Purified IgG Fraction Research Articles

Plant-Based Production of Recombinant Plasmodium Surface Protein Pf38 and Evaluation of its Potential as a Vaccine Candidate

Pf38 is a surface protein of the malarial parasite Plasmodium falciparum. In this study, we produced and purified recombinant Pf38 and a fusion protein composed of red fluorescent protein and Pf38 (RFP-Pf38) using a transient expression system in the plant Nicotiana benthamiana. To our knowledge, this is the first description of the production of recombinant Pf38. To verify the quality of the recombinant Pf38, plasma from semi-immune African donors was used to confirm specific binding to Pf38. ELISA measurements revealed that immune responses to Pf38 in this African subset were comparable to reactivities to AMA-1 and MSP119. Pf38 and RFP-Pf38 were successfully used to immunise mice, although titres from these mice were low (on average 1∶11.000 and 1∶39.000, respectively). In immune fluorescence assays, the purified IgG fraction from the sera of immunised mice recognised Pf38 on the surface of schizonts, gametocytes, macrogametes and zygotes, but not sporozoites. Growth inhibition assays using αPf38 antibodies demonstrated strong inhibition (≥60%) of the growth of blood-stage P. falciparum. The development of zygotes was also effectively inhibited by αPf38 antibodies, as determined by the zygote development assay. Collectively, these results suggest that Pf38 is an interesting candidate for the development of a malaria vaccine.

Dedicated removal of immunoglobulin (Ig)A, IgM, and Factor (F)XI/activated FXI from human plasma IgG

Adverse events can be associated with treating critically ill patients with immunoglobulin (Ig)G. Some adverse events are due to contaminants like IgA and activated Factor (F)XI. Therefore, new purification strategies are needed for dedicated removal of these contaminants without impairing IgG recovery. An immunoglobulin fraction containing IgG, IgM, and IgA was prepared by caprylic acid precipitation of cryoprecipitate-poor plasma. The capacities of the cation exchangers (S HyperCel and CM Ceramic HyperD F) and anion exchangers (HyperCel STAR AX and Q HyperCel) to remove IgA, IgM, and spiked FXI were tested following a design of experiment approach using microplates and chromatographic column scale-up. FXI removal was also evaluated using Mustang S chromatographic membranes. IgG/IgG subclasses, IgA, IgM, and FXI were assessed by enzyme-linked immunosorbent assay, and caprylic acid, by gas chromatography. Extensive removal of IgA and IgM, but not FXI, was achieved by a two-step chromatographic process combining S HyperCel used in the IgG binding and elution mode and HyperCel STAR AX used in the IgG flow-through mode, providing high IgG and IgG subclass recovery (>85%), high purity (>99.5%), and efficient removal of IgA (<0.5%) and IgM (undetectable). Twenty-six-fold FXI removal was achieved by processing the resulting purified IgG fraction through Mustang S cation-exchanger membranes at pH 6.0 and 12.7 mS/cm. Caprylic acid was removed by S HyperCel. Combining S HyperCel and HyperCel STAR AX extensively removed IgA and IgM, with good IgG recovery. Mustang® S membranes can be used for dedicated removal of FXI.

Human Stiff person syndrome IgG-containing high-titer anti-GAD65 autoantibodies induce motor dysfunction in rats

Stiff person syndrome (SPS) is an autoimmune CNS disorder characterized by muscle rigidity, spasms and anxiety. The majority of patients have high-titer autoantibodies (ab) against glutamate decarboxylase (GAD65). A pathogenic role of SPS-associated IgG with ab against GAD65 has been shown for anxiety-like behavior but not for the core motor signs.We repetitively injected the purified IgG fraction of an SPS patient with severe motor impairment but without anxious comorbidity containing high titers of anti-GAD65 ab (SPS-IgG) into the lateral ventricle (i.c.v.) or intrathecally (i.th.) at the spinal level in experimental rats. We analyzed the effects on motor and anxiety-like behavior. Non-SPS human IgG fractions served as controls.Animals injected i.c.v. with SPS-IgG showed stiffness-like behavior with impaired walking ability and reduced grip strength of the upper limbs as well as postural and sensorimotor dysfunction. Testing for anxiety-like behavior revealed no significant differences between SPS and control IgG-treated rats. IgG deposits were found only in rats treated with SPS-IgG and were localized predominantly in CNS structures involved in motor control including globus pallidus, internal capsule, striatum and anterior thalamus. Double immunofluorescence staining revealed that predominantly GABAergic interneurons were positive for i.c.v. injected SPS-IgG. Rats injected i.th. with SPS-IgG did not present obvious motor symptoms and had a normal synaptic transmission at the spinal level.We conclude that SPS-like motor dysfunction can be induced in rats by passive transfer of IgG from an SPS-patient with high titer of anti-GAD65 ab. GABAergic dysfunction in supraspinal motor pathways rather than in the spinal cord may lead to motor deficits observed in the rats contrasting observations made in SPS with amphiphysin antibodies.

