Autoantibodies against leucine-rich glioma inactivated 1 (LGI1) are found in patients with limbic encephalitis and focal seizures. Here, we generate patient-derived monoclonal antibodies (mAbs) against LGI1. We explore their sequences and binding characteristics, plus their pathogenic potential using transfected HEK293T cells, rodent neuronal preparations, and behavioural and electrophysiological assessments in vivo after mAb injections into the rodent hippocampus. In live cell-based assays, LGI1 epitope recognition was examined with patient sera (n = 31), CSFs (n = 11), longitudinal serum samples (n = 15), and using mAbs (n = 14) generated from peripheral B cells of two patients. All sera and 9/11 CSFs bound both the leucine-rich repeat (LRR) and the epitempin repeat (EPTP) domains of LGI1, with stable ratios of LRR:EPTP antibody levels over time. By contrast, the mAbs derived from both patients recognized either the LRR or EPTP domain. mAbs against both domain specificities showed varied binding strengths, and marked genetic heterogeneity, with high mutation frequencies. LRR-specific mAbs recognized LGI1 docked to its interaction partners, ADAM22 and ADAM23, bound to rodent brain sections, and induced internalization of the LGI1-ADAM22/23 complex in both HEK293T cells and live hippocampal neurons. By contrast, few EPTP-specific mAbs bound to rodent brain sections or ADAM22/23-docked LGI1, but all inhibited the docking of LGI1 to ADAM22/23. After intrahippocampal injection, and by contrast to the LRR-directed mAbs, the EPTP-directed mAbs showed far less avid binding to brain tissue and were consistently detected in the serum. Post-injection, both domain-specific mAbs abrogated long-term potentiation induction, and LRR-directed antibodies with higher binding strengths induced memory impairment. Taken together, two largely dichotomous populations of LGI1 mAbs with distinct domain binding characteristics exist in the affinity matured peripheral autoantigen-specific memory pools of individuals, both of which have pathogenic potential. In human autoantibody-mediated diseases, the detailed characterization of patient mAbs provides a valuable method to dissect the molecular mechanisms within polyclonal populations.

Objective. We have hypothesized that the process of monocyte to macrophage differentiation may alter the inflammatory response of mononuclear phagocytes to the uptake of monosodium urate monohydrate (MSU) crystals.Methods. Eight mouse monocyte/macrophage cell lines were arranged in increasing order of differentiation, as judged by expression of the macrophage markers F4/80 and BM 8 and by phagocytic capacity. Secretion of tumor necrosis factor ␣ (TNF␣) in response to MSU was measured by enzyme-linked immunosorbent assay.Results. The panel of monocyte/macrophage cell lines revealed a close linkage between the state of differentiation and the capacity of the cells to ingest MSU crystals. TNF␣ production, however, was not linked to phagocytic ability. Peak TNF␣ levels were synthesized by cells at an intermediate state of differentiation (3.2-14.1 ng/ml), whereas mature macrophages, which efficiently phagocytosed crystals, did not secrete TNF␣. Mature cell lines produced TNF␣ when stimulated with zymosan (5.9-6.2 ng/ml), but this was abolished by coincubation with MSU crystals. Suppression of the zymosan response was not due to apoptosis or steric hindrance by MSU crystals. Culture supernatants from mature macrophages did not stimulate endothelial cell activation, in contrast to MSU-treated cells at an earlier stage of differentiation, which stimulated intercellular adhesion molecule 1 expression on sEND endothelioma cells through the release of TNF␣ (inhibited 80.6% by anti-TNF␣).Conclusion. We demonstrated that phagocytosis and TNF␣ production are distinct events in the response of mononuclear phagocytes to urate crystals, and these events can be distinguished at the level of macrophage differentiation. The noninflammatory removal of urate crystals by mature macrophages defines a new pathway that may be important in controlling the development of acute gout in patients with hyperuricemia.Acute gouty arthritis is a self-limiting inflammatory response to the intraarticular deposition of monosodium urate monohydrate (MSU) microcrystals. Previous studies investigating the effect of MSU crystals in mononuclear phagocytes have focused primarily on the ability of peripheral blood monocytes or monocytic cell lines to secrete proinflammatory cytokines such as tumor necrosis factor ␣ (TNF␣), interleukin-1 (IL-1), . Investigators in our group have further shown that TNF␣ and IL-1 released by peripheral blood monocytes can activate vascular endothelial cell expression of E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1, thereby stimulating both the recruitment of leukocytes to the site of crystal deposition and the amplification of the inflammatory response (6-8).Hyperuricemia and precipitation of inflammatory microcrystals in synovial fluid per se are not always predictive of acute synovitis, as the severity of disease Supported by grants from the British Heart Foundation and Glaxo Wellcome PLC.

