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Rationale : The overwhelming majority of radioimmunoconjugates are produced via random conjugation methods predicated on attaching bifunctional chelators to the lysines of antibodies. However, this approach inevitably produces poorly defined and heterogeneous immunoconjugates because antibodies have several lysines distributed throughout their structure. To circumvent this issue, we have previously developed a chemoenzymatic bioconjugation strategy that site-specifically appends cargoes to the biantennary heavy chain glycans attached to C H 2 domains of the immunoglobulin's Fc region. In the study at hand, we explore the effects of this approach to site-specific bioconjugation on the Fc receptor binding and in vivo behavior of radioimmunoconjugates. Methods : We synthesized three desferrioxamine (DFO)-labeled immunoconjugates based on the HER2-targeting antibody pertuzumab: one using random bioconjugation methods (DFO- nss pertuzumab) and two using variants of our chemoenzymatic protocol (DFO- ss pertuzumab-EndoS and DFO- ss pertuzumab-βGal). Subsequently, we characterized these constructs and evaluated their ability to bind HER2, human FcγRI (huFcγRI), and mouse FcγRI (muFcγRI). After radiolabeling the immunoconjugates with zirconium-89, we conducted PET imaging and biodistribution studies in two different mouse models of HER2-expressing breast cancer. Results : MALDI-ToF and SDS-PAGE analysis confirmed the site-specific nature of the bioconjugation, and flow cytometry and surface plasmon resonance (SPR) revealed that all three immunoconjugates bind HER2 as effectively as native pertuzumab. Critically, however, SPR experiments also illuminated that DFO- ss pertuzumab-EndoS possesses an attenuated binding affinity for huFcγRI (17.4 ± 0.3 nM) compared to native pertuzumab (4.7 ± 0.2 nM), DFO- nss pertuzumab (4.1 ± 0.1 nM), and DFO- ss pertuzumab-βGal (4.7 ± 0.2 nM). ImmunoPET and biodistribution experiments in athymic nude mice bearing HER2-expressing BT474 human breast cancer xenografts yielded no significant differences in the in vivo behavior of the radioimmunoconjugates. Yet experiments in tumor-bearing humanized NSG mice revealed that 89 Zr-DFO- ss pertuzumab-EndoS produces higher activity concentrations in the tumor (111.8 ± 39.9 %ID/g) and lower activity concentrations in the liver and spleen (4.7 ± 0.8 %ID/g and 13.1 ± 4.0 %ID/g, respectively) than its non-site-specifically labeled cousin, a phenomenon we believe stems from the altered binding of the former to huFcγRI. Conclusion : These data underscore that this approach to site-specific bioconjugation not only produces more homogeneous and well-defined radioimmunoconjugates than traditional methods but may also improve their in vivo performance in mouse models by reducing binding to FcγRI., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Chronic active antibody-mediated rejection (CAMR) has unsatisfactory prognosis in spite of intensive standard antihumoral treatment. Efficacy of additional bortezomib in CAMR remains uncertain., Methods: A retrospective chart review was conducted among kidney transplant patients with biopsy-proven CAMR. Our standard CAMR protocol included plasma exchange, intravenous immunoglobulin, and rituximab. Repeated treatment was provided for refractory cases. Patients receiving at least 1 course of bortezomib were enrolled as the bortezomib group. Allograft outcome was compared among patients receiving repeated standard protocol alone and the bortezomib group., Results: Thirteen and 15 patients were assigned to the bortezomib and control groups, respectively. Repeated bortezomib protocol was given for 1, 2, 3, and 4 courses in 6, 4, 1, and 2 patients, respectively. With a median follow-up time after treatment of 41.8 (18.3-47.4) months, the bortezomib group had a lower rate of glomerular filtration rate declination (-4.20 ± 4.89 mL/min/y vs -12.33 ± 10.44 mL/min/y; P = .014), a higher rate of disappearance of donor specific antibodies (69.2% vs 25%; P = .03), a lower rate of allograft loss (15.4% vs 66.7%; P = .006), and better allograft survival (P = .006)., Conclusion: In CAMR, additional bortezomib treatment was more effective in eliminating donor specific antibodies and improving allograft survival than standard protocol treatment., (Copyright © 2019 Elsevier Inc. All rights reserved.)

[Fam-] trastuzumab deruxtecan (DS-8201a) is a HER2 (ERBB2)-targeting antibody-drug conjugate, composed of a HER2-targeting antibody and a topoisomerase I inhibitor, exatecan derivative, that has antitumor effects in preclinical xenograft models and clinical trials. Recently, [fam-] trastuzumab deruxtecan was reported to enhance antitumor immunity and was beneficial in combination with an anti-PD-1 antibody in a mouse model. In this study, the antitumor effect of [fam-] trastuzumab deruxtecan in combination with an anti-CTLA-4 antibody was evaluated. [Fam-] trastuzumab deruxtecan monotherapy had antitumor activity in an immunocompetent mouse model with EMT6 human HER2-expressing mouse breast cancer cells (EMT6-hHER2). [Fam-] trastuzumab deruxtecan in combination with the anti-CTLA-4 antibody induced more potent antitumor activity than that by monotherapy with either agent. The combination therapy increased tumor-infiltrating CD4+ and CD8+ T cells in vivo. Mechanistically, cured mice with treatment of [fam-] trastuzumab deruxtecan and an anti-CTLA-4 antibody completely rejected EMT6-mock cells similar to EMT6-hHER2 cells, and splenocytes from the cured mice responded to both EMT6-hHER2 and EMT6-mock cells as measured by interferon-gamma release. Taken together, these results indicate that antitumor immunity is induced by [fam-] trastuzumab deruxtecan and is facilitated in combination with anti-CTLA-4 antibody., Competing Interests: The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: all of the authors are paid employees of Daiichi Sankyo Co., Ltd. The authors would like to declare the following patent applications associated with this research: combination of antibody-drug conjugate and immune checkpoint inhibitor; PCT/JP2017/044426 [TNI, TW]. [fam-] trastuzumab deruxtecan is in clinical development. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Aim: Sensitization to HLA antigens creates an immunologic barrier, linked to an increased risk of antibody-mediated rejection and poorer graft survival, that remains a persistent and often impenetrable deterrent to transplantation. Desensitization can improve transplantation rates in broadly sensitized kidney transplant recipients. We aimed to compare the clinical outcomes of immunologic high-risk kidney recipients who had desensitization treatment with the outcomes of those who did not., Materials and Methods: We retrospectively evaluated patients who underwent desensitization protocol due to immunologic risk between 2010 and 2018. Living-donor transplantation patients with panel reactive antibody positivity, retransplantation, donor specific antibody, and/or single antigen bead positivity were included in the study. We excluded deceased-donor transplantation recipients. Demographic data (age, sex, etiology of end-stage renal disease, blood transfusions, pregnancy, etc), immunologic status (HLA-mismatch [HLA-MM], panel reactive antibody, donor specific antibody, etc), induction and maintenance of immunosuppressive medications, and complications (all-cause hospitalizations, episodes of acute rejections, etc) were noted. We compared data and clinical outcomes of patients who had desensitization (Group 1) with data and clinical outcomes of patients who had not had desensitization (Group 2)., Findings: There were 124 living-kidney donors (49 female, mean age 43.7 ± 12.2 years, mean body mass index [BMI] 25.8 ± 5.8 kg/m 2 , mean follow-up time 20.9 ± 14.6 months). Thirty-four of these patients (25 female, mean age 43.7 ± 12.5 years, mean follow-up time 26.1 ± 17.7 months, mean BMI 27 ± 6.5 kg/m 2 ) had desensitization treatment (rituximab+plasmapheresis for 19 patients, rituximab for 11 patients, rituximab+plasmapheresis+intravenous immunoglobulin for 4 patients). Ninety patients (24 female, mean age 43.7 ± 12.2 years, mean follow-up time 18.9 ± 12.9 months, mean BMI 25.3 ± 5.4 kg/m 2 ) had not had desensitization. There was no statistical difference between groups for age, sex, hepatitis serology, history of blood transfusion, history of pregnancy, or history of dialysis (P < .05 for all parameters). While scores for HLA-MM and HLA-relative intensity scale (RIS) were 2.7 ± 1.6 and 7.86 ± 6.2, respectively, in Group 1, in Group 2 the same scores were 2.1 ± 1.1 and 3.6 ± 2.5, respectively (P: .053 and .03). Delayed graft function, acute rejection episodes, and hospitalizations were similar between groups (P: .47, .29, and .34, respectively). Follow-up time and length of hospitalization were longer in Group 1 (P: .013 and .001, respectively). Total doses of ATG were higher in Group 1 patients (P: .007)., Conclusion: Despite the higher HLA-MM and RIS scores, clinical outcomes in desensitized patients were found to be similar to those in nondesensitized patients for acute rejection episodes and hospitalizations. Desensitization with rituximab in patients with high HLA-RIS scores can prevent acute rejection and hospitalization., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are a first-line therapy for rapidly killing tumors such as those associated with non-small cell lung cancer by blocking oncogenic receptor signaling, but tumor relapse often occurs. Here, we have observed that hypofractionated EGFR TKI treatment (HypoTKI) is more potent than standard hyperfractionated EGFR TKI treatment (HyperTKI), and its antitumor effect associated with preventing tumor relapse depends on T cells. HypoTKI triggers greater innate sensing for type I IFN and CXCL10 production through the Myd88 signaling pathway to enhance tumor-specific T cell infiltration and reactivation. We also demonstrate that timely programmed cell death ligand-1 (PD-L1) blockade can synergize with HypoTKI to control advanced large tumors and effectively limit tumor relapse without severe side effects. Our study provides evidence for exploring the potential of a proper combination of EGFR TKIs and immunotherapy as a first-line treatment for treating EGFR-driven tumors., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

