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SARS-CoV-2 and its variants have persisted in this ongoing COVID-19 pandemic. While the vaccines have greatly reduced the COVID-19 cases, hospitalizations, and death, about half of the world remain unvaccinated due to various reasons. Furthermore, the duration of the immunity gained from COVID-19 vaccination is still unclear. Therefore, there is a need for innovative prophylactic and treatment measures. In response to this need, we previously reported on the successful computer-aided development of potent VHH-based multispecific antibodies that were characterized in vitro. Here, we evaluated in vivo efficacy and safety of the lead trispecific VHH-Fc, ABS-VIR-001. Importantly, our data showed that ABS-VIR-001 treatment prevented SARS-CoV-2 infection and death when provided as an intranasal prophylaxis in a humanized ACE-2 mouse model. In addition, ABS-VIR-001 post-exposure treatment was shown to greatly reduce viral loads by as much as 50-fold. A detailed panel of metabolic and cellular parameters demonstrated that ABS-VIR-001 treatment was overall comparable to the PBS treatment, indicating a favorable safety profile. Notably, our inhibition studies show that ABS-VIR-001 continued to demonstrate unwavering efficacy against SARS-CoV-2 mutants, associated with key variants including Delta and Omicron, owing to its multiple epitope design. Lastly, we rigorously tested and confirmed the excellent thermostability of ABS-VIR-001 when heated to 45 °C for up to 4 weeks. Taken together, our study suggests that ABS-VIR-001 is an efficacious and durable prophylaxis and post-exposure treatment for COVID-19 with promising safety and manufacturability features for global distribution., (© 2022. The Author(s).)

Glycoforms are otherwise identical proteins with different glycosylation. A lectin, Sambucus sieboldiana agglutinin (SSA), specifically binds glycoforms having α2,6-sialyl residues. The binding is found to inhibit antigen-antibody reaction; e.g., SSA inhibits anti-transferrin antibody binding to α2,6-sialylated transferrin (Tf) (SSA inhibition). SSA inhibition is not observed with other Tf glycoforms, indicating that the inhibition is glycoform-specific. Here we describe the application of SSA inhibition to ELISA as a specific assay for quantifying α2,6-sialylated Tf.

Antibodies are useful for localizing glycoprotein antigens in histochemistry, but they do not differentiate glycoforms in tissue sections because conventional antibodies recognize only protein epitopes rather than glycans. Glycan epitopes are recognized by lectins, which are found, occasionally, to inhibit antigen-antibody reaction in a glycoform-specific manner (lectin inhibition). Here we describe the application of lectin inhibition to immunohistochemistry for visualizing a glycoform in a tissue section.

Drug-induced hypersensitivity immune reactions are exaggerated immunoinflammatory responses to allergenic components of the medications that occur in genetically susceptible subjects. The type of hypersensitivity immune response generated, whether antibody mediated or T cell mediated, or an immune complex reaction is determined by multiple factors, including the molecular characteristics of the allergen, the route of administration of the medication, the manner of presentation of the allergen by antigen-presenting cells to naïve T cells, the repertoire of the T cell receptors, and the cytokine profile within the microenvironment. This review deals with the clinical and histopathological aspects of adverse immunologically mediated oral mucosal reactions to systemic medication. We elaborate on diseases showing features of lichenoid tissue reaction/interface dermatitis-stomatitis, autoimmune vesiculobullous oral lesions, and immunoglobulin E- (IgE-) and immune complex-mediated oral reactions to drugs.

The influenza virus hemagglutinin (HA) glycoprotein mediates receptor binding and membrane fusion during viral entry in host cells. Blocking these key steps in viral infection has applications for development of novel antiinfluenza therapeutics as well as vaccines. However, the lack of structural information on how small molecules can gain a foothold in the small, shallow receptor-binding site (RBS) has hindered drug design against this important target on the viral pathogen. Here, we report on the serendipitous crystallization-based discovery of a small-molecule N -cyclohexyltaurine, commonly known as the buffering agent CHES, that is able to bind to both group-1 and group-2 HAs of influenza A viruses. X-ray structural characterization of group-1 H5N1 A/Vietnam/1203/2004 (H5/Viet) and group-2 H3N2 A/Hong Kong/1/1968 (H3/HK68) HAs at 2.0-Å and 2.57-Å resolution, respectively, revealed that N -cyclohexyltaurine binds to the heart of the conserved HA RBS. N -cyclohexyltaurine mimics the binding mode of the natural receptor sialic acid and RBS-targeting bnAbs through formation of similar hydrogen bonds and CH-π interactions with the HA. In H3/HK68, N -cyclohexyltaurine also binds to a conserved pocket in the stem region, thereby exhibiting a dual-binding mode in group-2 HAs. These long-awaited structural insights into RBS recognition by a noncarbohydrate-based small molecule enhance our knowledge of how to target this important functional site and can serve as a template to guide the development of novel broad-spectrum small-molecule therapeutics against influenza virus., Competing Interests: The authors declare no conflict of interest.

Objectives: Recently, daratumumab has been included in the therapeutic strategies for myeloma patients. This molecule is an antibody directed against CD38, strongly expressed on plasma cells. Nevertheless, as CD38 is also present on erythrocyte membrane, daratumumab interferes with immunohaematological tests, complicating the selection of compatible blood., Methods: A total of 14 patients treated by daratumumab have been followed in our transfusion laboratory. Among them, 11 have been transfused. Dithiotreitol (DTT) has been used to inhibit the daratumumab's interference, in the pre-transfusion tests (irregular antibody screening and cross-match)., Results: The red blood cell treatment with DTT has been very efficacious to inhibit the daratumumab's interference in 13 patients out of 14. Some precautionary measures had to be taken into account, especially the pH and the storage conditions. An extended pheno/genotype was an additional security element in the selection of compatible blood. To simplify and to optimize the laboratory practices, a decisional flow chart has been written., Conclusion: DTT red blood cell treatment is very useful and efficacious in the pre-transfusion tests of patients treated with daratumumab. It allows to avoid the selection of blood bags only on the basis of an extended pheno/genotype, what is more secure and more ethical with respect to other at higher risk patients. A clear decisional flow chart allows a quality assurance gait. Collaboration with physicians is essential., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Vaccination is a reliable method of prophylaxis and a crucial measure for public health. However, the majority of vaccines cannot be administered orally due to their degradation in the harsh gut environment or inability to cross the GI tract. In this study, we report the first proof-of-concept study of orally producible chemically programmed antibodies via specific conjugation of adaptor ligands to endogenous antibodies, in vivo. Pre-immuniztion with 2,4-dinitrophenyl (DNP), or the reactive hapten, 1,3-diketone (DK), or a novel reactive hapten, vinyl sulfone (VS) in mice, followed by oral administration of adaptor ligands composed of the hapten and biotin to the pre-immunized mice resulted in successful in vivo formation of the biotin-hapten-antibody complexes within 2h. Pharmacokinetic evaluations revealed that apparent serum concentrations of programmed antibodies were up to 144nM and that the serum half-lives reached up to 34.4h. These findings show promise for the future development of orally bioavailable drug-hapten-antibody complexes asa strategy to quickly and easily modulate immune targets for aggressive pathogens as well as cancer., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Acute antibody-mediated rejection (AMR) remains an important challenge in the field of kidney transplantation despite the advances in immunosuppressive strategies, and has been recognized as an important cause of allograft dysfunction and graft loss. The treatments of acute AMR are not standardized and poor evidence is currently available on the effectiveness of therapy regimens with combinations of drugs. Standard treatment for acute AMR is based on 3 cornerstones: removal of donor-specific antibodies (DSAs) from the bloodstream, reduction of DSAs synthesis, and inhibition of the interaction between DSAs and human leukocyte antigen antigens on donor's cells. In this paper, we discuss the mechanism of action of the principal drugs currently in use, the main published studies on this topic, and the future insights related to the treatment of acute AMR., (© 2017 S. Karger AG, Basel.)