Chronic inflammation leads to bone loss, and increased fracture rates have been reported in a number of human chronic inflammatory conditions. The study reported here investigates the skeletal effects of dosing a neutralizing antibody to the bone regulatory protein sclerostin in a mouse model of chronic colitis. When dosed prophylactically, an antibody to sclerostin (Scl-AbI) did not reduce the weight loss or histological changes associated with colitis but did prevent inflammation-induced bone loss. At the end of the experiment, Scl-AbI-treated animals had a significantly higher femoral BMD (+27%, p < 0.05) than control antibody (Cntrl-Ab)-treated animals. In a second experiment, treatment with Scl-AbI was delayed until colitis had developed, by which time the mechanical properties of femurs in colitic animals were significantly worse than those of healthy age-matched control mice (maximum load, -26%, p < 0.05; energy, -37%, p < 0.05; ultimate strength, -33%, p < 0.05; elastic modulus, 217%, p < 0.05). A short treatment with Scl-AbI halted bone loss and reversed the decline of both intrinsic and extrinsic mechanical properties of the femur such that, after 19 days of treatment, the bone mechanical properties in the Scl-AbI-treated animals were not significantly different from those of noncolitic age-matched controls. Serum markers of bone formation and resorption suggested that the antibody to sclerostin stimulated osteoblast activity and inhibited osteoclastmediated bone resorption.

Objective. Exposure to supraphysiologic levels of glucocorticoid drugs is known to have detrimental effects on bone formation and linear growth. Patients with sclerosteosis lack the bone regulatory protein sclerostin, have excessive bone formation, and are typically above average in height. This study was undertaken to characterize the effects of a monoclonal antibody to sclerostin (Scl-AbI) in mice exposed to dexamethasone (DEX).Methods. Young mice were concomitantly treated with DEX (or vehicle control) and Scl-AbI antibody (or isotype-matched control antibody [Ctrl-Ab]) in 2 independent studies. Linear growth, the volume and strength of the bones, and the levels of bone turnover markers were analyzed.Results. In DEX-treated mice, Scl-AbI had no significant effect on linear growth when compared to control treatment (Ctrl-Ab). However, in mice treated with DEX and Scl-ABI, a significant increase in trabecular bone at the femoral metaphysis (bone volume/total volume ؉117% versus Ctrl-Ab-treated mice) and in the width and volume of the cortical bone at the femoral diaphysis (؉24% and ؉20%, respectively, versus CtrlAb-treated mice) was noted. Scl-AbI treatment also improved mechanical strength (as assessed by 4-point bending studies) at the femoral diaphysis in DEXtreated mice (maximum load ؉60% and ultimate strength ؉47% in Scl-AbI-treated mice versus Ctrl-Abtreated mice). Elevated osteocalcin levels were not detected in DEX-treated mice that received Scl-AbI, although levels of type 5b tartrate-resistant acid phosphatase were significantly lower than those observed in mice receiving DEX and Ctrl-Ab.Conclusion. Scl-AbI treatment does not prevent the detrimental effects of DEX on linear growth, but the antibody does increase both cortical and trabecular bone and improves bone mechanical properties in DEX-treated mice.Glucocorticoid (GC)-based drugs have potent immunosuppressive and antiinflammatory properties and have assumed an important role in the treatment of many types of inflammatory and autoimmune conditions. However, drugs of this type are associated with a range of well-known side effects (1). One of the most serious problems associated with GC exposure is a deleterious effect on bone, which leads to a high proportion of patients who, after receiving long-term GC therapy, develop GC-induced osteoporosis and are susceptible to bone fractures (2). The detrimental effect of GCs on bone strength has been reported to involve many different mechanisms, including inhibition of osteoblastic bone formation, increased osteoclastic bone resorption, changes in calcium balance, and inhibition of the osteoanabolic action of sex steroids (3). More recently, it has also been proposed that GC exposure not only may cause changes to bone mass and bone architecture, but also may alter the localized material properties of bone (4).When administered to children or to growing

The pro‐inflammatory cytokine IL‐17A has been implicated in the immunopathology of inflammatory arthritis. IL‐17F bears 50% homology to IL‐17A and has recently been suggested to play a role in inflammation. We investigated the induction and cytokine profile of IL‐17F+ CD4+ T cells, and how IL‐17F may contribute to inflammation. Upon culture of healthy donor CD4+ T cells with IL‐1β, IL‐23, anti‐CD3, and anti‐CD28 mAb, both IL‐17A and IL‐17F‐expressing cells were detected. In comparison to IL‐17A+IL‐17F− CD4+ T cells, IL‐17F+IL‐17A− and IL‐17A+IL‐17F+ CD4+ T cells contained lower proportions of IL‐10‐expressing and GM‐CSF‐expressing cells and higher proportions of IFN‐γ‐expressing cells. Titration of anti‐CD28 mAb revealed that strong co‐stimulation increased IL‐17F+IL‐17A− and IL‐17A+IL‐17F+ CD4+ T cell frequencies, whereas IL‐17A+IL‐17F− CD4+ T cell frequencies decreased. This was partly mediated via an IL‐2‐dependent mechanism. Addition of IL‐17A, IL‐17F, and TNF‐α to synovial fibroblasts from patients with inflammatory arthritis resulted in significant production of IL‐6 and IL‐8, which was reduced to a larger extent by combined blockade of IL‐17A and IL‐17F than blockade of IL‐17A alone. Our data indicate that IL‐17A and IL‐17F are differentially regulated upon T cell co‐stimulation, and that dual blockade of IL‐17A and IL‐17F reduces inflammation more effectively than IL‐17A blockade alone.