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A small proportion of multiple sclerosis (MS) patients develop new disease activity soon after starting anti-CD20 therapy. This activity does not recur with further dosing, possibly reflecting deeper depletion of CD20-expressing cells with repeat infusions. We assessed cellular immune profiles and their association with transient disease activity following anti-CD20 initiation as a window into relapsing disease biology. Peripheral blood mononuclear cells from independent discovery and validation cohorts of MS patients initiating ocrelizumab were assessed for phenotypic and functional profiles using multiparametric flow cytometry. Pretreatment CD20-expressing T cells, especially CD20 dim CD8 + T cells with a highly inflammatory and central nervous system (CNS)-homing phenotype, were significantly inversely correlated with pretreatment MRI gadolinium-lesion counts, and also predictive of early disease activity observed after anti-CD20 initiation. Direct removal of pretreatment proinflammatory CD20 dim CD8 + T cells had a greater contribution to treatment-associated changes in the CD8 + T cell pool than was the case for CD4 + T cells. Early disease activity following anti-CD20 initiation was not associated with reconstituting CD20 dim CD8 + T cells, which were less proinflammatory compared with pretreatment. Similarly, this disease activity did not correlate with early reconstituting B cells, which were predominantly transitional CD19 + CD24 high CD38 high with a more anti-inflammatory profile. We provide insights into the mode-of-action of anti-CD20 and highlight a potential role for CD20 dim CD8 + T cells in MS relapse biology; their strong inverse correlation with both pretreatment and early posttreatment disease activity suggests that CD20-expressing CD8 + T cells leaving the circulation (possibly to the CNS) play a particularly early role in the immune cascades involved in relapse development.

AimsOcrelizumab is a humanized monoclonal antibody that selectively targets CD20-positive B cells and is indicated for treatment of patients with relapsing forms of multiple sclerosis (RMS) or primary progressive multiple sclerosis (PPMS). The pharmacokinetics and pharmacodynamics of ocrelizumab in patients with RMS or PPMS were assessed.MethodsA population pharmacokinetic model was developed based on data from the Phase II study and the Phase III studies OPERA I and OPERA II in patients with RMS. Data from the ORATORIO Phase III study in patients with PPMS became available after model finalization and was used for external model evaluation.ResultsThe ocrelizumab serum concentration vs time course was accurately described by a 2-compartment model with time-dependent clearance. Body weight was found to be the main covariate. The area under the concentration-time curve over the dosing interval was estimated to be 26% higher for patients with RMS weighing 90kg when compared with the 60-90kg group. The terminal half-life of ocrelizumab was estimated as 26days. The extent of B-cell depletion in blood, as the pharmacodynamic marker, was greater with increasing ocrelizumab exposure.ConclusionThe pharmacokinetics of ocrelizumab was described with pharmacokinetic parameters typical for an immunoglobulin G1 monoclonal antibody, with body weight as the main covariate. The pharmacokinetics and B-cell depletion in blood were comparable across the RMS and PPMS trials, and the extent of blood B-cell depletion was greater with higher exposure.

Background: Neurofilament light (NfL) chain is an established cerebrospinal fluid (CSF) biomarker for neuroaxonal injury. The highly sensitive Quanterix Simoa™ platform is evaluated for NfL measurement in both CSF and blood. There is a need to link historical ELISA data that use bovine NfL to that of Simoa using a recombinant human (rhuman) NfL standard. Results/Methodology: The Simoa NF-light® Advantage Kit was validated for CSF and qualified for serum and plasma, using both rhuman and bovine NfL calibrators. Matched CSF, serum and plasma samples from 112 multiple sclerosis patients were analyzed using both calibrators. Conclusion: In multiple sclerosis, there is a good correlation between blood and CSF NfL levels. A conversion factor of approximately 5:1 was established between bovine and rhuman NfL calibrators.

Interleukin-22 (IL-22) plays a role in epithelial barrier function and repair, and may provide benefits in conditions like inflammatory bowel disease. However, limited human data are available to assess the clinical effect of IL-22 administration. This study used a human intestinal cell line to identify an IL-22-dependent gene signature that could serve as a pharmacodynamic biomarker for IL-22 therapy. The response to IL-22Fc (UTTR1147A, an Fc-stabilized version of IL-22) was assessed in HT-29 cells by microarray, and the selected responsive genes were confirmed by qPCR. HT-29 cells demonstrated dose-dependent increases in STAT3 phosphorylation and multiple gene expression changes in response to UTTR1147A. Genes were selected that were upregulated by UTTR1147A, but to a lesser extent by IL-6, which also signals via STAT3. IL-1R1 was highly upregulated by UTTR1147A, and differential gene expression patterns were observed in response to IL-22Fc in the presence of IL-1β. An IL-22-dependent gene signature was identified that could serve as a pharmacodynamic biomarker in intestinal biopsies to support the clinical development of an IL-22 therapeutic. The differential gene expression pattern in the presence of IL-1β suggests that an inflammatory cytokine milieu in the disease setting could influence the clinical responses to IL-22.

