puromycin resistance marker. Our latently infected B cell lines, termed BrK.219, can be reactivated by triggering the B cell receptor (BCR) with antibodies to surface IgM, a stimulus imitating antigen recognition. Using this B cell model system we studied the mechanisms that mediate the reactivation of KSHV in B cells following the stimulation of the BCR and could identify phosphatidylinositol 3-kinase (PI3K) and X-box binding protein 1 (XBP-1) as proteins that play an important role in the BCR-mediated reactivation of latent KSHV.