Assessment of Batch to Batch Variation in Polyclonal Antithymocyte Globulin Preparations

Antithymocyte globulins (ATGs) are used to prevent and treat allograft rejection and graft versus host disease. They are purified IgG fractions derived from rabbits immunized with the Jurkat T-cell line (ATG-Fresenius) or thymus cells (Thymoglobulin). Differences not only in the amounts of leukocyte reactive antibodies but also in the antigens targeted by ATGs could potentially affect the clinical efficacy of different batches of these polyclonal antibody preparations. Four batches of ATG-Fresenius and Thymoglobulin were compared regarding their capacity to interact with human leukocytes from healthy donors and kidney transplant recipients. Using flow cytometric assays, we analyzed the reactivity of these ATG preparations with Jurkat cells and with primary leukocytes. In addition, ATGs derived from different batches were probed with a panel of cell lines expressing high levels of ATG antigens. Their ability to mediate complement-mediated lysis of human monocytes and lymphocytes was also compared. Binding studies to leukocyte antigens and functional analysis pointed to a high conformity among different batches in both ATG preparations. From our in vitro data, it can be expected that ATGs derived from different batches will not differ in their clinical efficacy. Furthermore, the methods described in this study allow for a reliable analysis of ATG batches.

Anti-phospholipid antibodies contribute to arteriosclerosis in patients with systemic lupus erythematosus through induction of tissue factor expression and cytokine production from peripheral blood mononuclear cells

IntroductionIn systemic lupus erythematosus (SLE) patients, the prevalence of arteriosclerosis obliterans (ASO) is high despite a lack of common risk factors for ASO. The main objective of this study was to investigate a possible direct role of anti-phospholipid antibodies (aPLs), which are frequently detected in SLE patients, in the pathogenesis of ASO. Materials and MethodsWe examined tissue factor (TF) expression on the monocyte surface by flow cytometric analysis in 89 SLE patients with or without ASO and/or aPLs and studied the in vitro effect of purified IgG fractions from plasma of SLE patients or normal healthy volunteers (aPLs(+) IgG, n=8; aPLs(−) IgG, n=6; Normal IgG, n=6) on the expression of TF and production of TNF-α and IL-1β in healthy peripheral blood mononuclear cells (PBMCs) or isolated monocytes. ResultsWe confirmed that high expression of monocyte TF was strongly associated with the prevalence of ASO and the presence of aPLs. Treatments of PBMCs with aPLs(−) IgG or normal IgG did not significantly increase expression of TF, TNF-α, and IL-1β messenger RNA (mRNA) and the production of TNF-α and IL-1β. However, stimulation of PBMCs with aPLs(+) IgG caused significant increase in expression of TF, TNF-α, and IL-1β mRNA. Moreover, aPLs(+) IgG stimulated PBMCs and significantly enhanced the production of TNF-α and IL-1β. ConclusionThese results suggest that IgG-aPLs cause persistently high TF expression and inflammatory cytokine production by interacting with peripheral blood monocytes and lymphocytes, which may be an important mechanism in the pathogenesis of ASO peculiar to SLE patients.