Suramin inhibits immune responses and protects cells against inflammatory cell injury. However, little is known about its mechanisms. Using an in vitro model of glomerular mesangial cell (MC) lysis induced by antibodies plus complement, we investigated the potential protective effects and mechanisms of suramin on immunologic cell injury. Exposure of rat MCs to anti-Thy-1 antibody plus complement or anti-MC rabbit serum caused complement-dependent cell lysis, which was blocked by suramin and its structural analogue NF023 and NF049, but not by PPADS, an antagonist of purinergic receptors. Addition of exogenous ATP also failed to affect MC lysis. Further analysis revealed that suramin interfered with antibody binding to cell membrane antigens and suppressed antibody-induced phosphorylation of several proteins, including p38. Inhibition of p38 with chemical inhibitor significantly attenuated cell injury. Collectively, our results indicate that suramin protects cells against antibody-initiated and complement-dependent cell injury through inhibition of antibody binding to cell surface antigens and suppression of p38 activation. Our study thus provides novel mechanistic insights into the actions of suramin and suggests that suramin might be used to treat certain immune diseases., (Copyright © 2016 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.)

We report use of U-shaped biconically tapered optical fibers (BTOF) as probes for label-free immunoassays. The tapered regions of the sensors were functionalized by immobilization of immunoglobulin-G (Ig-G) and tested for detection of anti-IgG at concentrations of 50 ng/mL to 50 µg/mL. Antibody-antigen reaction creates a biological nanolayer modifying the waveguide structure leading to a change in the sensor signal, which allows real-time monitoring. The kinetics of the antibody (mouse Ig-G)-antigen (rabbit anti-mouse IgG) reactions was studied. Hydrofluoric acid treatment makes the sensitive region thinner to enhance sensitivity, which we confirmed by experiments and simulations. The limit of detection for the sensor was estimated to be less than 50 ng/mL. Utilization of the rate of the sensor peak shift within the first few minutes of the antibody-antigen reaction is proposed as a rapid protein detection method.

Fab fragments (Fabs) maintain the ability to bind to specific antigens but lack effector functions due to the absence of the Fc portion. In the present study, we tested whether Fabs of an allergen-specific monoclonal antibody (mAb) were able to regulate asthmatic responses in mice. Asthmatic responses were induced in BALB/c mice by passive sensitization with anti-ovalbumin (OVA) polyclonal antibodies (pAbs) (day 0) and by active sensitization with OVA (days 0 and 14), followed by intratracheal (i.t.) challenge with OVA on day 1 and days 28, 29, 30 and 35. Fabs prepared by the digestion of an anti-OVA IgG1 (O1-10) mAb with papain were i.t. administered only once 30 min before antigenic challenge on day 1 or day 35. The results showed that i.t. administration of O1-10 Fabs with OVA markedly suppressed the early and/or late phases of asthmatic responses caused by passive and active sensitization. Similar results were obtained when Fabs of anti-OVA IgG2b mAb (O2B-3) were i.t. administered. In contrast, neither i.t. injection of intact 01-10/O2B-3 nor systemic injection of O1-10 Fabs suppressed the asthmatic responses. In vitro studies revealed that the capture of OVA by O1-10 Fabs prevented the subsequent binding of intact anti-OVA pAbs to the captured OVA. These results suggest that asthmatic responses may be down-regulated by the i.t. exposure to Fabs of an allergen-specific mAb via a mechanism involving the capture of allergen by Fabs in the respiratory tract before the interaction of intact antibody and allergen essential for the induction of asthmatic responses., (© 2013 John Wiley & Sons Ltd.)

Serum IgG is a potent inhibitor of monoclonal antibody (mAb) binding to the cell-surface Fcγ receptors (FcγRs), which mediate cytotoxic and phagocytic effector functions. Here, we show that this competition can be eliminated, selectively, by the introduction to serum of (i) an enzyme that displaces Fc from FcγRs and (ii) a modification present in the therapeutic mAb that renders it resistant to that enzyme. Specifically, we show that (i) EndoS (endoglycosidase S) cleaves only complex-type glycans of the type found on IgG but (ii) is inactive against an engineered IgG Fc with oligomannose-type glycans. EndoS thus reduces FcγR binding of serum IgG, but not that of engineered mAb. Introduction of both the engineered mAb and endoglycosidase in serum leads to a dramatic increase in FcγR binding compared to the introduction of mAb in serum alone. Antibody receptor refocusing is a general technique for boosting the effector signal of therapeutic antibodies., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The mouse monoclonal antibody (mAb) WN1 222-5 recognizes a carbohydrate epitope in the inner core region of LPS that is shared by all strains of Escherichia coli and Salmonella enterica and is able to neutralize their endotoxic activity in vitro and in vivo. Immunization of mice with mAb WN1 222-5 yielded several anti-idiotypic mAbs one of which (mAb S81-19) competitively inhibited binding of mAb WN1 222-5 to E. coli and Salmonella LPS. After immunization of rabbits with mAb S81-19, the serological responses towards LPS were characterized at intervals over two years. Whereas the serological response against the anti-idiotype developed as expected, the anti-anti-idiotypic response against LPS developed slowly and antibodies appeared after 200 d that bound to E. coli LPS of the R3 core-type and neutralized its TNF-α inducing capacity for human peripheral mononuclear cells. We describe the generation of a novel anti-idiotypic antibody that can induce LPS core-reactive antibodies upon immunization in rabbits and show that it is possible, in principle, to obtain LPS neutralizing antibodies by anti-idiotypic immunization against the mAb WN1 222-5. The mimicked epitope likely shares common determinants with the WN1 222-5 epitope, yet differences with respect to either affinity or specificity do exist, as binding to smaller oligosaccharides of the inner core was not observed.