HLA-DRB3*01:01 is a predisposing factor for human platelet antigen 1a (HPA-1a) immunization, which is responsible for most cases of fetal and neonatal alloimmune thrombocytopenia. The aim of this study was to investigate if the HLA-DRB3*01:01 allele imposes a dose-dependent effect on anti-HPA-1a levels and neonatal platelet counts. One hundred and thirty HPA-1a-immunized women were divided into 3 groups: HLA-DRB3*01:01 negative, HLA-DRB3*01:01 hemizygous or heterozygous, and HLA-DRB3*01:01 homozygous. The dose of the HLA-DRB3*01:01 allele was determined by sequencing exon 2 of the HLA-DRB3 gene followed by HLA-DRB3 and HLA-DRB1 typing of selected samples. Anti-HPA-1a levels at time of delivery and neonatal platelet counts were compared among groups. There was a significant dose-dependent effect of the HLA-DRB3*01:01 allele on anti-HPA-1a levels (global P value [ P global ] = .0032). Median (range) anti-HPA-1a levels were 1.5 IU/mL (0.0-19.0 IU/mL), 21.1 IU/mL (0.0-1967 IU/mL), and 43.7 IU/mL (1.0-980 IU/mL) in women with 0, 1, and 2 copies of the HLA-DRB3*01:01 allele, respectively. There was also a significant, but opposite, dose-dependent effect of the mother's HLA-DRB3*01:01 allele on the platelet count of the newborn ( P global = .0155). Median (range) neonatal platelet counts were 241 × 10 9 /L (59 × 10 9 /L to 393 × 10 9 /L), 107 × 10 9 /L (4 × 10 9 /L to 387 × 10 9 /L) and 32 × 10 9 /L (4 × 10 9 /L to 352 × 10 9 /L) for newborns of mothers with 0, 1, and 2 copies of the HLA-DRB3*01:01 allele, respectively. Thus, the HLA-DRB3*01:01 allele exhibits a dose-dependent impact on maternal anti-HPA-1a levels in HPA-1a-immunized women., (© 2019 by The American Society of Hematology.)

A 2-year longitudinal study of enzyme-linked immunosorbent assay (ELISA) antibodies against Phlebotomus perniciosus and Phlebotomus papatasi (Diptera: Psychodidae) sandfly saliva was performed in 32 Beagle dogs treated preventively with an imidacloprid-permethrin topical insecticide in an endemic area in Spain. Dogs were grouped into three sandfly exposure groups according to the time of inclusion in the study. Assays analysed immunoglobulin G (IgG) against salivary gland homogenates (SGH) of both species and recombinant P. papatasi rSP32 and P. perniciosus rSP03B proteins in serum. The dogs were participating in a Leishmania infantum (Kinetoplastida: Trypanosomatidae) vaccine trial and were experimentally infected with the parasite in the second year. No dog acquired natural L. infantum infections during the first year, but most developed anti-saliva antibodies, and median log-transformed optical densities (LODs) were seasonal, mimicking those of local sandflies. This indicates that the repellent efficacy of the insecticide used is below 100%. Multi-level modelling of LODs revealed variability among dogs, autocorrelation and differences according to the salivary antigen and the dog's age. However, dog seroprevalence, estimated using pre-exposure LODs as cut-offs, was relatively low. This, and the fact that dogs did not become naturally infected with L. infantum, would support the efficacy and usefulness of this imidacloprid-permethrin topical insecticide in canine leishmaniasis control., (© 2018 The Royal Entomological Society.)

Objective: To quantitatively assess the influence of reducing agents on biological macromolecules and on the possible repair of oxidative damage., Methods: Samples (antibody, enzyme, DNA, and diluted serum) were treated with reducing agents (ammonium ferrous sulfate, ascorbic acid, potassium iodide, and sodium hyposulfite) in the experimental group and with NaCl in the control group. Enzyme-linked immunosorbent assay and quantitative PCR were used to determine the activity of antibody, enzyme, and DNA. Native gel electrophoresis (Native-PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to determine protein structure. Reducing agents that had no inhibitory effect on biological macromolecules were selected. Antibodies were treated with oxidants to caused oxidative damage and then treated with reducing agents, and the possible repair of oxidative damage was assessed., Results: Certain concentrations of ammonium ferrous sulfate resulted in significant inhibition of antibody, enzyme, DNA, and diluted serum. Certain concentrations of ascorbic acid resulted in significant inhibition of antibody. Sodium hyposulfite and potassium iodide had no effect on antibody, enzyme, DNA, and diluted serum. The OD values in group A (in which HBsAb was treated by oxidation and then a reductant) were significantly higher than those in group B (HBsAb treated by oxidation)., Conclusion: Ammonium ferrous sulfate, ascorbic acid, sodium hyposulfite, and potassium iodide had different effects on antibody, enzyme, DNA, and diluted serum. The reduction in antibody activity due to an oxidant was partially repaired by a reductant.

Chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplant reflects a complex immune response resulting in chronic damage to multiple tissues. Previous studies indicated that donor B cells and the antibodies they produce play an important role in the development of cGVHD. To understand the pathogenic role of antibodies in cGVHD, we focused our studies on posttransplant production of immunoglobulin G antibodies targeting cell surface antigens expressed in multiple cGVHD affected tissues, due to their potential functional impact on living cells in vivo. Using plate-bound cell membrane proteins as targets, we detected a significantly higher level of antibodies reactive with these membrane antigens in patients who developed cGVHD, compared with those who did not and healthy donors. Plasma-reactive antibody levels increased significantly prior to the clinical diagnosis of cGVHD and were reduced following cGVHD therapies including prednisone, interleukin-2, or extracorporeal photophoresis. Using cell-based immunoprecipitation with plasma from cGVHD patients and mass spectrometry, we identified 43 membrane proteins targeted by these antibodies. The presence of antibodies in cGVHD patients' plasma that specifically target 6 of these proteins was validated. Antibodies reactive with these 6 antigens were more frequently detected in patients with cGVHD compared with patients without cGVHD and healthy donors. These results indicate that antibodies that target membrane antigens of living cells frequently develop in cGVHD patients and further support a role for B cells and antibodies in the development of cGVHD., (© 2017 by The American Society of Hematology.)

Unwanted antibody responses significantly impact human health, and current options for treating deleterious antibody responses largely rely on broad immunosuppressants that can compromise overall immunity. A desirable alternative is to induce antigen-specific immune tolerance. We have shown that co-presentation of antigen and ligands of B cell sialic acid-binding immunoglobulin-like lectins (Siglecs) on a liposomal nanoparticle induces antigen-specific tolerance. Although Siglec-engaging tolerance-inducing antigenic liposomes (STALs) induce robust B cell tolerance in naïve mice, the full potential of STALs requires long-term tolerance induction and suppression of an ongoing immune response. We hypothesized that STALs encapsulated with rapamycin (RAPA), an immunomodulator, could improve the efficacy of STALs and potentially enable their use in the context of immunological memory. Here, we showed that formulation of STALs with RAPA produced enhanced tolerance induction in naïve mice compared to STALs without RAPA but had minimal impact on inducing tolerance in previously sensitized mice. These findings indicate that the addition of immunomodulators to STALs could be beneficial in tolerance induction and support future development of STALs for the treatment of allergy and autoimmune diseases., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Belatacept is a biologic that targets CD80/86 and prevents its interaction with CD28 and its alternative ligand, cytotoxic T lymphocyte antigen 4 (CTLA-4). Clinical experience in kidney transplantation has revealed a high incidence of rejection with belatacept, especially with intensive regimens, suggesting that blocking CTLA-4 is deleterious. We performed a head to head assessment of FR104 (n=5), a selective pegylated Fab' antibody fragment antagonist of CD28 that does not block the CTLA-4 pathway, and belatacept (n=5) in kidney allotransplantation in baboons. The biologics were supplemented with an initial 1-month treatment with low-dose tacrolimus. In cases of acute rejection, animals also received steroids. In the belatacept group, four of five recipients developed severe, steroid-resistant acute cellular rejection, whereas FR104-treated animals did not. Assessment of regulatory T cell-specific demethylated region methylation status in 1-month biopsy samples revealed a nonsignificant trend for higher regulatory T cell frequencies in FR104-treated animals. Transcriptional analysis did not reveal significant differences in Th17 cytokines but did reveal higher levels of IL-21, the main cytokine secreted by CD4 T follicular helper (Tfh) cells, in belatacept-treated animals. In vitro, FR104 controlled the proliferative response of human preexisting Tfh cells more efficiently than belatacept. In mice, selective CD28 blockade also controlled Tfh memory cell responses to KLH stimulation more efficiently than CD80/86 blockade. Our data reveal that selective CD28 blockade and belatacept exert different effects on mechanisms of renal allograft rejection, particularly at the level of Tfh cell stimulation., (Copyright © 2016 by the American Society of Nephrology.)

A novel series (13) of isoxazoline functionalized coumarins was synthesized through 1,3-dipolar cyclization of nitrile oxides with Allylated coumarins. Synthesis of effective and target selective immunostimulators through conjugation of diversely substituted isoxazolines and 7-hydroxycoumarins is the focus of the present article. The proposed synthetic scheme was observed to be highly regiospecific yielding attempted conjugates in good yield (>90%). Kinetic resolution of the racemates was carried out by employing lipase B from Candida antarctica (CALB). The synthesized compounds were screened in vitro and in vivo for their biological activities viz. toxicity and impact on splenocyte proliferation (T- and B-cell proliferation), antibody production (HA titre), delayed-type hypersensitivity reaction (DTH), T-cell subtypes (CD4 and CD8), cytokine production (IL-2, IFN-γ, and IL-4) and NO (macrophage) production. Our results establish that isoxazoline functionalized coumarins exhibit excellent immune potentiating activity especially compounds 2, 4 and 8 whose activity is more than that of Levimasole as standard. The structure activity relations are explained in light of the structural/functional aspects of tested compounds. To the best of our knowledge the presented work is first of its kind and is presaged to prove very useful for the design and synthesis of bis-heterocycle based novel, therapeutically selective and effective immunopotentiators., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Objective: To demonstrate that ephrin-B2 (the ligand of EphB2 receptor) antagonizes the pathogenic effects of patients' N-methyl-D-aspartate receptor (NMDAR) antibodies on memory and synaptic plasticity., Methods: One hundred twenty-two C57BL/6J mice infused with cerebrospinal fluid (CSF) from patients with anti-NMDAR encephalitis or controls, with or without ephrin-B2, were investigated. CSF was infused through ventricular catheters connected to subcutaneous osmotic pumps over 14 days. Memory, behavioral tasks, locomotor activity, presence of human antibodies specifically bound to hippocampal NMDAR, and antibody effects on the density of cell-surface and synaptic NMDAR and EphB2 were examined at different time points using reported techniques. Short- and long-term synaptic plasticity were determined in acute brain sections; the Schaffer collateral pathway was stimulated and the field excitatory postsynaptic potentials were recorded in the CA1 region of the hippocampus., Results: Mice infused with patients' CSF, but not control CSF, developed progressive memory deficit and depressive-like behavior along with deposits of NMDAR antibodies in the hippocampus. These findings were associated with a decrease of the density of cell-surface and synaptic NMDAR and EphB2, and marked impairment of long-term synaptic plasticity without altering short-term plasticity. Administration of ephrin-B2 prevented the pathogenic effects of the antibodies in all the investigated paradigms assessing memory, depressive-like behavior, density of cell-surface and synaptic NMDAR and EphB2, and long-term synaptic plasticity., Interpretation: Administration of ephrin-B2 prevents the pathogenic effects of anti-NMDAR encephalitis antibodies on memory and behavior, levels of cell-surface NMDAR, and synaptic plasticity. These findings reveal a strategy beyond immunotherapy to antagonize patients' antibody effects. Ann Neurol 2016;80:388-400., (© 2016 American Neurological Association.)