Relationship between Complement Membrane Attack Complex, Chemokine (C-C Motif) Ligand 2 (CCL2) and Vascular Endothelial Growth Factor in Mouse Model of Laser-induced Choroidal Neovascularization

The present study investigated the interactions among the complement membrane attack complex (MAC), CCL2, and VEGF that occur in vivo during the development of choroidal neovascularization (CNV). We first investigated the sequential expression of MAC, CCL2, and VEGF during laser-induced CNV in C57BL/6 mice. Increased MAC deposition was detected at 1 h, CCL2 increased at 3 h, and VEGF was up-regulated at day 3 post-laser treatment. These results suggested that during laser-induced CNV, MAC, CCL2 and VEGF are formed and/or expressed in the following order: MAC → CCL2 → VEGF. To determine the cross-talk between MAC, CCL2, and VEGF during laser-induced CNV, neutralizing antibodies were injected both systemically and locally to block the bioactivity of each molecule. Blocking MAC formation inhibited CCL2 and VEGF expression and also limited CNV formation, whereas neutralization of CCL2 bioactivity did not affect MAC deposition; however, it reduced VEGF expression and CNV formation. When bioactivity of VEGF was blocked, CNV formation was significantly inhibited, but MAC deposition was not affected. Together, our results demonstrate that MAC is an upstream mediator and effect of MAC on the development of laser-induced CNV can be attributed to its direct effect on VEGF as well as its effect on VEGF that is mediated by CCL2. Understanding the interplay between immune mediators is critical to gain insight into the pathogenesis of CNV.

99 Phosphorylated Signaling Proteins as Biomarkers of Antibody-Mediated Heart Allograft Rejection

Purpose Ligation of class I molecules on endothelial cells (EC) by anti-HLA Ab stimulates phosphorylation of p70 S6K, S6RP, ERK1/2 and Akt that in turn mediate cell survival and proliferation. We postulate that the capacity of Ab to mediate these signals is dependent upon the HLA Ab titer and level of HLA antigen expression on the endothelium. Methods and Materials Purified IgG fractions of heart recipient sera containing HLA Ab were tested for their ability to induce phosphorylation of S6RP, Akt, S6K and ERK in primary EC bearing relevant HLA antigens. Phosphorylation was measured by Western blot and normalized to EC treated with IgG containing no HLA Ab. The Ab binding capacity (ABC) of IgG fractions to EC was measured by flow-cytometry. Phosphorylation of paraffin embedded endomyocardial biopsies was determined by immunohistochemical staining. Results DSA with ABC values >=14000 triggered higher levels of phosphorylation of S6K (T421/S424), S6RP (S235/6), and ERK1/2 (T202/Y204). In contrast, DSA with low ABC values = Conclusions These results provide mechanistic insights into how DSA contribute to the process of AMR. High levels of DSA activate cell proliferation pathways which may play a role in the development of transplant vasculopathy. Low levels of DSA may play a role in accommodation by activating cell survival pathways. The study also emphasizes the value of immunostaining of phosphorylated kinases, such as S6K, as biomarkers of AMR.

Immune mechanisms in heparin‐induced thrombocytopenia: no evidence for immunoglobulin M anti‐idiotype antibodies

Heparin-induced thrombocytopenia (HIT), which is caused by platelet (PLT)-activating immunoglobulin (Ig)G antibodies against platelet factor 4 (PF4)/heparin complexes, differs from other immune responses seen in immunohematology: IgG antibodies are formed as early as 5 days even without previous heparin exposure; antibodies are remarkably transient (<100 days); HIT is more frequent in postsurgery patients compared with medical patients despite administering the same type and dose of heparin; and increasing evidence implicates autoantibody-like reactivity of anti-PF4/heparin antibodies. We hypothesized that these unusual features could be caused by loss of regulatory anti-idiotype IgM antibodies due to disturbance (e.g., by surgery) of an idiotype-anti-idiotype network. Sera were obtained prospectively before heparin administration and during the immunization phase of HIT and also from patients with previous HIT after waning of antibodies to nondetectable levels. To detect inhibitory IgM anti-idiotype antibodies, we performed serum coincubation experiments and IgG purification by protein G and size filtration to exclude coprecipitating IgM. Sera (n = 3) containing known anti-PF4/heparin IgG or IgM antibodies and normal sera (n = 20) were processed as controls. Fifteen preimmune response sera (seroconverting in the PF4/heparin-IgG enzyme-linked immunosorbent assay only [n = 4] or additionally in a PLT activation assay [n = 5] or in both assays plus thrombosis [n = 6]) and four sera of previously immunized patients were included. Neither did the neat sera inhibit binding of anti-PF4/heparin antibodies nor did the purified IgG fractions show enhanced binding to PF4/heparin complexes. The atypical immunologic features of HIT do not appear to be caused by disruption of an idiotype (IgG)-anti-idiotype (IgM) network.