The ABCG2 multidrug transporter protein has been identified as a key player in cancer drug resistance and xenobiotic elimination, as its actively transported substrates include anticancer drugs, intermediates of heme metabolism, xenobiotics, and also drug conjugates. Several transported substrates at higher concentrations, and some anticancer agents even at low concentrations directly inhibit the ABCG2 transporter, thus it is difficult to provide estimation for pharmacologically important ABCG2-dependent interactions. In addition, as documented here, in mutant variants of the transporter, inhibitors of the wild-type ABCG2 may become actively transported substrates. In this paper we describe a rapid in vitro assay to identify transport modulation by measuring the cell surface interaction of a conformation sensitive monoclonal antibody (5D3) with ABCG2 in intact cells. As documented, in conjunction with membrane ATPase, transport and cytotoxicity measurements, this assay provides a reliable estimate of concentration-dependent modulation of ABCG2 by newly emerging pharmacophores. A high-throughput, 96-well plate assay platform is also provided., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In vitro exposure of Postnatal Day 4 (PND4) rat ovaries to the occupational chemical 4-vinylcyclohexene diepoxide (VCD) destroys specifically primordial and primary follicles via acceleration of atresia. Because oocyte-expressed c-kit (KIT) plays a critical role in follicle survival and activation, a direct interaction of VCD with KIT as its mechanism of ovotoxicity was investigated. PND4 rat ovaries were cultured with and without VCD (30 μM) for 2 days. When assessed by Western analysis or mobility shift detection, phosphorylated KIT (pKIT) was decreased (P < 0.05) by VCD exposure, while total KIT protein was unaffected. Anti-mouse KIT2 (ACK2) antibody binds KIT and blocks its signaling pathways, whereas anti-mouse KIT 4 (ACK4) antibody binds KIT but does not block its activity. PND4 rat ovaries were incubated for 2 days with and without VCD with and without ACK2 (80 μg/ml) or ACK4 (80 μg/ml). ACK2 decreased pKIT; however, ACK4 had no effect. Conversely, ACK2 did not affect a VCD-induced decrease in pKIT, whereas ACK4 further reduced it. Because ACK2 and ACK4 (known to directly bind KIT) affect VCD responses, these results support the fact that VCD interacts directly with KIT. The effect of these antibodies on VCD-induced follicle loss was measured after 8 days of incubation. ACK2 further reduced (P < 0.05) VCD-induced follicle loss, whereas ACK4 did not affect it. These findings demonstrate that VCD induces ovotoxicity by direct inhibition of KIT autophosphorylation of the oocyte. The data also further support the vital function of KIT and its signaling pathway in primordial follicle survival and activation, as well as its role in VCD-induced ovotoxicity.

The cell surface glycoprotein CD44 was implicated in the progression, metastasis and apoptosis of certain human tumors. In this study, we used atomic force microscope (AFM) to monitor the effect of curcumin on human hepatocellular carcinoma (HepG2) cell surface nanoscale structure. High-resolution imaging revealed that cell morphology and ultrastructure changed a lot after being treated with curcumin. The membrane average roughness increased (10.88 ± 4.62 nm to 129.70 ± 43.72 nm) and the expression of CD44 decreased (99.79 ± 0.16% to 75.14 ± 8.37%). Laser scanning confocal microscope (LSCM) imaging showed that CD44 molecules were located on the cell membrane. The florescence intensity in control group was weaker than that in curcumin treated cells. Most of the binding forces between CD44 antibodies and untreated HepG2 cell membrane were around 120-220 pN. After being incubated with curcumin, the major forces focused on 70-150 pN (10 μM curcumin-treated) and 50-120 pN (20 μM curcumin-treated). These results suggested that, as result of nanoscale molecular redistribution, changes of the cell surface were in response to external treatment of curcumin. The combination of AFM and LSCM could be a powerful method to detect the distribution of cell surface molecules and interactions between molecules and their ligands., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Imatinib and nilotinib are inhibitors that selectively target a set of protein tyrosine kinases, including abelson kinase (Abl), together with the chimeric oncoprotein, breakpoint cluster region-abelson kinase (Bcr-Abl), as well as stem cell factor receptor (KIT), platelet-derived growth factor receptor (PDGFR), discoidin domain receptor (DDR), and colony stimulating factor-1 receptor (CSF-1R). The aim of the present study was to investigate whether imatinib or nilotinib was effective against arthritis in the glucose-6-phosphate isomerase (GPI)-induced arthritis mouse model. Imatinib or nilotinib was administered orally to the arthritic mice at different time points. Efficacy was evaluated by visual scoring and by determining the production of anti-GPI antibody. Splenocytes from the arthritic mice were cultured with GPI in the presence of imatinib or nilotinib in vitro, and cytokine levels in the culture supernatants were analyzed. To investigate the effects of imatinib and nilotinib on T-cell proliferation, lymph node cells from the arthritic mice were cultured with GPI in the presence of imatinib or nilotinib in vitro. Interleukin (IL)-17 mRNA expression in the arthritic ankle joints from the onset of arthritis was analyzed by real-time polymerase chain reaction (PCR). The administration of imatinib from day 0 showed suppression of arthritis (P < 0.05), the administration of nilotinib from day 0 resulted in pronounced suppression of arthritis (P < 0.01), and that from day 7 showed significant inhibition of the progression of arthritis (P < 0.05). A reduction in anti-GPI antibodies was correlated with the therapeutic efficacy of imatinib, but not with that of nilotinib. Imatinib dose-dependently inhibited tumor necrosis factor (TNF)-α, IL-6, interferon (IFN)-γ, and IL-17 production by splenocytes in vitro, while nilotinib inhibited only IL-17 and IFN-γ production in a dose-dependent fashion. Imatinib at 3 μM exerted a mild antiproliferative effect on CD4+ T cells (P < 0.05), whereas imatinib at 10 μM and nilotinib at 3 and 10 μM demonstrated a marked antiproliferative effect (P < 0.01). The IL17 gene expression level on day 7 tended to be higher than that on day 14. These findings suggest that imatinib and nilotinib could prevent autoimmune arthritis, essentially via distinct mechanisms, in that imatinib inhibits both inflammatory and T-cell-derived cytokine production, whereas nilotinib suppresses T-cell-derived cytokine production. Imatinib and nilotinib could have therapeutic potential for rheumatoid arthritis (RA) and other inflammatory diseases.

Celiac disease is caused by an inappropriate immune response to incompletely digested gluten proteins. We investigated whether synthetic peptides with high affinity to wheat gliadin could be selected with a phage display technique and whether complexes between such peptides and gliadin could sustain gastric and pancreatic digestion. Two synthetic peptides, P61 and P64, were selected because of their high affinity to immobilized gliadin. They were allowed to form complexes with gliadin, whereafter the complexes were subjected to in vitro digestion with gastric and pancreatic enzymes. The digestion products were analyzed with Western blot and RP HPLC. The results showed that both peptides formed stable complexes with intact gliadin and that complexes between gliadin and peptide P64 partly resisted gastrointestinal digestion. The two peptides reduced the binding of serum anti-gliadin IgA antibodies by 12%, and 11.5%, respectively, and the binding of anti-gliadin antibodies of the IgG isotype by 13% and 10%. Thus peptides produced by a phage display technique could interact stably with gliadin partly masking epitopes for antibody binding. A combination of peptides of this kind may be used to block gliadin-immune system interactions., (Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.)