The nonapeptide fragment of the HLA-DR molecule, located in the exposed loop of the alpha-chain (164-172), having the VPRSGEVYT sequence, suppresses the immune response. Based on the three-dimensional structure of the HLA-DR superdimer, we designed a new cyclodimeric analog in which the two parallel peptide chains of VPRSGEVYT sequence are linked through their C-termini by spacer of (Gly5 )2 -Lys-NH2 and the N-termini are also linked by poly(ethylene glycol). The (VPRSGEVYTG5 )2 K-resin analog was synthesized using solid-phase peptide synthesis protocols. The cyclization was achieved by cross-linking the N-terminal positions of the dimeric peptide, attached to a MBHA resin, with alpha, omega-bis (acetic acid) poly(ethylene glycol), activated by esterification with pentafluorophenol. Our results demonstrate that the cyclodimerization of VPRSGEVYT results in enhanced immunosuppressive activity of the peptide. Mass spectrometry fragmentation analysis of the obtained cyclodimeric peptide is also presented. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd., (Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.)

Unlabelled: Rituximab, a monoclonal antibody against the surface antigen of B-lymphocytes CD20 is beeing used in the treatment of numerous hematological, hematooncological and autoimmune disorders. After administration of ritu-ximab, quick and almost complete depletion of B-lymphocytes with the exception of pre-B-lymphocytes and plasma cells occur. Neutropenia and low serum antibody levels in classes IgA, IgM and IgG may also develop. These changes usually persist for 6-12 months, rarely for several years. In the consequence, patients with the rituximab treatment are more prone to infections - usually of bacterial and viral origin. Concomitantly, rituximab treatment influences negatively postvaccination antibody production and therefore adequate preventive measures are necessary before the beginning of the treatment. The authors offer complex overview of actual literature, emphasize adequate education of patients as well as of healthcare providing staff and discuss the vaccination recommendation against preventable communicable diseases like influenza, pneumococcal diseases, tetanus, diphtheria and pertussis., Key Words: autoimmune disease - immunosupression - infectious complications - prevention - rituximab - vaccination.

Objective: Transcutaneous immunization (TCI) is a novel route of vaccination through application of a topical vaccine antigen on the skin. Phosphorylcholine (PC) is a structural component of a variety of pathogens and anti-PC immune responses protect mice against invasive bacterial diseases. The purpose of the study was to examine the effect of TCI using PC in BALB/c mice., Methods: TCI was performed in BALB/c mice using PC-keyhole limpet hemocyanin (KLH) plus cholera toxin (CT). Immunogenicity was evaluated by measuring PC-specific IgG and specific IgG1, IgG2a, IgM, IgA, and secretory IgA antibodies by ELISA. The concentrations of IL-4, IL-5, IL-10, IL-12, IL-13 and IFN-γ were also measured using ELISA for mouse., Results: Six months after immunization, IgG after TCI using PC plus CT was significantly higher than in controls, but this was not found for IgA. In saliva, secretory IgA antibodies decreased with a peak level at 2-3 months. IgG1 was significantly higher than IgG2 after TCI. Production of IL-4 from CD4(+) cells was significantly higher after TCI than in controls, whereas production of IFN-γ, IL-5, IL-12 and IL-13 was not detected in either group., Conclusion: These results suggest that TCI using PC plus CT with BALB/c mice is a simple approach for induction of systemic and mucosal immune responses that are shifted in the Th-2 direction., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: The use of xenogeneic chondrocytes may benefit the development of clinical tissue-engineering applications for cartilage repair. However, cartilage xenografts are slowly rejected by humoral and cellular mechanisms to which galactose α1,3-galactose (Gal) antigen and complement contribute. Accordingly, transgenic expression of human α1,2-fucosyltransferase (HT) in porcine cartilage helps to protect from the Gal-mediated immune response. Here, we aimed to assess the effect of the broadly used complement inhibitor cobra venom factor (CVF) in comparison with anti-C5 therapy in α1,3-galactosyltransferase knockout (Gal KO) mice transplanted with porcine cartilage., Methods: Gal KO mice grafted with control or HT-transgenic cartilage were left untreated or treated systemically with either anti-C5 antibody or CVF for 5 weeks. The degree of rejection was evaluated by use of histopathological analysis, and serum anti-Gal antibodies were measured in all cohorts., Results: The rejection process of control cartilage was well advanced by 5 weeks after transplantation in untreated Gal KO mice, whereas enhanced graft survival characterized by reduced cellular immune infiltrate was found in mice grafted with HT cartilage and/or treated with anti-C5. In contrast, CVF administration led to inconsistent results, with some grafts showing no improvement or even increased amounts of granulocytes. Regarding antibody titers, the anti-Gal immunoglobulin (Ig)M increased in the control transplant cohort and remained unchanged in the HT-graft recipients at 5 weeks after transplantation. Notably, a strong anti-Gal IgM response was readily detected in CVF-treated mice of both transplanted cohorts., Conclusions: CVF does not present advantages over anti-C5 therapy for preventing rejection of xenogeneic porcine cartilage., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Anti-NDMA receptor (NMDAR) encephalitis is an auto-immune condition. There is no uniformly agreed treatment strategy for the disorder in children. We report the use of intrathecal treatment with methotrexate and methylprednisolone in three children (one male, two females, age 10y, 11y, and 14y) with anti-NMDAR encephalitis, who did not respond to steroids, plasmapheresis, or rituximab. There was significant clinical improvement and stabilization of the anti-NMDAR antibody titers in cerebrospinal fluid (CSF) and blood in two patients. In the third patient, although anti-NMDAR antibody titers in CSF decreased, clinical recovery was less satisfactory. Intrathecal treatment with methotrexate and methylprednisolone seems to be a promising alternative treatment for some paediatric cases of resistant anti-NMDAR encephalitis., (© 2014 Mac Keith Press.)

Background: The metabolic syndrome is the most important risk factor for cardiovascular disease. The heat shock proteins (HSPs) are highly conserved families of proteins expressed by a number of cell types following exposure to stressful environmental conditions include several known risk factors for cardiovascular disease. Recent studies have shown the potential of constituents of saffron in the treatment of atherosclerosis. We aimed on investigating the effect of saffron on antibody titers to HSP in patients with metabolic syndrome., Methods: This was a randomized, placebo-controlled clinical trial. One-hundred and five subjects with metabolic syndrome were randomly allocated to one of the three groups: the case group received 100 mg/day saffron, the placebo control group received a capsule of placebo and a non-placebo control group received no capsule, for 12 weeks., Results: Antibodies against heat shock proteins 27, 60, 65 and 70 were determined in all patients before (week 0) and after (week 6 and 12) intervention. At 12 weeks, saffron produced a significantly decrease in AntiHSP27, 70 levels. Saffron can decrease AntiHSP27, 70 levels in patients with metabolic syndrome., Conclusions: The results of this study indicate the efficacy of saffron in the improvement of some markers of autoimmunity HSPs in patients with metabolic syndrome.

