Deficiency of activating Fcγ-receptors reduces hepatic clearance and deposition of IC and increases CIC levels in mercury-induced autoimmunity

Background

Inorganic mercury (Hg) induces a T-cell dependent, systemic autoimmune condition (HgIA) where activating Fcγ-receptors (FcγRs) are important for the induction. In this study we examined the influence of activating FcγRs on circulating levels and organ localization of immune complexes (IC) in HgIA.

Methods and Principal Findings

Mercury treated BALB/c wt mice showed a significant but modest increase of circulating IC (CIC) from day 12 until day 18 and day 35 for IgG2a- and IgG1- CIC, respectively. Mercury-treated mice lacking the trans-membrane γ-chain of activating FcγRs (FcRγ^−/−) had significantly higher CIC levels of both IgG1-CIC and IgG2a-CIC than wt mice during the treatment course. The hepatic uptake of preformed CIC was significantly more efficient in wt mice compared to FcγR^−/− mice, but also development of extrahepatic tissue IC deposits was delayed in FcRγ^−/− mice. After 35 days of Hg treatment the proportion of immune deposits, as well as the amounts was significantly reduced in vessel FcRγ^−/− mice compared to wt mice.

Conclusions

We conclude that mice lacking functional activating FcγRs respond to Hg with increased levels and altered quality of CIC compared with wt mice. Lack of functional activating FcγRs delayed the elimination of CIC, but also significantly reduced extrahepatic tissue localization of CIC.     

RNAi-mediated CD40-CD154 interruption promotes tolerance in autoimmune arthritis

Introduction

We have previously demonstrated that ex vivo inhibition of costimulatory molecules on antigen-pulsed dendritic cells (DCs) can be useful for induction of antigen-specific immune deviation and suppression of autoimmune arthritis in the collagen induced arthritis (CIA) model. The current study evaluated a practical method of immune modulation through temporary systemic inhibition of the costimulatory molecule CD40.

Methods

Mice with collagen II (CII)-induced arthritis (CIA) were administered siRNA targeting the CD40 molecule. Therapeutic effects were evaluated by clinical symptoms, histopathology, Ag-specific T cell and B cell immune responses.

Results

Systemic administration of CD40-targeting siRNA can inhibit antigen-specific T cell response to collagen II, as well as prevent pathogenesis of disease in both a pre- and post-immunization manner in the CIA model. Disease amelioration was associated with suppression of Th1 cytokines, attenuation of antibody production, and upregulation of T regulatory cells.

Conclusions

These studies support the feasibility of transient gene silencing at a systemic level as a mechanism of resetting autoreactive immunity.     

FcγRIIIa expression on monocytes in rheumatoid arthritis: role in immune-complex stimulated TNF production and non-response to methotrexate therapy

Objective

The expression of FcγRIIIa/CD16 may render monocytes targets for activation by IgG-containing immune complexes (IC). We investigated whether FcγRIIIa/CD16 was upregulated in rheumatoid arthritis (RA), associated with TNF production in response to IC-stimulation, and if this predicted response to methotrexate therapy.

Methods

FcγRIIIa/CD16 expression on CD14^low and CD14⁺⁺ monocytes was measured by flow cytometry in healthy controls and RA patients (early and long-standing disease). Intracellular TNF-staining was carried out after in vitro LPS or heat-aggregated immunoglobulin (HAG) activation. FcγRIIIa/CD16 expression pre- and post-steroid/methotrexate treatment was examined.

Results

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in long-standing RA patients compared to controls was demonstrated (p = 0.002) with intermediate levels in early-RA patients. HAG-induced TNF-production in RA patients was correlated with the percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 (p<0.001). The percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 at baseline in early DMARD-naïve RA patients was negatively correlated with DAS28-ESR improvement 14-weeks post-methotrexate therapy (p = 0.003) and was significantly increased in EULAR non-responders compared to moderate (p = 0.01) or good responders (p = 0.003). FcγRIIIa/CD16 expression was not correlated with age, presence of systemic inflammation or autoantibody titers.