Background:   A hypoallergen of the major cat allergen Fel d 1, recombinant (r) Fel d 1 (DTE III), was previously shown to have retained T-cell reactivity and strongly reduced IgE-binding capacity compared to unmodified rFel d 1. Here, we evaluated the therapeutic capacity of rFel d 1 (DTE III) in a mouse model for cat allergy., Methods:   Mice were subcutaneously (s.c.) sensitized with rFel d 1 and subsequently treated (s.c.) with 50 or 200 μg rFel d 1 (DTE III), or 50 μg rFel d 1, prior to intranasal challenge with cat dander extract. Airway hyperreactivity (AHR), cells and cytokines in bronchoalveolar lavage fluid, splenocyte in vitro response, and serum immunoglobulins were analyzed. Seven cat-allergic patients and ten healthy controls were tested for skin prick test (SPT) reactivity to rFel d 1 (DTE III) and rFel d 1., Results:   Mice treated with 50 and 200 μg rFel d 1 (DTE III), and 50 μg rFel d 1, produced increased serum levels of rFel d 1-specific IgG1 and IgG2a compared to sham-treated mice. IgG from all treatment groups could block binding of patients' IgE to rFel d 1. The 200 μg rFel d 1 (DTE III) treatment tended to reduce AHR. All mice tolerated treatment with rFel d 1 (DTE III), in contrast to only four of ten treated with rFel d 1. Compared to rFel d 1, the hypoallergen showed a tendency of reduced SPT reactivity., Conclusion:   The rFel d 1 (DTE III) hypoallergen might be a promising candidate for application in immunotherapy of cat allergy with improved safety and efficacy., (© 2010 John Wiley & Sons A/S.)

Pemphigus foliaceus (PF) is an autoimmune skin blistering disease mediated by pathogenic autoantibodies against the desmosomal core glycoprotein desmoglein-1 (Dsg1). This study demonstrated that the O-glycan-specific plant lectin jacalin binds Dsg1 and inhibits the interaction of Dsg1/PF IgG. N-glycosylation is not involved in the interaction of Dsg1/jacalin or Dsg1/PF IgG. Subcutaneous injection of jacalin into neonatal mice drastically reduced PF IgG deposition at the epidermal cell surface and blocked PF IgG-induced skin blisters, both clinically and histologically. Interestingly, another plant lectin, peanut agglutinin, which shares the same carbohydrate specificity toward the O-linked carbohydrate structure known as Thomsen-Friedenreich antigen (TF antigen, Galβ1-3GalNAcα-O-Ser/Thr), also bound Dsg1 and blocked the skin blistering. In contrast, the plant lectin vicia villosa-B4 (VVL-B4), which shares the carbohydrate specificity toward the O-linked monosaccharide known as Thomsen-nouveau antigen (GalNAc-α1-O-Ser/Thr), did not bind Dsg1 and did not show a protective effect against the disease induced by the autoantibodies. Collectively, these results suggest that the binding of jacalin to O-linked TF carbohydrate motifs on Dsg1 impairs the Dsg1/PF autoantibody interactions and abrogates its pathogenicity in vivo. TF-specific binding ligands may have a potential therapeutic value for PF.

Chronic and complex autoimmune diseases, currently treated palliatively with immunosuppressives, require multi-targeted therapy for greater effectiveness. The naturally occurring polyphenol curcumin has emerged as a powerful "nutraceutical" that interacts with multiple targets to regress diseases safely and inexpensively. Up to 8 g/day of curcumin for 18 months was non-toxic to humans. However, curcumin's utility is limited by its aqueous insolubility. We have demonstrated a heat-mediated 12-fold increase in curcumin's aqueous solubility. Here, we show by SDS-PAGE and surface plasmon resonance that heat-solubilized curcumin binds to proteins. Based on this binding we hypothesized that heat-solubilized curcumin or turmeric would prevent autoantibody targeting of cognate autoantigens. Heat-solubilized curcumin/turmeric significantly decreased binding of autoantibodies from Sjögren's syndrome (up to 43/70%, respectively) and systemic lupus erythematosus (up to 52/70%, respectively) patients as well as an animal model of Sjögren's syndrome (up to 50/60%, respectively) to their cognate antigens. However, inhibition was not specific to autoimmunity. Heat-solubilized curcumin/turmeric also inhibited binding of commercial polyclonal anti-spectrin to spectrin (50/56%, respectively). Thus, we suggest that the multifaceted heat-solubilized curcumin can ameliorate autoimmune disorders. In addition, the non-toxic curcumin could serve as a new protein stain in SDS-PAGE even though it is less sensitive than the Coomassie system which involves toxic chemicals.

Background: Using recombinant human monoclonal thyroglobulin antibodies expressed as Fab molecules (TgAb-Fab), we have recently confirmed the restriction of Tg epitopes in Hashimoto's thyroiditis (HT)., Objective: To investigate Tg epitopes of serum TgAb in HT adults and HT juveniles from a geographically isolated area (Sardinia)., Design and Patients: Serum TgAb of 39 Sardinian HT adults, 53 Sardinian HT juveniles and 45 non-Sardinian HT adults were evaluated. The binding of serum TgAb to Tg in ELISA was inhibited by four recombinant human TgAb-Fab, identifying Tg epitopic regions A-D. The percentage of Tg binding inhibition was calculated comparing the binding of serum TgAb in presence of each TgAb-Fab with that in its absence., Results: In the whole cohort of 137 patients, A region TgAb-Fab induced the highest levels of inhibition (55.3 +/- 17.8%) (mean +/- SD). Lower levels of inhibition were induced by TgAb-Fab of regions B (27.8 +/- 25.8%), C (26.8 +/- 24.6%) and D (17.5 +/- 18.4%). In Sardinian HT adults inhibition by TgAb-Fab of regions A, B and C were comparable to Sardinian HT juveniles; the marginal D region TgAb-Fab induced a slightly higher inhibition (22.1 vs. 13.8%; P = 0.034) in the former than in the latter group. In Sardinian and non-Sardinian HT adults inhibitions by the four TgAb-Fab were similar., Conclusions: In HT, the Tg epitope pattern of serum TgAb was similar in juveniles and adults from a geographically restricted area and in two adult populations from different geographical areas. Thus, in HT, neither age nor genetic background appear to influence B-cell epitopes.

Crosslinking of ultrapure hemoglobin, crystalline catalase, and superoxide dismutase resulted in a soluble nanodimensional complex of polyhemoglobin-catalase-superoxide dismutase. A less expensive and more convenient way is to crosslink bovine stroma-free hemolysate (stroma-free hemolysate) that already contains hemoglobin, catalase, and superoxide dismutase into polyhemoglobin with catalase and superoxide dismutase activities (stroma-free polyhemolysate) [21]. The objective of the present study is to evaluate the immunological properties of this stroma-free polyhemolysate. Each of three groups of rats received weekly subcutaneous injections of one of the stroma-free polyhemolysate, stroma-free hemolysate, and saline for four weeks. One week after the four cycles of weekly immunization, serum and plasma were collected for C3a complement activation tests and Ouchterlony antibody-antigen precipitation tests, respectively. Results show that stroma-free polyhemolysate retained significant antioxidant enzyme activity. The C3a complement activation test and Ouchterlony test show that four weekly subcutaneous injections of bovine stroma-free polyhemolysate did not result in any immunological reaction in rats when tested this way.