To assess inhibition mechanisms of a Phellinus igniarius (PI) extract on cancer, C57BL/6 mice were orally treated with PI extractive after or before implanting H22 (hepatocellular carcinoma ) or B16 (melanoma) cells. Mice were orally gavaged with different doses of PI for 36 days 24h after introduction of H22 or B16 cells. Mice in another group were orally treated as above daily for 42 days and implanted with H22 cells on day 7. Then the T lymphocyte, antibody, cytokine, LAK, NK cell activity in spleen, tumor cell apoptosis status and tumor inhibition in related organs, as well as the expression of iNOS and PCNA in tumor tissue were examined. The PI extract could improve animal immunity as well as inhibit cancer cell growth and metastasis with a dose-response relationship. Notably, PI's regulation with the two kinds of tumor appeared to occur in different ways, since the antibody profile and tumor metastasis demonstrated variation between animals implanted with hepatocellular carcinoma and melanoma cells.

The effect of ionizing radiation (1 Gy) on the immunological characteristics of spleen in the mice that consumed essential oils of oregano, clove bud and the mixture of lemon oil with ginger extract at low doses with drinking water for 6 months was studied. It was found that the essential oils increased the content of antibody forming lymphocyte cells in the spleen. The maximal effect in comparison with control was found for essential oil of clove bud.

Background: The role of the complement system in antibody-mediated rejection has been investigated in relation to circulating complement interacting with renal microvascular endothelium, resulting in the formation of peritubular capillary C4d. However, the possible importance of local complement synthesis is less clear. The aim of this study was to determine whether human vascular endothelium could produce C4 in response to stimulation in vitro., Methods: Human microvascular endothelial cells and glomerular endothelial cells were stimulated with endotoxins, cytokines, and human leukocyte antigen-specific antibodies. Synthesis of complement was investigated using western blotting and indirect immunofluorescence. De novo C4 synthesis was confirmed by using C4 small interfering RNA., Results: Glomerular and microvascular endothelium, both produce C3 and C4 complement protein. Complement synthesis was stimulant-specific-C3 was produced mainly after stimulation with lipopolysaccharide whereas C4 synthesis occurred on treatment with gamma interferon. Culture with human leukocyte antigen-specific antibodies resulted in a significant increase of C4 protein synthesis by both cell lines., Conclusions: We have shown for the first time that human microvascular endothelium can be stimulated to synthesize C4 in vitro. The implications of this for clinical transplantation, especially in the context of antibody-mediated rejection, its histological interpretation and as a potential target for therapy would have to be determined by further studies.

This study was undertaken to assess the effect of simvastatin in the reduction of panel-reactive antibody (PRA) levels in patients with end-stage renal disease (ESRD) awaiting renal transplantation. During the 1-year period from September 2007 to August 2008, 60 ESRD patients with PRA levels more than 30% were enrolled into the study. Each patient was given oral simvastatin 10 mg/day as the initial dose. After one month, in those patients who did not respond, the dose was increased to 20 mg/day. Again, at the end of two months, if there was no response, the dose was further raised to 40 mg/day of simvastatin. The appropriate response to the treatment was defined as serum PRA levels getting reduced to less than 30%. Among the 60 patients who were enrolled in the study, three cases were excluded due to side-effects of the drug and 57 patients remained in our study. The mean age was 42.8 ± 28.2 years (range 18-75). Among these, 34 (59.6%) were females. Before the study, the PRA levels were between 30 and 100 (mean ± SD: 60.4% ± 20%). After the administration of simvastatin, a significant decrease in PRA levels was seen in our patients (P < 0.0001). In patients who had baseline PRA less than 40%, the PRA dropped to below 30% in 69.2% of the cases with 10 mg simvastatin. In those who had baseline PRA more than 40%, a similar reduction was seen only in 15.9% of the cases. However, in those who did not respond initially to 10 mg of simvastatin, statistically significant reductions were seen after the administration of 20 mg and, if needed, 40 mg of simvastatin (P = 0.001). In conclusion, simvastatin can be a useful drug in ESRD patients to lower the PRA levels.

Rejection is one of the most frequent causes of graft loss after a kidney transplant. In this context, in the last few years the essential role of antibodies in the anti-graft immune response has become more evident. Antibody-mediated damage has been classified into four histological patterns: hyperacute rejection, caused by the presence of pre-existing donor-specific antibodies directed against HLA or non-HLA antigens; acute antibody-mediated rejection, usually due to antibodies elicited following transplantation; chronic antibody-mediated rejection, which can develop months or years after the first appearance of circulating antibodies; and Cd4 deposition without morphologic evidence of active rejection, previously described as ''accommodation''. In recent years, thanks to the development of specific desensitization protocols, it has become possible to transplant patients sensitized to donor HLA antigens. Recently, besides consolidated protocols which include immunoglobulin administration associated or not with plasmapheresis, novel approaches of therapeutic apheresis with specific removal of antibodies and bortezomib, an agent that can efficiently decrease donor-specific antibody levels, have been developed. As far as the treatment of antibody-mediated rejection is concerned, different immunosuppressive strategies have been used. These include the combination of immunoglobulin administration and plasmapheresis with or without the use of an anti-CD20 monoclonal antibody. More recently, an innovative therapy with eculizumab has proved to be very effective against acute antibody-mediated rejection. The debate regarding the cause-effect relationship between the development of an early post-transplant humoral immune response in patients with stable graft function and premature graft loss remains open to discussion. Clinical studies are underway to provide an adequate answer to this question. In conclusion, comprehension of the fundamental role of antibodies and the consolidation of desensitization techniques together with early treatment of antibody-mediated rejection remain important objectives to improve long-term allograft survival.

Purpose: High-mobility group proteins are chromatin-binding factors with key roles in nuclear homeostasis. Evidence indicates that extracellularly released high-mobility group box 1 protein (HMGB1) behaves as a cytokine, promoting inflammation and disease pathogenesis. HMGB1 release occurs during endophtalmitis or uveoretinitis., Methods: The authors investigated the presence of HMGB1 in tear fluid of patients with different inflammatory disorders of the external eye., Results: Data demonstrate that HMGB1 content is close to detection limit in tears of control subjects but highly increased (about 15-fold) in patients with conjunctivitis or blepharitis. The authors also report that 18β-glycyrrhetic acid impairs antibody recognition of HMGB1, suggesting direct binding to the protein. Accordingly, 18β-glycyrrhetic acid prevented HMGB1-dependent COX2 expression and cluster formation in primary cultures of human macrophages., Conclusion: Together, these findings suggest that HMGB1 contributes to inflammatory disorders of the external eye, and 18β-glycyrrhetic acid may scavenge the protein and inhibit its detrimental effects.

The objective of this project was to study the interaction between HR1 and HR2, the stability of the complex formed, and to characterize the antibodies produced against monomeric HR1 and HR2 peptides as well as the HR1-HR2 complex. In this work, HR1 was mimicked by peptide N36, and HR2 was mimicked by peptide C34L and its analogues C34M2, C34M3, and C34D. Whereas C34M2 and C34M3 are partially composed of D-amino acids, C34D has same sequence as C34L, but is assembled entirely of D-amino acids. Using CD analysis, SPR assays, and gel filtration chromatography, we demonstrate the physical interaction between N36 and C34L and its analogues C34M2 and C34M3, but not C34D. We show that the HR1-HR2 complex is formed rapidly (<1 min) and remains stable, as demonstrated by its inability, in contrast to each free peptide, to inhibit the formation of syncytia. To generate antibodies with predetermined specificity against the transiently exposed intermediate that corresponds to the six-helix bundle structure, purified preformed HR1-HR2 complex was used, in parallel with monomeric HR1 and HR2 peptides, as immunogens in mice. Although the produced antibodies recognize total HIV-1 envelope glycoproteins in ELISA, they are unable to neutralize HIV-1-mediated fusion at 37 °C. However, if the incubation with these antibodies is carried out at 27 °C, a temperature that allows stabilization of the transient intermediate complex, anti-peptide antibodies are able to bind their corresponding domains in HeLa cells expressing HIV-1 gp41 in co-culture with HeLa CD4-CCR5/CXCR4 during the dynamic mechanism of membrane fusion. In agreement with the latter results, these antibodies, if previously incubated for 2 h at 27 °C, are able to strongly neutralize HIV-1 entry by membrane fusion, as shown by their ability to block the formation of syncytia.