Conclusion

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in RA may result in a cell that has increased responsiveness to IC-stimulation. This monocyte subset may contribute to non-response to methotrexate therapy.     

IdeS: a bacterial proteolytic enzyme with therapeutic potential

Background

IdeS, a proteinase from Streptococcus pyogenes, cleaves immunoglobulin (Ig)G antibodies with a unique degree of specificity. Pathogenic IgG antibodies constitute an important clinical problem contributing to the pathogenesis of a number of autoimmune conditions and acute transplant rejection. To be able to effectively remove such antibodies is therefore an important clinical challenge.

Methodology/Principal Findings

IdeS was found to specifically and efficiently cleave IgG in human blood in vitro (20 µg of IdeS caused a complete degradation of IgG in one ml of human whole blood in 15 minutes) and to clear IgG from the blood stream of rabbits in vivo (no IgG was detected six hours following an intravenous injection of 5 mg of IdeS) without any side effects. In a mouse model of immune thrombocytopenic purpura (ITP), polyclonal IgG antibodies against platelet surface antigens were used to induce a lethal disease. These profoundly thrombocytopenic animals were treated and cured by a single injection of IdeS.

Conclusions/Significance

Novel information is provided concerning the IgG-cleaving activity of IdeS in vitro and in vivo. The highly specific and rapid elimination of IgG in vivo, the dramatic effect in a mouse model of ITP, and the lack of side effects in the treated animals, indicate that IdeS could also be used to treat IgG-driven diseases in humans.     

Cholestatic Liver Disease after Rituximab and Adalimumab and the Possible Role of Cross-Reacting Antibodies to Fab 2 Fragments

Background

Millions of patients are treated with therapeutic monoclonal antibodies (Tmabs) for miscellaneous diseases. We investigated sera from six patients who received immune globulin, from one patient with refractory anti-neutrophil-cytoplasmic antibody (ANCA)-associated granulomatosis with polyangiitis (GPA) who developed two episodes of acute cholestatic liver disease, one after treatment with rituximab and a second after adalimumab and a healthy control group.

Methods

Three sera from the patient and six sera from patients who received immune globulin were analyzed for antibodies to rituximab and adalimumab by ELISA. Additionally, sera from the patients and from nine healthy blood donors were coated with the Fab fragment of an unrelated humanized monoclonal antibody, with human Fc proteins as well as a mouse IgG globulin.

Results

Viral serology for hepatitis A, B, C and autoantibodies specific for autoimmune liver disorders were negative. In all three sera from the patient antibodies to rituximab could be detected, but also antibodies to adalimumab were present even at time points when the patient had not yet received adalimumab, indicating cross reactivity between both substances. Testing against an unrelated human Fab fragment revealed positive results, indicating that the patient had antibodies against human Fab fragments in general. The Fc proteins were negative, and patients’ sera did also not react with mouse IgG globulins. Remarkably, 2 out of 5 patients which were treated with immune globulin had antibodies against human Fab fragments in general whereas in none of the samples from healthy controls antibodies to Fab fragment could be detected.

Conclusion

This is the first study demonstrating cholestatic liver disease induced by two different Tmabs. Cross - reacting antibodies to Fab2 fragments in general are probably involved. Further studies must show if these Fab2 antibodies in general are related with drug-induced side effects and accelerated drug clearance in patients on Tmab therapy.     

The Ratio of Treg/Th17 Cells Correlates with the Disease Activity of Primary Immune Thrombocytopenia

Background

Primary immune thrombocytopenia (ITP) is an autoimmune heterogeneous disorder that is characterized by decreased platelet count. Regulatory T (Treg) cells and T helper type 17 (Th17) cells are two subtypes of CD4⁺ T helper (Th) cells. They play opposite roles in immune tolerance and autoimmune diseases, while they share a common differentiation pathway. The imbalance of Treg/Th17 has been demonstrated in several autoimmune diseases. In this study, we aimed to investigate the ratio of the number of Treg cells to the number of Th17 cells in ITP patients and evaluate the clinical implications of the alterations in this ratio.