It has yet to be established whether chlorogenic acid (CGA), a common xenobiotic with potential exposure risk to humans, is associated with immune-mediated hypersensitivity reactions (HRs). The primary limitation in evaluating this potential relationship is the lack of an effective animal model for use in predicting the immunosensitizing potential of low molecular weight compounds (LMWCs). Currently, the popliteal lymph node assay (PLNA) is considered a very promising tool for assessing immunosensitizing potential of LMWCs. To determine whether CGA may possess an intrinsic capacity to stimulate or dysregulate immune responses, and if so, what mechanisms may be involved, we characterized the popliteal lymph node reaction induced by CGA in naive female BALB/c mice using both a direct PLNA (d-PLNA) and a reporter antigen PLNA (RA-PLNA) method. Our results show that CGA failed to induce immunoreactivity following a single subcutaneous injection either alone or when combined with TNP-OVA or TNP-Ficoll. These results indicated that CGA lacks the intrinsic capacity to sensitize or stimulate immune responses in BALB/c mice. Moreover, these results suggest that exposure to CGA may not represent a safety concern for humans and that removal of CGA from Traditional Chinese Medicine Injections may not significantly decrease the prevalence of HRs., (2010 Elsevier Ltd. All rights reserved.)

Silencing the antigenic response of red blood cells (RBCs) is a prerequisite toward the development of universal blood transfusion. Using a two-dimensional (2D) model whereby nonfixed RBCs are adsorbed on a human fibronectin (HFN)-coated surface, we demonstrate that the layer-by-layer (LbL) assembly technique of biocompatible polyelectrolytes can be employed to achieve the immunocamouflage of RBCs against the Anti-A antibody while maintaining the integrity and viability of the cells. The multilayered film consisted of a protecting shell (P-shell), containing five bilayers of chitosan-graft-phosphorylcholine (CH-PC) and sodium hyaluronate (HA), covered by a camouflage shell (C-shell) made up of five bilayers of poly-(L-lysine)-graft-poly(ethylene glycol) (PLL-PEG) and alginate (AL). Control experiments in which RBCs were coated by (CH-PC/HA)(10) bilayers indicated that the two polyelectrolytes alone did not prevent immunorecognition. The LbL film formation on RBCs and model substrates was monitored by quartz crystal microbalance with dissipation factor (QCM-D) and analyzed through zeta-potential measurements, atomic force microscopy (AFM), and optical microscopy. Antibody interaction with the coated RBCs was investigated by QCM-D, fluorescence microscopy, and hemolysis assays. Results from these measurements demonstrated that the hybrid LbL system built-up with different sets of polyelectrolytes was able to protect the RBCs from hemolysis and recognition by the Anti-A antibody.

The synthesis of a carba-analogue corresponding to the trisaccharide repeating unit of Streptococcus pneumoniae type 19F capsular polysaccharide, where a residue of carba-L-rhamnose has been inserted into the natural trisaccharide in place of L-rhamnose, is described. The conformational properties of the analogue were investigated with the aid of molecular dynamics simulations and were strictly analogous to those of the natural compound. The biological activity of the carba-analogue was comparable to that of the corresponding natural repeating unit, thus suggesting that this compound, more stable to hydrolysis, is a good mimic of the natural structure.

The overwhelming majority of antibodies useful for formalin fixed, paraffin embedded (FFPE) tissues require antigen retrieval to reverse the effect of formalin fixation and re-establish immunoreactivity. How this reversal happens is poorly understood. We developed a new experimental model for studying the mechanism of formalin fixation and antigen retrieval. Epitope mapping studies on nine antibodies useful for FFPE tissues revealed that each consisted of a contiguous stretch of amino acids in the native protein (linear epitope). Small peptides representing the epitopes of antibodies to human epidermal growth factor receptor type (HER2), estrogen, and progesterone receptors were attached covalently to glass microscope slides in a peptide array. Most peptides retained immunoreactivity after formalin fixation. Immunoreactivity was completely abrogated for all peptides, however, if an irrelevant large protein was present during formalin-induced cross-linking. We hypothesize that cross-linking the irrelevant protein to the peptide epitopes sterically blocked antibodies from binding. Antigen retrieval dissociates irrelevant proteins and restores immunoreactivity. Because the epitopes for clinical antibodies require only primary protein structure, the fact that antigen retrieval probably denatures the secondary and tertiary structure of the protein is irrelevant. The same mechanism may occur in tissue samples subjected to formalin fixation and antigen retrieval.

The physicochemical and biological properties of fulvic acid extracted and purified from excess sludge and solubilized excess sludge were studied. Solubilization was introduced to improve the recovery rate of fulvic acid from the sludge. The structural features of fulvic acid from excess sludge and solubilized excess sludge were characterized by using an elemental analysis, Fourier transform infrared spectroscopy and (1)H-nuclear magnetic resonance spectroscopy, and were compared with fulvic acid extracted from peat which had an inhibitory effect on the type I allergy in our previous study. The results show that they had a higher aliphatic characteristic with lower oxygen group content than fulvic acid from peat, and that the aliphatic characteristic was further strengthened by the use of solubilization. The biological properties of fulvic acid from excess sludge and solubilized excess sludge showed an inhibitory effect on beta-hexosaminidase release at the antigen-antibody binding stage and antigen-receptor binding stage by using rat basophilic leukemia cells.

In an attempt to exploit the large geometry changes associated with azobenzene photo-isomerization for the modulation of antibody-antigen interaction, we introduced in the backbone of the FLAG peptide (DYKDDDDK), an azobenzene unit to photo-modulate its conformational states and consequently its interaction with the monoclonal anti-FLAG-tag antibody M1. The FLAG-tag system is an established technique for purifying and detecting the corresponding fusion proteins. In this context, conflicting evidence has been presented regarding the necessity of calcium for stable binding. Using surface plasmon resonance, we showed that not the initial recognition but certainly the stability of the complex improves in the presence of calcium. Subsequently, we substituted two or three of the central aspartate residues for an artificial, azobenzene-based, photo-responsive amino acid. Four structural isomers of the artificial amino acid were considered, in total twelve FLAG-tag analogues were synthesized. Two showed significant differences in their ability to bind to the antibody in their cis versus their trans state. Interestingly, these two peptides are the two shortest of the twelve photo-peptides investigated. Finally, it was shown that for these two FLAG-analogues switching between cis and trans states is possible in the presence of the antibody.

Background: Myasthenia gravis (MG) is an autoimmune disease affecting approximately 40,000 patients in the United States. One of the major mechanisms of disease pathology in MG is the binding, internalization, and eventual destruction of acetylcholine receptors (AChR) at the neuromuscular junction by cross-linking AChR-specific autoantibodies. This process, known as antigenic modulation, ultimately attenuates the ability of muscle cells to contract in response to signals from neurons, leading to muscle weakness and fatigue. For this reason, antigenic modulation of the AChR on cultured cells has become an important diagnostic tool for assessing the pathogenicity of AChR-specific autoantibodies. Traditionally, these assays have been done using radiolabeled AChR ligands such as (125)I alpha-bungarotoxin to determine relative AChR number. Here, we present a high-throughput immunofluorescent flow cytometry-based assay that can be used to quantify AChR levels on the cell surface and assess the efficacy of molecules designed to rescue antigenic modulation., Methods: AChR levels were quantified on human muscle cells before and after treatment with AChR antibodies via immunofluorescent labeling with the AChR monoclonal antibodies, mAb210 and mAb B3, followed by flow cytometry of EDTA-treated cells., Results: Using a novel, flow cytometry-based assay, antigenic modulation of the AChR was demonstrated on human cells using both AChR-specific monoclonal antibody and MG patient serum. The degree of antigenic modulation was dose responsive to antibody levels and could be reversed by preincubating antibodies with soluble AChR alpha subunit extracellular domain., Summary: A rapid, nonradioactive assay was developed to determine the potential of AChR-specific antibodies in the serum of MG patients to bind and down-regulate the AChR. This assay can be used to assess the ability of putative therapeutics that rescue antigenic modulation and could be developed for the treatment of MG., ((c) 2008 Clinical Cytometry Society.)