A simple method for evaluating dengue vaccine effectiveness in mice based on levels of viremia caused by intraperitoneal injection of infected culture cells

A simple dengue vaccine evaluation system was established using a model of dengue type 2 virus (DENV2) infection in immunocompetent mice. Mice are usually non-permissive hosts, and artificial viremia was therefore created by intraperitoneal injection of K562 cells infected with DENV2. Plasma virus titers were approximately 4–5 log 10 focus-forming units/ml at 10 h after injection of 1 × 10 7 K562 cells into ICR, ddY and BALB/c mice. ICR mice immunized with an experimental vaccine against DENV2 showed reduced levels of viremia, associated with neutralizing antibody titers. Similarly, ICR mice passively immunized with purified IgG fractions of monoclonal antibodies possessing neutralizing activities also had reduced levels of viremia. However, the degree of viremia reduction differed according to the antibody species. Although some mice developed neurologic symptoms and/or died within 21 days of K562 injection, viremia reduction was considered to be a reliable indicator of the protective capacities of candidate dengue vaccines.

HPA-1a alloantibodies reduce endothelial cell spreading and monolayer integrity

Maternal human platelet antigen (HPA)-1a alloantibodies causing neonatal alloimmune thrombocytopenia can bind also to endothelium, via the β3-integrin (CD61). The aim of this study was to investigate the effect of HPA-1a Abs on endothelial cell function, with emphasis on monolayer integrity. We used a CD61 mAb as a model for the HPA-1a alloantibodies and confirmed the results with purified IgG fractions from HPA-1a alloimmunized women. The effect of these antibodies was examined by monitoring the adhesion, spreading, and monolayer integrity of primary HUVECs with conventional adhesion assays as well as electrical cell-substrate impedance sensing. We found that both the mAb CD61 and the HPA-1a antibodies caused a significant reduction in HUVEC spreading. Moreover, addition of the mAb CD61 and the HPA-1a antibodies prior to or following formation of a stable endothelial monolayer negatively affected endothelial monolayer integrity, which was accompanied by a redistribution of junctional proteins. Our data suggest that HPA-1a alloantibodies have a direct effect on endothelial cell spreading and monolayer integrity, which could contribute to the increased bleeding tendency in children with neonatal alloimmune thrombocytopenia.

Moderate Pharmacological Platelet Count Reduction Limits Thrombus Formation without Hemostatic Impairment in Primates