Methods

Thirty adult patients with newly diagnosed ITP enrolled in this study. Twelve patients had been clinically followed up for 12 months. The percentages of CD4⁺CD25^hiFoxp3⁺ Treg cells and CD3⁺CD4⁺IL-17-producing Th17 cells in these patients and healthy controls (n = 17) were longitudinally analyzed by flow cytometry.

Results

The percentage of Treg cells in ITP patients was significantly lower than that of healthy controls, and the percentage of Th17 cells increased significantly at disease onset. The ratio of Treg/Th17 correlated with the disease activity.

Conclusion

The ratio of Treg/Th17 might be relevant to the clinical diversity of ITP patients, and this Treg/Th17 ratio might have prognostic role in ITP patients.     

Expression of high-affinity IgE receptor on human peripheral blood dendritic cells in children

Background

In a mouse model of viral induced atopic disease, expression of FcεRI on dendritic cells is critical. While adult human conventional (cDC) and plasmacytoid (pDC) dendritic cells have been shown to express FcεRI, it is not known if this receptor is expressed in childhood and how its expression is governed by IgE.

Methods

Following informed consent of subjects (n = 27, aged 12–188 months), peripheral blood was stained for surface expression of CD19, ILT7, CD1c, IgE, FcεRI and analyzed by flow cytometry (cDC: CD19⁻ ILT7⁻ CD1c⁺; pDC: CD19⁻ ILT7⁺ CD1c⁻). Total and specific serum IgE levels to food and inhalant allergens were determined by ImmunoCAP, and the relationship between FcεRI expression on dendritic cells and sensitization, free IgE, cell bound IgE, and age was determined.

Results

Independent of sensitization status, FcεRI expression was noted on cDC and pDC as early as 12 months of age. Serum IgE level correlated with expression of FcεRI on cDC, but not pDC. Based on the concentration of IgE, a complex relationship was found between surface bound IgE and expression of FcεRI on cDC. pDC exhibited a linear relationship of FcεRI expression and bound IgE that was consistent through all IgE concentrations.

Conclusions

In children, FcεRI expression on cDC and pDC is modulated differently by serum and cell bound IgE. IgE governance of FcεRI expression on cDC depends upon a complex relationship. Further studies are needed to determine the functional roles of FcεRI on cDC and pDC.     

The Effectiveness of an Online Support Group for Members of the Community with Depression: A Randomised Controlled Trial

Background

Internet support groups (ISGs) are popular, particularly among people with depression, but there is little high quality evidence concerning their effectiveness.

Aim

The study aimed to evaluate the efficacy of an ISG for reducing depressive symptoms among community members when used alone and in combination with an automated Internet-based psychotherapy training program.

Method

Volunteers with elevated psychological distress were identified using a community-based screening postal survey. Participants were randomised to one of four 12-week conditions: depression Internet Support Group (ISG), automated depression Internet Training Program (ITP), combination of the two (ITP+ISG), or a control website with delayed access to e-couch at 6 months. Assessments were conducted at baseline, post-intervention, 6 and 12 months.

Results

There was no change in depressive symptoms relative to control after 3 months of exposure to the ISG. However, both the ISG alone and the combined ISG+ITP group showed significantly greater reduction in depressive symptoms at 6 and 12 months follow-up than the control group. The ITP program was effective relative to control at post-intervention but not at 6 months.

Conclusions

ISGs for depression are promising and warrant further empirical investigation.