Proteolytic enzymes can be extremely useful for the identification of clinically significant antibodies in blood grouping; however, they can also be destructive to certain agglutination reactions. ABO antibodies are the most clinically significant. A review of the literature reveals little research involving proteolytic enzymes and IgM antibodies. This study investigates the effects of various concentrations of the proteolytic enzyme bromelain on the agglutination of reagent A1 and B cells with donor plasma during ABO reverse grouping on the Olympus PK 7300 automated serology analyser. The optimum bromelain concentration improved or enhanced antigen-antibody reactions, causing a reduction in ABO failures. The results were analysed using an ANOVA test. An anomalous result was obtained at 0% bromelain where less ABO failures were generated than expected. Although most results were not statistically significant, the optimum bromelain concentration for ABO reverse grouping indicated was 0.25%. This work also highlighted the fact that ABO failures can be attributed to other factors such as sensitivity of reagent cells and antibody avidity. This study identified limitations and problems with the methods used and presents recommendations for future research which may assist in the clarification of the role of proteolytic enzymes in ABO reverse grouping.

Most current techniques employed to improve antigen-antibody signals in Western blotting and in immunohistochemistry rely on sample processing prior to staining (e.g., microwaving) or using a more robust reporter (e.g., a secondary antibody with biotin-streptavidin). We have developed and optimized a new approach intended to stabilize the complexes formed between antigens and their respective primary antibodies by cupric ions at high pH. This technique improves the affinity and lowers cross-reactivity with nonspecific bands of approximately 20% of antibodies tested (5/25). Here we report that this method can enhance antigen-antibody specificity and can improve the utility of some poorly reactive primary antibodies.

In this article, we present a systematic study on IgG and Fab fragment of anti-IgG molecules using fluorescence auto- and cross-correlation spectroscopy to investigate their diffusion characteristics, binding kinetics, and the effect of small organic molecule, urea on their binding. Through our analysis, we found that the diffusion coefficient for IgG and Fab fragment of anti-IgG molecules were 37+/-2 microm2s(-1) and 56+/-2 microm2s(-1), respectively. From the binding kinetics study, the respective forward (k(a)) and backward (k(d)) reaction rates were (5.25+/-0.25)x10(6)M(-1)s(-1) and 0.08+/-0.005 s(-1), respectively and the corresponding dissociation binding constant (K(D)) was 15+/-2 nM. We also found that urea inhibits the binding of these molecules at 4M concentration due to denaturation.

Background and Objectives: Enzymatic treatment of red blood cells is thought to reduce the cell zeta (zeta) potential, effectively decreasing the distance between cells to less than the length of an immunoglobulin G antibody binding site, and resulting in agglutination of cells. However, the zeta potential given by Smoluchowski's formula is based on theories of the electrophoresis of hard colloidal particles. A theory has recently been developed for the electrophoresis of colloidal particles covered with polyelectrolytes, which we call 'soft particles'., Materials and Methods: The electrophoretic mobility of red blood cell treated with papain and neuraminidase was measured as the electrolyte concentration of the medium using phosphate buffer. The results were analysed via the formula for 'soft particles'. This mobility formula involved two parameters, the fixed charge density (ZN) and parameter 1/lambda characterizing the 'softness' of the cell surface layer., Results: The best-fit curves of 0.1 units neuraminidase-treated red blood cells indicated that ZN decreased by 76% and 1/lambda decreased by 8% compared to intact red blood cells. In contrast, in 5 units of papain-treated red blood cells ZN decreased by 45% and 1/lambda decreased by 33% compared to intact red blood cells., Conclusion: The present study shows that the change in ZN for neuraminidase-treated cells was very large, but the cells did not become agglutinable. Papain-treated cells had changes in both ZN and 1/lambda, and the cells became agglutinable. 1/lambda is one of the important factors for agglutination.

The acute motor axonal neuropathy (AMAN) variant of Guillain-Barré syndrome (GBS) is associated with anti-GD1a and anti-GM1 IgG antibodies. The basis of preferential motor nerve injury in this disease is not clear, however, because biochemical studies demonstrate that sensory and motor nerves express similar quantities of GD1a and GM1 gangliosides. To elucidate the pathophysiology of AMAN, we have developed several monoclonal antibodies (mAbs) with GD1a reactivity and reported that one mAb, GD1a-1, preferentially stained motor axons in human and rodent nerves. To understand the basis of this preferential motor axon staining, several derivatives of GD1a were generated by various chemical modifications of N-acetylneuraminic (sialic) acid residues (GD1a NeuAc 1-amide, GD1a NeuAc ethyl ester, GD1a NeuAc 1-alcohol, GD1a NeuAc 1-methyl ester, GD1a NeuAc 7-alcohol, GD1a NeuAc 7-aldehyde) on this ganglioside. Binding of anti-GD1a mAbs and AMAN sera with anti-GD1a Abs to these derivatives was examined. Our results indicate that mAbs with selective motor axon staining had a distinct pattern of reactivity with GD1a-derivatives compared to mAbs that stain both motor and sensory axons. The fine specificity of the anti-GD1a antibodies determines their motor selectivity, which was validated by cloning a new mAb (GD1a-E6) with a chemical and immunocytochemical binding pattern similar to that of GD1a-1 but with two orders of magnitude higher affinity. Control studies indicate that selective binding of mAbs to motor nerves is not due to differences in antibody affinity or ceramide structural specificity. Since GD1a-reactive mAb with preferential motor axon staining showed similar binding to sensory- and motor nerve-derived GD1a in a solid phase assay, we generated computer models of GD1a based on binding patterns of different GD1a-reactive mAbs to different GD1a-derivatives. These modelling studies suggest that critical GD1a epitopes recognized by mAbs are differentially expressed in motor and sensory nerves. The GD1a-derivative binding patterns of AMAN sera resembled those with motor-specific mAbs. On the basis of these findings we postulate that both the fine specificity and ganglioside orientation/exposure in the tissues contribute to target recognition by anti-ganglioside antibodies and this observation provides one explanation for preferential motor axon injury in AMAN.

In this study, we evaluated the profile of anti-Paracoccidioides brasiliensis immunoglobulin isotypes in serum from patients with the acute and chronic forms of paracoccidioidomycosis, using the whole Paracoccidioides brasiliensis antigen and the antigen treated with sodium metaperiodate. All the immunoglobulin isotypes present in the serum from patients with the acute and chronic forms of paracoccidioidomycosis presented higher reactivity towards the whole antigen than to the antigen treated with metaperiodate (P < 0.05). The reactivity of IgG and IgM to the antigen treated with metaperiodate was greater in serum from patients with the acute form of the disease (P < 0.05), while IgA was more reactive in serum from patients with the chronic form (P < 0.05). There was greater reactivity of IgG1 and IgG2 to the whole antigen and the antigen treated with metaperiodate in the serum from patients with paracoccidioidomycosis than there was in serum from patients with other parasitic infections (P < 0.05). Furthermore, IgG1 from patients with the acute form recognized the 19kDa, 27kDa and 31kDa antigens in the western blot test. Thus, the results suggest that modifications to the epitopes of Paracoccidioides brasiliensis antigens may help to improve the immunodiagnosis of paracoccidioidomycosis.