G-protein-coupled receptors are important molecular targets in drug discovery. These receptors play a pivotal role in physiological signaling pathways and are targeted by nearly 50% of currently available drugs. Mounting evidence suggests that G-protein-coupled receptors form dimers, and various studies have shown that dimerization is necessary for receptor maturation, signaling, and trafficking. However, the physiological implications of dimerization in vivo have not been well explored because detection of GPCR dimers in endogenous systems has been a challenging task. One exciting new approach to this challenge is the generation of antibodies against specific G-protein-coupled receptor dimers. Such antibodies could be used as tools for characterization of heteromer-specific function; as reagents for their purification, tissue localization, and regulation in vivo; and as probes for mapping their functional domains. In addition, such antibodies could serve as alternative ligands for G-protein-coupled receptor heteromers. Thus, heteromer-specific antibodies represent novel tools for the exploration and manipulation of G-protein-coupled receptor-dimer pharmacology., (2010 Mount Sinai School of Medicine.)

The duration of humoral immunity in patients treated with immunosuppressive drugs is poorly defined. The objective of the study was to investigate the effect of infliximab on the levels of antiviral antibodies against poliomyelitis, rubella and measles in rheumatoid arthritis (RA) patients. Fifty-two consecutive RA patients being treated with 3 mg/kg infliximab were prospectively studied. The antiviral antibody profiles for measles, rubella and three serotypes of poliomyelitis were tested on the day of the first infusion of infliximab and 6 months later. The study group comprised 36 women and 16 men (mean age 54 years, range 33-81) with a mean disease duration of 15 +/- 9 years. Forty-two (81%) patients were being treated with methotrexate and 22 (42%) were receiving prednisone. All patients had baseline protective levels of antibodies against measles and the three strains of polio, while 48 (92%) patients had protective antibodies against rubella. No significant change in the levels of antiviral antibodies was observed after 6 months of treatment with infliximab: from 3.67 at baseline to 3.87 IU/ml for measles, 169.50-197.0 IU/ml for rubella. No change was noticed for the geometric mean concentrations of antibodies against strains of poliomyelitis: 366-478 IU/ml for the Mahoney polio strain, 906-845 IU/ml for the MEF strain and 175-196 IU/ml for the Sauket strain. Patients with longstanding RA conserve long-term immunity to common viruses despite the use of immunosuppressive drugs. Levels of antiviral antibodies against measles, rubella and polio remain stable under treatment with infliximab.

We report 3 children who developed persistent antibody depletion and abnormal response to bacteriophage after rituximab treatment for autoimmune cytopenias. Whether these patients have developed immunodeficiency secondary to an underlying disease process, to rituximab, or both, is not understood. Rituximab is an efficacious drug for a number of pediatric conditions. However, some patients who receive the drug have prolonged suppression or absence of B-cell function. Families should be counseled of this possibility prior to therapy. Patients should have baseline measurement of quantitative immunoglobulins and specific antibody levels and should be monitored for long term changes in immune function after rituximab.

Haemophilia B is an X-linked disorder resulting in coagulation factor IX deficiency. Patients with severe deficiency (<1% factor IX activity) may have significant bleeding complications similar to patients with haemophilia A or factor VIII deficiency. The development of inhibitory antibodies to the missing coagulation factor is a major complication in patients with haemophilia. While the incidence of inhibitors in patients with haemophilia A is higher than that in haemophilia B, the occurrence of allergic and or anaphylactic reactions with the development of inhibitors is unique to haemophilia B patients. Since haemophilia B is a rare bleeding disorder and the incidence of inhibitors is an even rarer entity, a registry was established by Dr Indira Warrier under the auspices of the FVIII/FIX subcommittee of the International Society of Thrombosis and Haemostasis, to gather information on the occurrence and characteristics of patients with inhibitors and also the incidence of allergic and anaphylactic reactions in this group of patients. This is the first report from this registry and helps us to gather some insight on haemophilia B patients with inhibitors and complications related to inhibitor development and difficulties with immune tolerance.

The ligand-dependent transcription factor aryl hydrocarbon receptor (AhR) has been implicated in various immune functions. Our previous studies have shown that AhR activation by exposure of ovalbumin (OVA)-immunized mice to the potent ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases immunization-induced IFN-gamma production in the spleen and suppresses the production of T(h)2 cytokines and OVA-specific antibodies. In the present study, we used transgenic (Tg) mice that express a constitutively active mutant of aryl hydrocarbon receptor (CA-AhR) specifically in T-lineage cells to clarify the role of AhR activation in T cells in these reactions. The results of this study clearly demonstrated that AhR activation only in the T cells augments IFN-gamma production upon OVA immunization. By contrast, production of T(h)2 cytokines and antibodies were not significantly suppressed by CA-AhR in the T cells. These results suggest that suppression of T(h)2 cytokines and antibodies production require AhR activation not only in T cells but also in other cell types as caused by TCDD exposure. Alternatively, these results may indicate that IFN-gamma augmentation and T(h)2 cytokines and antibodies suppression depend on different ways of functions of AhR in the T cells and that CA-AhR does not replicate the suppressive effect of TCDD-activated AhR on T(h)2 cytokines and antibodies. Expression of CA-AhR in the T cells was also shown to increase the percentage of CD25(+) cells among CD4(+) cells in the thymus and spleen. Thus, studies using T-cell-specific CA-AhR Tg mice provide a way to dissect the role of AhR in individual cell types and how the AhR functions.

To examine whether grape seed proanthocyanidin extract (GSPE) which is known to act as an antioxidant has therapeutic effect on collagen-induced arthritis (CIA) in mice, an animal model of rheumatoid arthritis. Mice were treated with an intraperitoneal injection of GSPE (10, 50, or 100 mg/kg) or saline. Clinical, histological, and biochemical parameters were assessed. The effects of GSPE on osteoclastogenesis were determined by tartrate-resistant acid phosphatase (TRAP) staining of the inflamed joints and bone-marrow cells cultured with the receptor activator of nuclear factor B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Intracellular levels of hydrogen peroxide were determined using carboxy-dichlorodihydrofluorescein diacetate. GSPE treatment significantly attenuated the severity of CIA in a dose-dependent manner and reduced the histology scores for synovial inflammation, cartilage erosion, bone erosion, and the number of TRAP+ osteoclasts. GSPE treatment significantly reduced the numbers of tumor necrosis factor alpha (TNF-alpha)- or interleukin 17 (IL-17)-producing cells in the synovial tissue and the spontaneous production of TNF-alpha and IL-17 by splenocytes compared with those in the control mice. The serum levels of type-II-collagen-specific IgG2a and plasma levels of 8-isoprostane in the GSPE-treated mice were significantly lower than those in the control mice. GSPE dose-dependently suppressed osteoclastogenesis in vitro. GSPE significantly reduced hydrogen peroxide production by anti-CD3-monoclonal-antibody-stimulated CD4+ splenocytes. These results indicate that intraperitoneal injection of GSPE attenuated CIA in mice. GSPE may be useful in the treatment of rheumatoid arthritis.