Trial Registration

Controlled-Trials.com ISRCTN65657330     

Integrated analyses of copy number variations and gene differential expression in lung squamous-cell carcinoma

Background

Although numerous efforts have been made, the pathogenesis underlying lung squamous-cell carcinoma (SCC) remains unclear. This study aimed to identify the CNV-driven genes by an integrated analysis of both the gene differential expression and copy number variation (CNV).

Results

A higher burden of the CNVs was found in 10–50 kb length. The 16 CNV-driven genes mainly located in chr 1 and chr 3 were enriched in immune response [e.g. complement factor H (CFH) and Fc fragment of IgG, low affinity IIIa, receptor (FCGR3A)], starch and sucrose metabolism [e.g. amylase alpha 2A (AMY2A)]. Furthermore, 38 TFs were screened for the 9 CNV-driven genes and then the regulatory network was constructed, in which the GATA-binding factor 1, 2, and 3 (GATA1, GATA2, GATA3) jointly regulated the expression of TP63.

Conclusions

The above CNV-driven genes might be potential contributors to the development of lung SCC.     

Detection of Circulating B Cells Producing Anti-GPIb Autoantibodies in Patients with Immune Thrombocytopenia

Background

We previously reported that an enzyme-linked immunospot (ELISPOT) assay for detecting anti-GPIIb/IIIa antibody-secreting B cells is a sensitive method for identifying patients with immune thrombocytopenia (ITP). Here we assessed the clinical significance of measuring circulating B cells producing antibodies to GPIb, another major platelet autoantigen.

Methods

Anti-GPIb and anti-GPIIb/IIIa antibody-producing B cells were simultaneously measured using ELISPOT assays in 32 healthy controls and 226 consecutive thrombocytopenic patients, including 114 with primary ITP, 25 with systemic lupus erythematosus (SLE), 30 with liver cirrhosis, 39 with post-hematopoietic stem cell transplantation (post-HSCT), and 18 non-ITP controls (aplastic anemia and myelodysplastic syndrome).

Results

There were significantly more circulating anti-GPIb and anti-GPIIb/IIIa antibody-producing B cells in primary ITP, SLE, liver cirrhosis, and post-HSCT patients than in healthy controls (P<0.05 for all comparisons). For diagnosing primary ITP, the anti-GPIb ELISPOT assay had 43% sensitivity and 89% specificity, whereas the anti-GPIIb/IIIa ELISPOT assay had 86% sensitivity and 83% specificity. When two tests were combined, the sensitivity was slightly improved to 90% without a reduction in specificity. In primary ITP patients, the anti-GPIb antibody response was associated with a low platelet count, lack of Helicobacter pylori infection, positive anti-nuclear antibody, and poor therapeutic response to intravenous immunoglobulin.

Conclusion

The ELISPOT assay for detecting anti-GPIb antibody-secreting B cells is useful for identifying patients with ITP, but its utility for diagnosing ITP is inferior to the anti-GPIIb/IIIa ELISPOT assay. Nevertheless, detection of the anti-GPIb antibody response is useful for subtyping patients with primary ITP.     

Type II collagen antibody response is enriched in the synovial fluid of rheumatoid joints and directed to the same major epitopes as in collagen induced arthritis in primates and mice

Introduction

Antibodies towards type II collagen (CII) are detected in patients with rheumatoid arthritis (RA) and in non-human primates and rodents with collagen induced arthritis (CIA). We have previously shown that antibodies specific for several CII-epitopes are pathogenic using monoclonal antibodies from arthritic mice, although the role of different anti-CII epitopes has not been investigated in detail in other species. We therefore performed an inter-species comparative study of the autoantibody response to CII in patients with RA versus monkeys and mice with CIA.

Methods

Analysis of the full epitope repertoire along the disease course of CIA was performed using a library of CII triple-helical peptides. The antibody responses to the major CII epitopes were analyzed in sera and synovial fluid from RA patients, and in sera from rhesus monkeys (Macaca mulatta), common marmosets (Callithrix jacchus) and mice.