Neutralizing of TNF-alpha has been proved effective in treatment of some autoimmune diseases, e.g. rheumatoid arthritis and Crohn's disease. Low molecular weight synthetic peptides can mimic the binding sites of TNF-alpha receptors and block the activity of TNF-alpha. In order to stabilize the conformation, increase the affinity and bioactivity, in this study, heavy chain variable region of human antibody was used as a scaffold to simultaneously display three peptides, which were designed on the interaction between TNF-alpha and it's neutralizing monoclonal antibody. On the basis of the structural character and physical-chemical property of the families of seven kinds of heavy chain variable regions (VH) in human antibodies, the fifth type of VH was screened as scaffold to display the antagonist peptide. Based on the computer-guided molecular design method, a novel domain antibody against TNF-alpha (named as ATD5) was designed as TNF-alpha antagonist. The theoretical study showed that ATD5 was more stable than displayed antagonist peptide. The binding activity with TNF-alpha was higher than free peptides. After expression and purification in Escherichia coli, ATD5 could bind directly with TNF-alpha and inhibit the binding of TNF-alpha to its two receptors, TNFR1 and TNFR2. ATD5 could also reduce the TNF-alpha-mediated cytotoxicity and inhibit TNF-alpha-mediated caspase activation on L929 cells in a dose dependent manner. The activity of ATD5 was significantly stronger than three peptides displayed by ATD5. This study provides a novel strategy for the development of new TNF-alpha inhibitors. This study demonstrates that it is possible to screen potential antagonists of TNF-alpha using in vitro analysis systems in combination with the computer-aided modeling method.

Background: Olive pollen is an important cause of allergy in Mediterranean countries. To date, 10 allergens (Ole e 1 to Ole e 10) have been isolated and characterized. Animal models of olive pollen allergy are suitable tools for testing the efficacy and safety of new forms of immunotherapy., Objectives: To characterize the immune response in mice sensitized with olive pollen extract and to compare it with that of allergic patients., Methods: BALB/c mice were sensitized by 4 intraperitoneal injections of olive pollen extract in aluminum hydroxide. The allergic state was proved by measuring serum specific IgG1 and total IgE antibody levels. The IgG1 responses to olive pollen allergens were assayed by immunoblotting and enzyme-linked immunosorbent assay. Competition experiments between human IgE and mouse IgG1 binding to olive pollen allergens were performed., Results: Sensitization with olive pollen extract induced high levels of specific IgG1 and total IgE in all tested animals. Immunoblotting experiments showed that the mouse IgG1 binding pattern to pollen extract was complex and heterogeneous, as occurs with human IgE. High IgG1 antibody levels to the major olive pollen allergens described for humans were detected in serum samples from sensitized mice, whereas minor olive pollen allergens induced no significant IgG1 response. Coincubation of mouse serum samples with a cocktail of Ole e 1, Ole e 9, and Ole e 10 resulted in a significant decrease (60%) in IgG1 binding to olive pollen extract. Specific mouse IgG1 strongly inhibited human IgE binding to olive pollen allergens., Conclusions: This mouse model of olive pollen sensitization mimics immunologic features of human pollinosis and could be a useful tool for designing novel forms of immunotherapy for olive pollen allergy based on allergen cocktails.

Even though antigen retrieval is highly denaturing, it paradoxically restores immunoreactivity after formalin fixation. It is unclear how this happens. We address this question using a peptide array to model formalin fixation and antigen retrieval. The peptides are linear stretches based on the native protein sequence, containing antibody epitopes of HER-2, estrogen receptor, progesterone receptor, and Ki-67. Of the 7 peptides, 6 retain their immunoreactivity after formalin fixation. However, if formalin fixation is performed in the presence of an irrelevant protein, immunoreactivity is abrogated, regardless of the peptides' amino acid composition. Fixation of an external protein around the antibody epitope prevents antibody binding. Antigen retrieval restores immunoreactivity. These findings demonstrate that native protein conformation is not relevant during antigen retrieval. Moreover, the loss and recovery of immunoreactivity associated with fixation and antigen retrieval, respectively, can be accounted for completely with a model of steric interference by adjacent proteins.

Purpose of Review: Drug hypersensitivity reactions are relatively rare but may result in severe morbidity and fatalities. Due to the idiosyncratic nature and multifactorial etiology of these reactions, development of a single animal model to study the immunosensitizing mechanisms of all drugs is impossible. This hampers the development of predictive screening models that are urgently needed to assess the immunostimulating capacity of newly developed drugs. The present review will focus on recent findings on mechanisms of drug hypersensitivity reactions obtained with murine models, and on the use of these models as potential screening tools to assess the immunostimulating capacity of drugs., Recent Findings: Mechanisms of drug-induced sensitization versus tolerance appear dependent on generally accepted immunological paradigms. For instance, co-stimulatory signaling by antigen-presenting cells is decisive in drug-induced immunosensitization and both T cells and antigen-presenting cells are important for the induction of tolerance to orally administered drugs. From recent studies it has been hypothesized that expression of stress-associated transcription factors and the expression of costimulatory molecules or cytokine production within hours or days after the initial exposure may be representative of drug-induced hypersensitivity reactions and may thus be used as predictive parameters to screen for immunosensitizing drugs., Summary: The development of animal models to study mechanisms of drug hypersensitivity reactions is still in its infancy. Much effort has been made, however, to search for early indicators of immunostimulation in murine animal models that may eventually appear useful in a tiered strategy to assess drug-induced sensitization.

Interventions with heparin therapies have increased fetal survival in patients with antiphospholipid syndrome (APS). In this review, several physiological and pharmacological effects of heparin are described. These different mechanisms of action could explain the beneficial effects of heparin in the treatment of recurrent pregnancy loss in patients with APS. In addition to direct effects of heparin on the coagulation cascade, heparin might protect pregnancies by reducing the binding of antiphospholipid antibodies, reducing inflammation, facilitating implantation and or inhibiting complement activation.

The capability of certain drugs to cause immune-mediated drug hypersensitivity reactions in susceptible individuals has initiated a search for pre-clinical screening tools to identify immunosensitizing drugs. Since most drugs are taken orally, hazard assessment of their immunosensitizing potential should include oral exposure models. In this study, the predictive value of the reporter antigen (RA) approach was investigated in combination with oral or intraperitoneal (ip) exposure to a selection of allergenic drugs, i.e., D-penicillamine (D-Pen), Diclofenac (DF), or Nevirapine (Nevi). The RA trinitrophenyl-Ovalbumin (TNP-OVA) was used to assess the capacity of the drugs to stimulate systemic immune responses to a bystander antigen, whereas the RA TNP-Ficoll was used to indicate whether the drugs were able to induce specific anamnestic T-cell responses. TNP-OVA was injected (ip) in C3H/HeOuJ mice that were subsequently exposed (orally or ip) to one of the drugs via different exposure protocols. All three model drugs used resulted in delayed type hypersensitivity reactions to TNP-OVA after ip and oral exposure. In addition, TNP-specific serum antibody levels were increased after ip exposure to Nevi, and after both oral and ip exposure to D-Pen and DF. These data indicate that the present drugs are able to stimulate immune responses to bystander antigens. Responses to TNP-Ficoll were measured in the popliteal lymph node of BALB/c mice three weeks after they received a single oral dose of D-Pen or DF. Results of this approach show that orally pre-treated mice responded with enhanced responses (TNP-specific IgG1 and IFN-gamma production) to sub-optimal doses of D-Pen or DF in a drug-specific manner. Data with TNP-Ficoll indicate that these drugs stimulate systemic formation of specific T cells. Together, the RA-approach allows assessment of systemic sensitization upon oral and/or ip exposure to the selected drugs. To further evaluate the utility of these models, more drugs, including non-allergenic drugs and those that require metabolic conversion to become allergenic need to be studied in the present models.