Despite the established contribution of platelets to thrombotic cardiovascular disorders, documented in part by the effectiveness of platelet function inhibitors and the increased risk of thrombosis associated with high normal and supranormal platelet counts, relationships between circulating platelet count and thrombotic events remain largely undefined. Since the initiation and propagation of arterial, platelet-dependent thrombus must depend upon platelet count, albeit in a manner that could be nonlinear, we hypothesized that reducing platelet count within the normal range would produce an anti-thrombotic benefit with minimal effects on hemostasis. To test this hypothesis, we reduced the platelet count in baboons (n=4) by targeting the megakaryocyte growth and development factor thrombopoietin (TPO). A polyclonal anti-TPO autoantibody (anti-TPOab) was purified from the serum of a baboon that developed thrombocytopenia following recombinant TPO injections. The IC50 of the purified IgG fraction was found to be 0.76 μg/ml, determined using a proliferation assay with a TPO-dependent cell line. An i.v. bolus of the anti-TPO antiserum, 30–35 ml infused into baboons, resulted in a transient, >60% decrease in the circulating platelet count after 2–3 weeks. Other blood cell counts were unaffected vs. baseline values. The effect of platelet count reduction on thrombogenesis was evaluated using an established baboon arteriovenous (AV) shunt thrombosis model. Accumulation of 111-Indium-labeled platelets and 125-Iodine-labeled fibrinogen were measured within a 4 mm i.d. thrombogenic vascular graft segment that was deployed into a chronic AV shunt for 60 min. Blood flow was maintained at 100 ml/min, producing an arterial wall shear rate of 265 sec−1. Standard template bleeding times (BTs) were used to assess hemostatic impairment at various platelet counts. Platelet count reductions, ranging from 46–61% (normal levels averaging 352,000 ± 61,000 platelets/μl), reduced platelet deposition onto the graft surface by 46–68% (vs. control values of 4.1 ± 0.9 x 109 platelets deposited, n=9). Similarly, thrombus fibrin accumulation was reduced by 14–39% (vs. control values of 2.2 ± 0.4 mgs of deposited fibrin). Thrombus formation was not affected acutely by anti-TPOab administration, but correlated directly with circulating platelet numbers. As expected, BTs were not significantly prolonged until platelet counts fell below ~100,000 cells/μl. In contrast, single dose aspirin (32 mg/kg) at normal platelet counts did not significantly reduce graft associated platelet deposition in this model but doubled the BTs to 6.8 ± 2.6 min (vs. control values of 3.4 ± 0.9 min). With further reduction in platelet counts to 90,000 ± 30,000 platelets/μL, BTs were only slightly prolonged (5.6 ± 1.7 min, n=5). When platelet counts averaged 74,000 ± 20,000 platelets/μl in animals given ASA, BTs averaged only 9.4 ± 2.7 min (n=5). Thus ASA produced a hemostatic impairment that was approximately fixed (i.e., a BT prolongation of 3–4 min) and not disproportionately prolonged at reduced platelet counts. Thus specific lowering of the platelet count by pharmacologic inhibition of megakaryocytopoiesis may be an effective anti-thrombotic strategy in populations currently treated with conventional anti-platelet agents. Since direct inhibitors of platelet function produce a significant risk of bleeding, inhibition of platelet production may represent a safer approach for reducing the pro-thrombotic capacity of the circulating platelet pool.

Novel Antibodies to the Donor Stem Cell Population CD34+/VEGFR-2+ Are Associated With Rejection After Hematopoietic Stem Cell Transplantation

Reconstitution of hematopoiesis after hematopoietic stem cell transplantation (HSCT) occurs through a reservoir of donor CD34+ HSC. CD34+/VEGFR-2+ is a primitive, quiescent subpopulation of HSC known to generate hematopoietic or endothelial progeny in vitro and in vivo. We hypothesized that donor-specific antibodies to the CD34+/VEGFR-2+ stem cells may be associated with rejection after HSCT. We studied 30 patients without and 11 with rejections after HSCT and 20 nontransplanted healthy individuals. Ninety-three sera taken pre and posttransplantation from patients receiving HSCT were studied for the presence of donor CD34+/VEGFR-2+ cell-specific antibodies. We provide evidence that significantly higher numbers of patients with rejections 9/11 (81%), whereas 1/30 (3%) (P=0.001) without rejections had antibodies against donor CD34+/VEGFR-2+ cells, but not CD34-/VEGFR-2- cells. In eight transplantations, antibodies against donor CD34+/VEGFR-2+ cells were detected already before transplantation. Purified IgG fractions from patients with rejections but not controls significantly decreased the ability of these cells to form hematopoietic and endothelial colonies. In multivariate analysis, antibodies against CD34+/VEGFR-2+ cells proved to be the most significant risk factor for rejection. These novel findings document a high rate of rejections in patients with donor-specific antibodies to CD34+/VEGFR-2+ HSC. These antibodies may significantly and commonly contribute to HSCT rejections.

Inhibitor(s) of natural anti-cardiolipin autoantibodies.