Results

Many CII epitopes including the major C1, U1, and J1 were associated with established CIA and arginine residues played an important role in the anti-CII antibody interactions. The major epitopes were also recognized in RA patients, both in sera and even more pronounced in synovial fluid: 77% of the patients had antibodies to the U1 epitope. The anti-CII immune response was not restricted to the anti-citrulline protein antibodies (ACPA) positive RA group.

Conclusion

CII conformational dependent antibody responses are common in RA and are likely to originate from rheumatoid joints but did not show a correlation with ACPA response. Importantly, the fine specificity of the anti-CII response is similar with CIA in monkeys and rodents where the recognized epitopes are conserved and have a major pathogenic role. Thus, anti-CII antibodies may both contribute to, as well as be the consequence of, local joint inflammation.     

Dengue virus infection-enhancing activity in serum samples with neutralizing activity as determined by using FcγR-expressing cells

Background

Progress in dengue vaccine development has been hampered by limited understanding of protective immunity against dengue virus infection. Conventional neutralizing antibody titration assays that use FcγR-negative cells do not consider possible infection-enhancement activity. We reasoned that as FcγR-expressing cells are the major target cells of dengue virus, neutralizing antibody titration assays using FcγR-expressing cells that determine the sum of neutralizing and infection-enhancing activity, may better reflect the biological properties of antibodies in vivo.

Methods and Findings

We evaluated serum samples from 80 residents of a dengue endemic country, Malaysia, for neutralizing activity, and infection-enhancing activity at 1∶10 serum dilution by using FcγR-negative BHK cells and FcγR-expressing BHK cells. The serum samples consisted of a panel of patients with acute DENV infection (31%, 25/80) and a panel of donors without acute DENV infection (69%, 55/80). A high proportion of the tested serum samples (75%, 60/80) demonstrated DENV neutralizing activity (PRNT₅₀≥10) and infection-enhancing activity. Eleven of 18 serum samples from patients with acute secondary DENV infection demonstrated neutralizing activity to the infecting serotype determined by using FcγR-negative BHK cells (PRNT₅₀≥10), but not when determined by using FcγR-expressing cells.

Conclusion

Human serum samples with low neutralizing activity determined by using FcγR-negative cells showed DENV infection-enhancing activity using FcγR-expressing cells, whereas those with high neutralizing activity determined by using FcγR-negative cells demonstrate low or no infection-enhancing activity using FcγR-expressing cells. The results suggest an inverse relationship between neutralizing antibody titer and infection-enhancing activity, and that neutralizing activity determined by using FcγR-expressing cells, and not the activity determined by using FcγR-negative cells, may better reflect protection to DENV infection in vivo.     

IgE mediates killing of intracellular Toxoplasma gondii by human macrophages through CD23-dependent, interleukin-10 sensitive pathway

Background

In addition to helminthic infections, elevated serum IgE levels were observed in many protozoal infections, while their contribution during immune response to these pathogens remained unclear. As IgE/antigen immune complexes (IgE-IC) bind to human cells through FcεRI or FcεRII/CD23 surface molecules, the present study aimed to identify which functional receptor may be involved in IgE-IC interaction with human macrophages, the major effector cell during parasite infection.

Methodology/Principal Findings

Human monocyte-derived macrophages were infected with Toxoplasma gondii before being incubated with IgE-IC. IgE receptors were then identified using appropriate blocking antibodies. The activation of cells and parasiticidal activity were evaluated by mediator quantification and direct counting of infected macrophages. RNAs were extracted and cell supernatants were also collected for their content in tumor necrosis factor (TNF)-α, interleukin-10 (IL-10) and nitrites. Sera from symptomatic infected patients were also tested for their content of IgE, IL-10 and nitrites, and compared to values found in healthy donors. Results showed that IgE-IC induced intracellular elimination of parasites by human macrophages. IgE-mediated effect was FcεRI-independent, but required cross-linking of surface FcεRII/CD23, cell activation and the generation of nitric oxide (NO). Although TNF-α was shown to be produced during cell activation, this cytokine had minor contribution in this phenomenon while endogenous and exogenous IL-10 down-regulated parasite killing. Inverse relationship was found between IL-10 and NO expression by infected human macrophages at both mRNA and mediator levels. The relationship between these in vitro data and in vivo levels of various factors in T. gondii infected patients supports the involvement of CD23 antigen and IL-10 expression in disease control.