Background: Japanese cedar (Cryptomeria japonica) pollinosis is one of the most prevalent allergic diseases in Japan. Only three C. japonica allergens, Cry j 1, Cry j 2, and CJP-6, have been characterized. The full IgE-binding spectrum of C. japonica pollen allergens demonstrates that many allergens remain to be identified., Objective: The aim of this study was to characterize a novel allergen with a high frequency of IgE binding., Methods: The cDNA coding for a high-frequency IgE-binding protein, designated CJP-4, was cloned from the total mRNA of C. japonica pollen. The corresponding native allergen was purified by affinity precipitation with colloidal chitin and gel chromatography. The IgE-binding ability of purified native CJP-4 was characterized by ELISA and ELISA inhibition., Results: The CJP-4 cDNA encoded 281 amino acids with significant sequence homology to class IV chitinases. Purified native CJP-4, migrated as a homogeneous 34-kDa protein on SDS-PAGE, revealed endochitinase activity on native PAGE. The purified protein displayed the ability to bind IgE from all patients tested (31/31) in ELISA, whereas Cry j 1 bound to IgE at a 71% frequency (22/31). Pre-incubation with latex C-serum completely inhibited the reaction of pooled sera IgE from patients with C. japonica pollinosis and/or latex allergy to purified CJP-4., Conclusion: We identified CJP-4 as a novel and fourth C. japonica chitinase allergen with high IgE-binding frequency. The competitive IgE-binding profile between C. japonica chitinase and latex C-serum indicated that C. japonica chitinase should be an important pan-allergen in C. japonica pollen.

Water-in-oil (W/O) emulsions are known as most effective adjuvants to generate high and durable antibody responses to vaccine antigens following a single immunization. However, their structural requirements remain poorly understood. Here we addressed the significance of certain pharmaceutical characteristics including water/oil ratios--ranging from 60/40 to 30/70 (w/w(%))--droplet size and type of oil, i.e. non-metabolizable (mineral oil) versus metabolizable (Miglyol 840). Stability of emulsions was accomplished by the use of a polymeric emulsifier. Distinct W/O emulsions were formulated with inactivated (i) infectious bronchitis virus (iIBV) and Newcastle disease virus (iNDV), and evaluated in immunized chickens for magnitude and duration of in vivo antiviral antibody formation and local reactions. A high mineral oil content proved most effective for antibody response formation. In general, a larger droplet size evoked higher antibody responses for both oil types. Inoculum residues proved lower using biodegradable Miglyol, when compared to mineral oil, for all emulsion variants. Especially water-to-oil ratio and droplet size may provide useful parameters for improving (antiviral) antibody production by W/O emulsions.

In the theory of traditional Chinese medicine (TCM), eqi ([Symbol: see text]) circulates at the superficial portion of the body to guard against exopathogen. Gyokuheifusan (GHS; [Symbol: see text]), containing Astragalus Root, Atractylodes Rhizome, and Saposhnikovia Root, is a TCM formula to treat the insufficiency of eqi by invigorating qi and consolidating the superficial resistance. In this study, we evaluated the effect of GHS on murine antibody production against ovalbumin (OVA) used as exopathogen. Balb/c mice were sensitized with OVA and alum via intraperitoneal (i.p.) injection or intranasal (i.n.) infusion daily for 7 d. GHS was orally administered daily at the dose of 10-times amount of human daily dosage from 3 d before the sensitization for 14 d. Fourteen d after the final sensitization, the blood was collected, and the concentrations of OVA-specific or non-specific immunoglobulins were measured. When OVA was sensitized i.p., the concentration of OVA-specific IgG, IgG1, IgG2a and IgA in the sera significantly increased by GHS-treatment. When OVA was sensitized i.n., GHS significantly reduce the concentration of OVA-specific IgG and IgG1 in the sera. Non-specific immunoglobulins were not changed by GHS-treatment. It is suggested that GHS could stimulate immune responses when antigen had already been invaded into the inside of the body, and that GHS might consolidate the resistance of nasal mucosa to protect from the invasion of OVA, then OVA-specific antibodies in sera might be hypocritically suppressed. The present study might provide the experimental evidence for TCM theory.

Vaginally applied antimicrobial compounds (microbicides) are being developed as an alternative method for preventing the spread of sexually transmitted diseases. In addition to identifying compounds effective against a spectrum of sexually transmitted pathogens, it will be important to ensure that these compounds are safe. Avoiding toxicity, inflammatory responses, or alteration of the function of resident immune cells are important considerations for the development of vaginally applied microbicides. Studies were performed with two classes of candidate microbicide compounds to determine if they would interfere with the recognition of antigen by CD4(+) and CD8(+) T lymphocytes. The presence of nontoxic concentrations of the anionic detergent cholic acid or the sulfated polymer lambda carrageenan did not inhibit recognition of immune peptide by antigen-specific T cells. However, antigen recognition by both CD4(+) and CD8(+) T lymphocytes was inhibited in the presence of the naphthalene sulfonate polymer PRO 2000. Brief (4-h) exposure of antigen-presenting cells or T cells to PRO 2000 did not result in inhibition of antigen uptake and processing by antigen-presenting cells or the ability of specific T cells to respond to antigen stimulation, suggesting that the inhibition was temporary. Binding of antibodies specific for CD18, CD8, and CD3 was impaired in the presence of PRO 2000, suggesting that the mechanism by which this microbicide inhibits T cell recognition of antigenic peptide may involve masking or internalization of surface proteins involved in T cell signaling or stabilizing T cell-antigen-presenting cell interactions. The assays described in this study represent a useful means to screen candidate topical microbicide compounds for inappropriate interactions with immune cells and may be useful for prioritization of candidate microbicide compounds.

While studying the serologic characteristics of certain monoclonal blood group antibodies, we observed a hybridoma clone (5A-11E10) with anti-N-like serologic specificity that was dependent on the presence of the bicarbonate anion. The diluted cell culture supernatant preferentially agglutinated M-N+ RBCs by immediate spin. This supernatant also agglutinated M-N+ RBCs that had been treated with trypsin or sialidase (to remove N-reactivity), suggesting anti-'N' activity. Anti-'N' specificity was confirmed by the supernatant's non-reactivity with N+ RBCs treated with papain (to remove 'N' reactivity) or with ('N'-negative) M+N-U- RBCs. The requirement for bicarbonate in the MoAb's formulation was not a function of pH. Both sodium and ammonium bicarbonate supported agglutination, but neither sulfate nor carbonate was effective.