IgG fractions were purified on Sepharose anti-human IgG column from eight sera of healthy donors, having no anti-cardiolipin (aCL) activity as measured by anti-cardiolipin ELISA assay (aCL-ELISA). All the IgG fractions, after elution with 4.9 M MgCl2, reacted with CL. The antigen-binding characteristics of the IgG fractions purified from normal human serum (NHS) were similar to those of IgG fractions purified from sera of four patients with the anti-phospholipid syndrome (APLS). Competition assay confirmed the specificity of the binding of the purified IgG fractions to CL. The same results have been achieved with IgG fractions purified on Sepharose Protein-A column. The binding to CL was completely inhibited by either whole NHS and sera from various animal species, or by beta 2-glycoprotein I (beta 2-GPI). Our results support the notion of the existence of both natural anti-CL antibodies and serum inhibitor(s) in sera of healthy individuals. It is conceivable that in part the pathogenesis of APLS entails defects in the natural inhibitors of aCL antibodies.

Mesangial autoantigens in IgA nephropathy: matrix synthesis and localization

Primary IgA nephropathy, a chronic nephritis with variable prognosis, is characterized by mesangial immunoglobulin A, frequently with codeposition of other immunoglobulin isotypes and complement components accompanying matrix expansion typically preceding glomerular scarring. Glomerular immunoglobulin G, when present, is localized to the mesangial periphery found variably in repeat biopsies. IgG anti-mesangial cell autoantibodies (IgG-MESCA) in sera of patients with IgA nephropathy, specific by F(ab')(2) binding to 48- and 55-kD autoantigen(s) could account for these deposits, but their in vivo localization, and the functional role in promoting scarring is unknown. A specific monoclonal antibody raised previously to these human mesangial cell autoantigen fractions, in this study localized to similar glomerular sites, reinforcing the view that immunoglobulin G deposition in vivo is a result of antibody-autoantigen binding. The propensity for immunoglobulin G more than other isotypes to enhance inflammation prompted study of its functional role in vitro. Using cultured human mesangial cells in a complement-free tritiated glycosaminoglycan synthesis single outcome assay, purified IgG fractions from patient sera increased matrix production in a dose-dependent manner compared with controls. At a constant total IgG concentration, matrix synthesis was proportional to the titre of IgG-MESCA. Autoreactive IgG stimulated matrix synthesis when compared with controls or IgA fractions. These findings are consistent with IgG-MESCA autoantibodies enhancing mesangial matrix synthesis in vitro, which suggests that in IgA nephropathy, similar prosclerotic autoimmune mechanisms might operate. Recombinant TGFbeta(1) also induced matrix synthesis, raising the possibility that both autoimmune mechanisms and those TGFbeta(1)-dependent are functional or inter-related. The pathogenesis of glomerular scarring and loss in IgA nephropathy may include, in part, these mechanisms.

Encapsulation of biomolecules for bioanalytical purposes: Preparation of diclofenac antibody-doped nanometer-sized silica particles by reverse micelle and sol–gel processing

In recent years, the sol–gel technique has attracted increasing interest as a unique approach to immobilize biomolecules for bioanalytical applications as well as biochemical and biophysical studies. For this purpose, crushed biomolecule-doped sol–gel glass monoliths have been widely used. In the present work, for the first time, the encapsulation of anti-diclofenac antibodies in silica nanoparticles was carried out by a combination of reverse micelle and sol–gel technique. Cyclohexane was used for the preparation of the microemulsion as organic solvent, while surfactant Igepal CO-520 was found to be the optimal stabilizer. The antibody source was a purified IgG fraction originating from a polyclonal rabbit antiserum. Tetramethyl orthosilicate (TMOS) was used as precursor. Rather uniform, monodispersed and spherical silica particles of about 70 nm diameter size were fabricated, as was demonstrated by transmission electron microscopy (TEM) and scanning electron microscopy/energy dispersive X-ray fluorescence analysis (SEM/EDX). The biological activity of the encapsulated antibodies was evaluated by incubation of the nanoparticles with a diclofenac standard solution and analysis of the filtrate and followed washing solutions by a highly sensitive enzyme-linked immunosorbent assay (ELISA), using non-doped particles as blanks. While only about 6% of the added diclofenac was nonspecifically retained by the blank, the corresponding amount of about 66% was much higher with the antibody-doped particles. An obvious advantage of this approach is the general applicability of the developed technique for a mild immobilization of different antibody species.