Post mortem examinations of AN-1792-vaccinated humans revealed this therapy produced focal senile plaque disruption. Despite the dispersal of substantial plaque material, vaccination did not constitute even a partial eradication of brain amyloid as water soluble amyloid-beta (Abeta) 40/42 increased in the gray matter compared to sporadic Alzheimer's disease (AD) patients and total brain Abeta levels were not decreased. Significant aspects of AD pathology were unaffected by vaccination with both vascular amyloid and hyper-phosphorylated tau deposits appeared refractory to this therapy. In addition, vaccination resulted in the consequential and drastic expansion of the white matter (WM) amyloid pool to levels without precedent in sporadic AD patients. Although vaccination disrupted amyloid plaques, this therapy did not enhance long-term cognitive function or necessarily halt neurodegeneration. The intricate involvement of vascular pathology in AD evolution and the firm recalcitrance of vessel-associated amyloid to antibody-mediated disruption suggest that immunization therapies might be more effective if administered on a prophylactic basis before vascular impairment and well ahead of any clinically evident cognitive decline. Amyloid-beta is viewed as pathological based on the postmortem correlation of senile plaques with an AD diagnosis. It remains uncertain which of the various forms of this peptide is the most toxic and whether Abeta or senile plaques themselves serve any desirable or protective functions. The long-term cognitive effects of chronic immunotherapy producing a steadily accumulating and effectively permanent pool of disrupted Abeta peptides within the human brain are unknown. In addition, the side effects of such therapy provided on a chronic basis could extend far beyond the brain. Eagerly seeking new therapies, critical knowledge gaps should prompt us to take a more wholistic perspective viewing Abeta and the amyloid cascade as aspects of complex and many-faceted physiological processes that sometimes end in AD dementia.

MEP HyperCel resin, one of the Protein-A mimetic columns, is designed to bind antibodies at physiological pH and elutes the bound antibodies at mildly acidic pH. We have tested aqueous arginine solution for washing and elution of the resin. To our surprise, bound antibody and Fc-fusion protein eluted at pH 7.0 using 1M arginine solution. Various solvent additives were then examined at pH 7.0. Among the tested additives, urea and arginine were the only additives that were effective in elution. Thus, urea and arginine at low concentrations were effectively used for washing the resin. NaCl and MgCl(2) at 0.1-1M and ethanol at 5-20% were not effective. Based on these observations, it appears that protein binds to MEP resin through both polar and hydrophobic interactions with some contribution of electrostatic interaction, which can be simultaneously reduced by arginine or urea. On the other hand, Mabsorbent, another Protein-A mimetic column, appears to be more non-specific and non-selective.

Neutralising antibodies develop in 15% of interferon-beta (IFNbeta)-treated patients, causing the reduction of the clinical effects of the treatment. This is the first study that shows that switching patients from IFNbeta to glatiramer acetate (GA) in case of neutralising antibodies (NAb) positivity is effective in reducing relapse rate and in delaying the time to first relapse. In conclusion, our data suggest the use of GA in NAb-positive patients.

Frondoside A, a major triterpene glycoside from North Atlantic commercially harvested sea cucumber Cucumaria frondosa, possesses strong immunomodulatory properties in subtoxic doses. Frondoside A stimulates lysosomal activity of mouse macrophages in vivo at a maximal effective stimulatory dose of 0.2 microg per mouse and is maintained over 10 days. This glycoside also shows a 30% stimulation of lysosomal activity in mouse macrophages in vitro at concentrations of 0.1-0.38 microg/mL. Frondoside A enhances macrophage phagocytosis of the bacterium Staphylococcus aureus in vitro at a maximal effective concentration of 0.001 microg/mL. Frondoside A stimulates reactive oxygen species formation in macrophages in vitro at a maximal effective concentration of 0.001 microg/mL. Frondoside A stimulates an increase in the number of antibody plaque-forming cells (normally B-cells in spleen) in vivo with a maximal stimulatory effect at a concentration of 0.2 microg per mouse (stimulatory index, 1.86). Frondoside A has a weak effect upon immunoglobulin (Ig) M production after immunization with sheep erythrocytes in mice. Frondoside A does not stimulate Ig production in mice and does not significantly enhance the ovalbumin-stimulated IgM and IgG antibody levels in ovalbumin-immunized mice. Hence frondoside A is an immunostimulant of cell-based immunity including phagocytosis without a significant effect on amplification of humoral immune activity or adjuvant properties. Therefore, frondoside A may provide curative and/or preventive treatment options against diseases wherein a depleted immune status contributes to the pathological processes.

Background: Gene therapy strategies directed at expressing factor (F)VIII in megakaryocytes has potential advantages in the treatment of hemophilia A. Among these is that platelet (p) FVIII may be effective in the presence of circulating anti-FVIII inhibitors., Objective: We examined in a murine transgenic model whether pFVIII could correct the coagulation defect in FVIII(null) mouse in the presence of circulating inhibitors., Methods: FVIII(null) mice that were transgenic for pFVIII (pFVIII/FVIII(null)) were compared with FVIII(null) mice receiving infused FVIII in a FeCl(3) carotid injury model in the presence of anti-FVIII inhibitors., Results: After injury, pFVIII/FVIII(null) mice were significantly more resistant to circulating inhibitors than after plasma FVIII correction in both an acute and chronic models of inhibitor exposure even although in the chronic model, significant amounts of inhibitor were stored within the platelets. Furthermore, bleeding in the pFVIII mice in the presence of inhibitors was not as a result of the development of thrombocytopenia., Conclusion: In FVIII(null) mice, pFVIII provides improved, but limited, protection in the presence of inhibitors of approximately 6-fold greater Bethesda Units per mL relative to infused FVIII. Our findings differ from a recent report using a tail-clip exsanguination assay on the degree of efficacy of pFVIII in the presence of inhibitors. We propose that this difference in outcome is as a result of the sensitivity of the tail-vein exsanguination model to low levels of pFVIII.

Many autoantibodies have variable-region sequences indicating their production in an affinity-matured antibody response involving germinal centers (GC). Plasma cells from GC can be long-lived, do not express CD20 and thus should not be depleted by a therapeutic monoclonal Ab against human CD20 - Rituximab. Nevertheless, autoantibody titers often fall following Rituximab treatment. To test if this reflects exclusive production by short-lived plasma cells in extrafollicular Ab responses, we monitored, after Rituximab treatment, levels of natural Ab and Ab against extrinsic antigens that do not induce productive GC. Eleven patients with active vasculitis and anti-proteinase-3 (PR3) Ab were assessed before and during 5 months after Rituximab therapy. Blood B cells were undetectable within 2 wk, and all patients achieved clinical remission. Levels of natural Ab - isohemagglutinins and anti-phosphorylcholine Ab - and Ab levels against thymus-independent and thymus-dependent extrinsic antigens were little affected. By contrast, 5 months after Rituximab, IgG autoantibody against PR3 had fallen to a median of 22% of pretreatment values. While the kinetics of this fall do not suggest an intrinsically short lifespan of autoantibody-producing cells, the data are consistent with Rituximab causing loss of sites within inflammatory tissues that selectively sustain autoantibody-producing cells.