Conclusion

Thus, IgE may be considered as immune mediator during antiprotozoal activity of human macrophages through its ability to trigger CD23 signaling. Increased cell activation by IgE-IC may also account for chronic inflammatory diseases observed in some patients.     

Statins accelerate the onset of collagen type II-induced arthritis in mice

Introduction

Statins (hydroxymethylglutaryl coenzyme A reductase inhibitors) are effective in reducing the risk of cardiovascular morbidity and mortality in patients with hyperlipidemia, hypertension, or type II diabetes. Next to their cholesterol-lowering activity, statins have immunomodulatory properties. Based on these properties, we hypothesized that statin use may eventually lead to dysregulation of immune responses, possibly resulting in autoimmunity. We have recently shown in an observational study that statin use was associated with an increased risk of developing rheumatoid arthritis. Our objective was to investigate whether a causal relationship could be established for this finding.

Methods

The mouse collagen type II (CII)-induced arthritis (CIA) model was used, with immunization, challenge, and euthanasia at days 0, 21, and 42, respectively. Statins were given orally before (day -28 until day 21) or after (day 21 until day 42) CIA induction. Atorvastatin (0.2 mg/day) or pravastatin (0.8 mg/day) was administered. Arthritis was recorded three times a week. Serum anti-CII autoantibodies and cytokines in supernatants from Concanavalin-A-stimulated lymph node cells and CII-stimulated spleen cells were measured.

Results

Statin administration accelerated arthritis onset and resulted in 100% arthritic animals, whereas only seven out of 12 nonstatin control animals developed arthritis. Atorvastatin administration after CIA induction resulted in earlier onset than atorvastatin administration before induction, or than pravastatin administration before or after induction. The arthritic score of animals given pravastatin before CIA induction was similar to that of the nonstatin controls, whereas the other groups that received statins showed higher arthritic scores. Atorvastatin administration, especially before CIA induction, increased anti-CII autoantibody production. IL-2 and IL-17 production by lymph node and spleen cells was higher in CIA animals than in PBS controls, but was not affected by statin administration. While IFNγ production was not affected by CIA induction, atorvastatin administration before CIA induction increased the production of this cytokine.

Conclusion

These data support previous results from our observational studies, indicating a role for statins in the induction of autoimmunity.     

Mapping the binding between the tetraspanin molecule (Sjc23) of Schistosoma japonicum and human non-immune IgG

Background

Schistosomal parasites can establish parasitization in a human host for decades; evasion of host immunorecognition including surface masking by acquisition of host serum components is one of the strategies explored by the parasites. Parasite molecules anchored on the membrane are the main elements in the interaction. Sjc23, a member of the tetraspanin (TSP) family of Schistosoma japonicum, was previously found to be highly immunogenic and regarded as a vaccine candidate against schistosomiasis. However, studies indicated that immunization with Sjc23 generated rapid antibody responses which were less protective than that with other antigens. The biological function of this membrane-anchored molecule has not been defined after decades of vaccination studies.

Methodology and Principal Findings

In this study, we explored affinity pull-down and peptide competition assays to investigate the potential binding between Sjc23 molecule and human non-immune IgG. We determined that Sjc23 could bind human non-immune IgG and the binding was through the interaction of the large extra-cellular domain (LED) of Sjc23 (named Sjc23-LED) with the Fc domain of human IgG. Sjc23 had no affinity to other immunoglobulin types. Affinity precipitation (pull-down assay) in the presence of overlapping peptides further pinpointed to a 9-amino acid motif within Sjc23-LED that mediated the binding to human IgG.