Human Chagasic Serum Contains Antibodies Capable of Inhibiting Trypanosoma cruzi Egress From Tissue Culture Cells

Previous studies at our laboratory have shown that an antibody (antiegressin) present in the serum of chronically infected mice is capable of inhibiting the egress of Trypanosoma cruzi from infected BALB/c fibroblasts. We have used this in vitro system to evaluate whether human chagasic serum is also capable of inhibiting T. cruzi egress. BALB/c fibroblasts were infected with tissue culture-derived parasites. Five-percent solutions of the individual human serum samples in culture medium were added to the wells, and the number of parasites released was determined at day 5 after infection. The cells cultured with serum from infected individuals released between 37% and 72% fewer parasites than those cultured with control serum. A similar reduction in parasite egress resulted from incubation with the protein-A purified IgG fraction from 3 of these human samples. Immunocytochemical staining employing antineuraminidase antibodies supported the notion that the reduction in parasite levels is due to inhibition at the point of parasite egress. These results indicate that human serum of individuals infected with T. cruzi is capable of inhibiting release of the parasite from infected tissue culture cells and that the phenomenon of egress-inhibition may be relevant during infection of human subjects.

A mimotope peptide-based anti-cancer vaccine selected by BAT monoclonal antibody

Combinatorial phage display peptide libraries are employed to identify small molecules which bind with high affinity to receptor molecules and which mimic the interaction with natural ligands. We used a synthetic combinatory phage display peptide library to screen for peptides that bind BAT monoclonal antibody, an immune modulatory and anti-tumor antibody, to serve as the basis for an anti-cancer vaccine. Two distinct mimotopes, peptides A and B, were isolated, with repeated Proline, Arginine, and Isoleucine amino acids. Mimotope binding was determined by direct binding and by inhibition of BAT binding to the peptide bound phages and to Daudi cells. Immunization of mice with the peptides induced cellular and humoral responses. Cellular response was manifested by significant increase in cytolitic activity. Humoral response was manifested by production of specific antibodies. Serum purified IgG fraction contained anti-peptide antibodies that identified BAT binding mimotopes and competed with BAT binding on Daudi cells. These “BAT like” antibodies exhibited similar immune stimulatory properties to BAT. Immunization of mice with the peptides prevented tumor growth. These finding are the basis for the development of an anti-cancer vaccine.

Paraneoplastic stiff-person syndrome: passive transfer to rats by means of IgG antibodies to amphiphysin

Stiff-person syndrome (SPS) with antibodies to amphiphysin is a paraneoplastic disorder of the central nervous system with a putative autoimmune pathogenesis. Proof of a causal role of the antibodies is still lacking for this and all other antibody-associated paraneoplastic syndromes of the central nervous system. We obtained the plasma filtrate of a patient with breast cancer and SPS that responded to therapeutic plasmapheresis. The purified IgG fraction included high-titre antibodies to the synaptic protein amphiphysin. In a cotransfer design, this IgG fraction was injected intraperitoneally into female Lewis rats that had received encephalitogenic T-helper (Th) lymphocytes specific for myelin basic protein, to induce an immune-mediated leaky blood-brain barrier. The rats were followed up with behavioural tests, video photography, and electromyography. The injection of the IgG fraction including antibodies to amphiphysin resulted in a dose-dependent stiffness with spasms resembling human SPS. Control IgG injected into rats that had received the same encephalitogenic Th cells had no effect. IgG binding was demonstrated in the central nervous system of rats that showed signs of the disorder. These experiments support the hypothesis of a pathogenetic role of antibodies to amphiphysin, thus adding paraneoplastic SPS to the group of antibody-mediated autoimmune disorders. These findings provide a strong argument for a direct pathogenetic role of anti-amphiphysin in this type of SPS and support therapeutic attempts to eliminate these autoantibodies by plasmapheresis. The experimental approach used could help to elucidate the role of autoantibodies in other paraneoplastic syndromes, such as SPS with antibodies to glutamic acid decarboxylase, and others including anti-Hu-associated subacute cerebellar degeneration and limbic encephalitis.