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases of animals and humans. Many TSEs are initiated by prion replication in the lymphoreticular system (LRS). The cellular and molecular prerequisites for prion trafficking within the LRS are not fully understood. Here we have manipulated CD40 and its ligand to investigate whether genetic or pharmacological ablation of germinal center B cells (GCBs), which migrate into and out of germinal centers, influences prion pathogenesis. In contrast to previous reports, no alteration of prion pathogenesis was detected in mice lacking CD40L and in mice treated with anti-CD40L antibodies. These results suggest that GCBs alone do not impact peripheral splenic prion transport, replication efficiency, or neuroinvasion, and point to other mechanisms affecting prion transport from lymphoreticular sites of replication to the nervous system.

Background: Mounting a humoral response to foreign proteins is a normal function of immunity, but an undesirable outcome for protein-based therapeutic biologics. "Neutralizing antibodies" (NAbs) is the term given for antibodies against a biologic that reduce its bioavailability. Biologic therapeutics used to treat patients with multiple sclerosis (MS) have been associated with the risk of seroconversion to neutralizing antibody-positive status. Therefore, it is important to understand the causes of immunogenicity, methods for assessing antibody status, and the clinical impact of NAbs, particularly in patients who appear to be nonresponsive to treatment., Review Summary: Factors such as protein structure, contaminants, administration route, and patient characteristics affect immunogenicity of biologics used to treat MS. Binding assays and bioassays can be used for the detection and quantification of NAbs to these agents. Clinical studies have demonstrated that NAbs to interferon beta therapy develop between 3 and 18 months after treatment initiation, and their presence affects treatment efficacy on clinical, imaging, and immunologic parameters. Binding antibodies to glatiramer acetate have been detected, but the clinical significance of these antibodies remains unclear. NAbs to natalizumab have also been identified and appear to at least partially abrogate the therapeutic response., Conclusion: When considering treatment options for patients with MS, the development of NAbs and their effects on clinical efficacy should be considered. Standardization of antibody assays and the development of guidelines for the management of patients with NAbs are important areas of future research.

Background: Little is known about the effects of chemotherapy on patient antibody titers to vaccine-preventable infectious diseases; thus, there is no standard protocol for revaccinating post-chemotherapy patients., Procedures: To assess losses of detectable antibody titers due to chemotherapy, we retrospectively examined antibody titers for tetanus, varicella, measles, mumps, rubella, hepatitis B, and polio in 109 pediatric sarcoma patients. We also evaluated revaccination data to determine current practices and efficacy of revaccination. We limited our sample to osteosarcoma, Ewing sarcoma, and rhabdomyosarcoma patients to control for the chemotherapy regimen patients received., Results: Patients had pre-treatment detectable antibody titer that fell within the range of healthy children's antibody titers. However, 71% of patients had post-chemotherapy negative titers for at least one infectious disease. Patients most commonly had negative titers for hepatitis B (64%). Few patients had negative titers for measles (14%), mumps (9%), rubella (4%), polio 1 (0%), polio 2 (2.9%), polio 3 (4.8%), tetanus (5%), or varicella (11%). Revaccinations most frequently administered were hepatitis B and polio., Conclusions: Our findings suggest that post-chemotherapy patients may need to be revaccinated against certain vaccine-preventable diseases including hepatitis B, tetanus, varicella, polio, measles, mumps, and rubella. Larger studies need to be performed to establish guidelines for revaccinating post-chemotherapy pediatric patients., ((c) 2007 Wiley-Liss, Inc.)

Diseases that affect the intestine may have hepatic manifestations, but the mechanisms involved in establishing hepatic disease secondarily remain poorly understood. We previously reported that IL-10 knockout (KO) mice developed severe necrotizing hepatitis following oral infection with Trichinella spiralis. In this study, we used this model of intestinal inflammation to further examine the role of IL-10 in regulating hepatic injury. Hepatic damage was induced by migrating newborn larvae. By delivering the parasite directly into the portal vein, we demonstrated that an ongoing intestinal immune response was necessary for the development of hepatitis. Intestinally derived CD4+ cells increased in the livers of IL-10 KO mice, and Ab-mediated blockade of MAdCAM-1 inhibited the accumulation of CD4+alpha(4)beta(7)+ cells in the liver. Moreover, adoptive transfer of intestinally primed CD4+ T cells from IL-10 KO mice caused hepatitis in infected immunodeficient animals. Conversely, transfer of wild-type donor cells reduced the severity of hepatic inflammation in IL-10 KO recipients, demonstrating regulatory activity. Our results revealed that IL-10 prevented migration of intestinal T cells to the liver and inhibited the development of hepatitis.

We have tested by [(35)S]-GTPgammaS binding the intrinsic activity of three full agonists (serotonin, 5-methoxytryptamine and 5-methoxy-2-methyl-N,N-dimethyltryptamine) on rat 5-HT(6) receptors cloned in HEK-293 cells, using the scintillation proximity assay. Serotonin and 5-methoxytryptamine are soluble in water, while the agonist 5-methoxy-2-methyl-N,N-dimethyltryptamine is soluble in dimethyl sulfoxide (DMSO). In [(35)S]-GTPgammaS binding 5-HT and 5-methoxytryptamine were able to increase basal binding, while 5-methoxy-2-methyl-N,N-dimethyltryptamine surprisingly showed an inverse agonist activity. So we have tested 5-HT and 5-methoxytryptamine in the presence of DMSO: in this condition the two agonists behaved as antagonists. This interfering effect of DMSO was not observed when GTP-europium filtration binding was used in place of scintillation proximity assay using [(35)S]-GTPgammaS. In addition, DMSO did not affect [(3)H]-5HT binding or cAMP accumulation in cloned HEK-293 cells expressing rat 5-HT(6) receptors. In conclusion, we demonstrated that DMSO, the most common solvent used to dissolve compounds insoluble in water, interferes with the method of scintillation proximity assay using [(35)S]-GTPgammaS. DMSO does not affect basal signal, nor the GTPgammaS binding itself, as indicated by the experiments with GTP-europium. Therefore its interfering effect is likely to occur at the binding of antibodies in the scintillation proximity assay.

In searching for binding partners of the intracellular domain of the immunoglobulin superfamily adhesion molecule CHL1, we identified the clathrin-uncoating ATPase Hsc70. CHL1 gene ablation resulted in reduced targeting of Hsc70 to the synaptic plasma membrane and synaptic vesicles, suggesting CHL1 as a synapse-targeting cue for Hsc70. CHL1 accumulates in presynaptic membranes and, in response to synapse activation, is targeted to synaptic vesicles by endocytosis. CHL1 deficiency or disruption of the CHL1/Hsc70 complex results in accumulation of abnormally high levels of clathrin-coated synaptic vesicles with a reduced ability to release clathrin. Generation of new clathrin-coated synaptic vesicles in an activity-dependent manner is inhibited when the CHL1/Hsc70 complex is disrupted, resulting in impaired uptake and release of FM dyes in synaptic boutons. Abnormalities in clathrin-dependent synaptic vesicle recycling may thus underlie brain malfunctions in humans and mice that carry mutations in the CHL1 gene.