Conclusion and Significance

S. japonicum parasites cloak themselves through interaction with human non-immune IgG, and a member of the tetraspanin family, Sjc23, mediated the acquisition of human IgG via the interaction of a motif of 9 amino acids with the Fc domain of the IgG molecule. The consequence of this interaction will likely benefit parasitism of S. japonicum by evasion of host immune recognition or immunoresponses. This is the first report that an epitope of schistosomal ligand and its immunoglobulin receptor are defined, which provides further evidence of immune evasion strategy adopted by S. japonicum.     

High affinity allele for the gene of FCGR3A is risk factor for HIV infection and progression

Background

We investigated the genetics of Fc receptors, which function as activating receptors on immune cells and help to control HIV through antibody-mediated cellular cytotoxicity. Thus, Fc receptors may be important for virus immunity but might also promote immune hyperactivation that would enhance infection.

Methodology/Principal Findings

We measured abundance of low and high activity alleles in two Fc receptor genes, FCGR2A and FCGR3A, for persons with HIV disease, natural virus suppressors (HIV+, without disease) and healthy controls to show whether genotypes were associated with infection and disease. Individuals homozygous for the high activity allele of FCGR3A (158VV) were predominantly found among HIV progressors and this group was also skewed toward higher allele frequencies for the V158 variant. Both of the HIV positive groups (progressors and natural virus suppressors) had significantly higher frequencies of the V158 allele compared with uninfected controls. There were no apparent associations among FCGR2A alleles and HIV status.

Conclusions/Significance

Our results indicate that high activity alleles of FCGR3A may be risk factors for HIV infection or progression and we need to understand how allelic variants affect the balance between virus control and immune activation.     

PDL241, a novel humanized monoclonal antibody,reveals CD319 as a therapeutic target for rheumatoid arthritis

Introduction

Targeting the CD20 antigen has been a successful therapeutic intervention in the treatment of rheumatoid arthritis (RA). However, in some patients with an inadequate response to anti-CD20 therapy, a persistence of CD20^- plasmablasts is noted. The strong expression of CD319 on CD20^- plasmablast and plasma cell populations in RA synovium led to the investigation of the potential of CD319 as a therapeutic target.

Methods

PDL241, a novel humanized IgG₁ monoclonal antibody (mAb) to CD319, was generated and examined for its ability to inhibit immunoglobulin production from plasmablasts and plasma cells generated from peripheral blood mononuclear cells (PBMC) in the presence and absence of RA synovial fibroblasts (RA-SF). The in vivo activity of PDL241 was determined in a human PBMC transfer into NOD scid IL-2 gamma chain knockout (NSG) mouse model. Finally, the ability of PDL241 to ameliorate experimental arthritis was evaluated in a collagen-induced arthritis (CIA) model in rhesus monkeys.

Results

PDL241 bound to plasmablasts and plasma cells but not naïve B cells. Consistent with the binding profile, PDL241 inhibited the production of IgM from in vitro PBMC cultures by the depletion of CD319⁺ plasmablasts and plasma cells but not B cells. The activity of PDL241 was dependent on an intact Fc portion of the IgG₁ and mediated predominantly by natural killer cells. Inhibition of IgM production was also observed in the human PBMC transfer to NSG mouse model. Treatment of rhesus monkeys in a CIA model with PDL241 led to a significant inhibition of anti-collagen IgG and IgM antibodies. A beneficial effect on joint related parameters, including bone remodeling, histopathology, and joint swelling was also observed.

Conclusions

The activity of PDL241 in both in vitro and in vivo models highlights the potential of CD319 as a therapeutic target in RA.     