Molecular and Genetic Studies Imply Akt-mediated Signaling Promotes Protein Kinase CβII Alternative Splicing via Phosphorylation of Serine/Arginine-rich Splicing Factor SRp40

Insulin regulates alternative splicing of PKCbetaII mRNA by phosphorylation of SRp40 via a phosphatidylinositol 3-kinase pathway (Patel, N. A., Chalfant, C. E., Watson, J. E., Wyatt, J. R., Dean, N. M., Eichler, D. C., and Cooper, D. C. (2001) J. Biol. Chem. 276, 22648-22654). Transient transfection of constitutively active Akt2 kinase promotes PKCbetaII exon inclusion. Serine/arginine-rich (SR) RNA-binding proteins regulating the selection of alternatively spliced exons are potential substrates of Akt kinase because many of them contain RXRXX(S/T) motifs. Here we show that Akt2 kinase phosphorylated SRp40 in vivo and in vitro. Mutation of Ser86 on SRp40 blocked in vitro phosphorylation. In control Akt2(+/+) fibroblasts, insulin treatment increased the phosphorylation of endogenous SR proteins, but their phosphorylation state remained unaltered by insulin in fibroblasts from Akt2(-/-) mice. Levels of PKCbetaII protein were up-regulated by insulin in Akt2(+/+) cells; however, only very low levels of PKCbetaII were detected in Akt2(-/-) cells and did not change following insulin treatment. Endogenous PKCbetaI and -betaII mRNA levels in Akt2(+/+) and Akt2(-/-) gastrocnemius muscle tissues were compared using quantitative real time PCR. The results indicated a 54% decrease in the expression of PKCbetaII levels in Akt(-/-), whereas PKCbetaI levels remained unchanged in both samples. Further, transfection of Akt2(-/-) cells with a PKCbetaII splicing minigene revealed defective betaII exon inclusion. Co-transfection of the mutated SRp40 attenuated betaII exon inclusion. This study provides in vitro and in vivo evidence showing Akt2 kinase directly phosphorylated SRp40, thereby connecting the insulin, PI 3-kinase/Akt pathway with phosphorylation of a site on a nuclear splicing protein promoting exon inclusion. This model is upheld in Akt2-deficient mice with insulin resistance leading to diabetes mellitus.

Autoantibodies to neutrophil cytoplasmic antigens (ANCA) do not bind to polymorphonuclear neutrophils in blood

Autoantibodies to neutrophil cytoplasmic antigens (ANCA), particularly to proteinase 3 (PR3), are found in the majority of patients with systemic Wegener's granulomatosis. The autoantibodies are widely used as diagnostic markers. Their role in the development and progression of the disease, however, is still under investigation. The primary target of ANCA, PR3, is located in the cytoplasm of polymorphonuclear neutrophils (PMN) or monocytes and is translocated to the cell surface upon stimulation. In patients with Wegener's granulomatosis PR3 is up-regulated most prominently during active disease. Despite the fact that both autoantibodies to PR3 and PMN expressing PR3 are present in patients with Wegener's granulomatosis, there is no evidence for binding of the autoantibodies to PMN. The present study was designed to analyze binding characteristics of autoantibodies to PR3 on PMN. PMN of patients with active Wegener's granulomatosis (N= 10) were tested for autoantibody binding. Despite high autoantibody titer and PR3 expression on the PMN, no surface-bound IgG was found on PMN ex vivo. When ANCA-containing plasma from patients was incubated with isolated PMN, stimulated to express PR3, again no specific binding of the autoantibody could be detected. Also keeping the samples on ice did not allow surface detection of IgG, ruling out degradation or internalization of the autoantibodies. Only when purified IgG fractions were used, binding to PMN was seen in 14 of 25 patients. Already 1% of plasma, however, was sufficient to greatly reduce the IgG binding. Reduced binding of the IgG fraction was also seen when a larger reaction volume was used. Our data indicate that autoantibodies to PR3 have a rather low affinity for surface-associated PR3 on PMN. This, in turn, argues against the hypothesis that ANCA contributes to the pathogenesis of the disease by stimulating viable PMN in whole blood.