Effect of Soluble ICAM-1 on a Sj?gren's Syndrome-like Phenotype in NOD Mice Is Disease Stage Dependent

Introduction

Intercellular adhesion molecule-1 (ICAM-1) is involved in migration and co-stimulation of T and B cells. Membrane bound ICAM-1 is over expressed in the salivary glands (SG) of Sjögren''s syndrome (SS) patients and has therefore been proposed as a potential therapeutic target. To test the utility of ICAM-1 as a therapeutic target, we used local gene therapy in Non Obese Diabetic (NOD) mice to express soluble (s)ICAM-1 to compete with membrane bound ICAM-1 for binding with its receptor. Therapy was given prior to and just after the influx of immune cells into the SG.

Methods

A recombinant serotype 2 adeno associated virus (rAAV2) encoding ICAM-1/Fc was constructed and its efficacy tested in the female NOD mice after retrograde instillation in SG at eight (early treatment) and ten (late treatment) weeks of age. SG inflammation was evaluated by focus score and immunohistochemical quantification of infiltrating cell types. Serum and SG tissue were analyzed for immunoglobulins (Ig).

Results

Early treatment with ICAM-1/Fc resulted in decreased average number of inflammatory foci without changes in T and B cell composition. In contrast, late treated mice did not show any change in focus scores, but immunohistochemical staining showed an increase in the overall number of CD4+ and CD8+ T cells. Moreover, early treated mice showed decreased IgM within the SGs, whereas late treated mice had increased IgM levels, and on average higher IgG and IgA.

Conclusions

Blocking the ICAM-1/LFA-1 interaction with sICAM-1/Fc may result in worsening of a SS like phenotype when infiltrates have already formed within the SG. As a treatment for human SS, caution should be taken targeting the ICAM-1 axis since most patients are diagnosed when inflammation is clearly present within the SG.     

Effect of sclerostin-neutralising antibody on periarticular and systemic bone in a murine model of rheumatoid arthritis: a microCT study

Introduction

Patients with chronic inflammatory diseases have increased bone loss and bone fragility and are at increased risk of fracture. Although anti-resorptive drugs are effective in blocking inflammation-induced bone loss, they are less effective at rebuilding bone. We have previously shown that treatment with sclerostin antibody (Scl-AbI) builds bone and can prevent or restore bone loss in a murine model of inflammatory bowel disease. In this study, we tested the effect of Scl-AbI in a murine model of rheumatoid arthritis (the collagen-induced arthritis model, CIA). We hypothesised that sclerostin blockade can protect and restore bone both locally and systemically without affecting progression of inflammation.

Methods

CIA was induced in male DBA/1 mice, which were treated with either PBS or Scl-AbI (10 mg/kg, weekly) prophylactically for 55 days or therapeutically for 21 days (starting 14 days post onset of arthritis). Systemic inflammation was assessed by measuring the serum concentration of anti-CII IgG1, IgG2a and IgG2b by ELISA. Changes in bone mass and structure, either at sites remote from the joints or at periarticular sites, were measured using DEXA and microCT. Bone focal erosion was assessed in microCT scans of ankle and knee joints.

Results

Circulating anti-CII immunoglobulins were significantly elevated in mice with CIA and there were no significant differences in the levels of anti-CII immunoglobulins in mice treated with PBS or Scl-ABI. Prophylactic Scl-AbI treatment prevented the decrease in whole body bone mineral density (BMD) and in the bone volume fraction at axial (vertebral body) and appendicular (tibial proximal metaphysis trabecular and mid-diaphysis cortical bone) sites seen in PBS-treated CIA mice, but did not prevent the formation of focal bone erosions on the periarticular bone in the knee and ankle joints. In the therapeutic study, Scl-AbI restored BMD and bone volume fraction at all assessed sites but was unable to repair focal erosions.

Conclusions

Sclerostin blockade prevented or reversed the decrease in axial and appendicular bone mass in the murine model of rheumatoid arthritis, but did not affect systemic inflammation and was unable to prevent or repair local focal erosion.