We appreciate the comment on our article1 by Dr. Hahn, who asked about the vaccination sequence of Pneumovax 23 (PPSV23) followed by Prevnar (PCV13), as used in the OCR1 subgroup of the VELOCE study. The goal of VELOCE was to evaluate, in a research setting, the ability of MS patients—who were B-cell depleted after ocrelizumab treatment—to raise antibody (IgG) responses to a range of vaccines that act through different immune response pathways. VELOCE used Pneumovax (23-PPV), in all patients, to assess responses to the unconjugated S. pneumoniae capsular polysaccharide chains, described to reflect an immune response pathway that is T-cell independent (i.e., mainly dependent on the presence of B cells). The study then explored, in the OCR1 subgroup, the potential boosting effect of Prevnar (13-PCV), a polysaccharide/protein conjugate vaccine that acts through a T-cell dependent response pathway. The VELOCE design was not meant to suggest a particular order or timeframe for clinical vaccine use. We recommend that for clinical use, readers follow their country's most current vaccination guidelines, such as the recently updated CDC recommendations for the use of pneumococcal vaccines in the United States: [cdc.gov/vaccines/vpd/pneumo/hcp/recommendations.html][1]. [1]: https://www.cdc.gov/vaccines/vpd/pneumo/hcp/recommendations.html

ObjectiveThe phase IIIb A Study to Evaluate the Effects of Ocrelizumab on Immune Responses in Participants With Relapsing Forms of Multiple Sclerosis (VELOCE) study (NCT02545868) assessed responses to selected vaccines in ocrelizumab (OCR)-treated patients with relapsing multiple sclerosis.MethodsPatients were randomized 2:1 into the OCR group (n = 68; OCR 600 mg) or control group (n = 34; interferon beta or no disease-modifying therapy). All received tetanus toxoid (TT)-containing vaccine, Pneumovax (23-valent pneumococcal polysaccharide vaccine [23-PPV]), and keyhole limpet hemocyanin (KLH). The OCR group was subdivided into OCR1 (n = 33) and OCR2 (n = 35) at randomization. The OCR1 group received Prevnar (13-valent conjugate pneumococcal vaccine) 4 weeks after 23-PPV; the OCR2 and control groups received influenza vaccine. Vaccinations started 12 weeks after OCR initiation (OCR group) or on day 1 (control group).ResultsPositive response rate to TT vaccine at 8 weeks was 23.9% in the OCR vs 54.5% in the control group. Positive response rate to ≥5 serotypes in 23-PPV at 4 weeks was 71.6% in the OCR and 100% in the control group. Prevnar did not enhance response to pneumococcal serotypes in common with Pneumovax. Humoral response to KLH was decreased in the OCR vs control group. Seroprotection rates at 4 weeks against 5 influenza strains ranged from 55.6% to 80.0% in the OCR2 group and 75.0% to 97.0% in the control group.ConclusionPeripherally B-cell–depleted OCR recipients mounted attenuated humoral responses to clinically relevant vaccines and the neoantigen KLH, suggesting that use of standard nonlive vaccines while on OCR treatment remains a consideration. For seasonal influenza vaccines, it is recommended to vaccinate patients on OCR because a potentially protective humoral response, even if attenuated, can be expected.Classification of evidenceThis study provides Class II evidence confirming that the humoral response to nonlive vaccines in patients with relapsing multiple sclerosis after OCR treatment is attenuated compared with untreated or interferon beta–treated patients, but they can still be expected to be protective.Clinicaltrials.gov identifierNCT02545868.

IL-17 has been implicated in the pathogenesis of multiple sclerosis (MS). Here, we show that blockade of IL-17A, but not IL-17F, attenuated experimental autoimmune encephalomyelitis (EAE). We further show that IL-17A levels were elevated in the CSF of relapsing-remitting MS (RRMS) patients and that they correlated with the CSF/serum albumin quotient (Qalb), a measure of blood-brain barrier (BBB) dysfunction. We then demonstrated that the combination of IL-17A and IL-6 reduced the expression of tight junction (TJ)-associated genes and disrupted monolayer integrity in the BBB cell line hCMEC/D3. However, unlike IL-17A, IL-6 in the CSF from RRMS patients did not correlate with Qalb. These data highlight the potential importance of targeting IL-17A in preserving BBB integrity in RRMS.

Although Interleukin‐22 (IL‐22) is produced by various leukocytes, it preferentially targets cells with epithelial origins. IL‐22 exerts essential roles in modulating various tissue epithelial functions, such as innate host defense against extracellular pathogens, barrier integrity, regeneration, and wound healing. Therefore, IL‐22 is thought to have therapeutic potential in treating diseases associated with infection, tissue injury or chronic tissue damage. A number of in vitro and in vivo nonclinical studies were conducted to characterize the pharmacological activity and safety parameters of UTTR1147A, an IL‐22 recombinant fusion protein that links the human cytokine IL‐22 with the Fc portion of a human immunoglobulin. To assess the pharmacological activity of UTTR1147A, STAT3 activation was evaluated in primary hepatocytes isolated from human, cynomolgus monkey, minipig, rat, and mouse after incubation with UTTR1147A. UTTR1147A activated STAT3 in all species evaluated, demonstrating that all were appropriate nonclinical species for toxicology studies. The nonclinical safety profile of UTTR1147A was evaluated in rats, minipigs, and cynomolgus monkeys to establish a safe clinical starting dose for humans in Phase I trials and to support clinical intravenous, subcutaneous and/or topical administration treatment regimen. Results demonstrate the cross‐species translatability of the biological response in activating the IL‐22 pathway as well as the translatability of findings from in vitro to in vivo systems. UTTR1147A was well tolerated in all species tested and induced the expected pharmacologic effects of epidermal hyperplasia and a transient increase in on‐target acute phase proteins. These effects were all considered to be clinically predictable, manageable, monitorable, and reversible.

Most treatments for epithelial injury target hematopoietic mechanisms, possibly causing immunosuppression. Interleukin (IL)-22 promotes tissue regeneration, acting directly on epithelial cells. UTTR1147A, a human IL-22Fc (immunoglobulin G (IgG)4) fusion protein, activates IL-22 signaling. This phase I placebo-controlled trial of single, ascending, i.v. (1-120 μg/kg) and s.c (3-120 μg/kg) doses of UTTR1147A analyzed its effects on safety, tolerability, pharmacokinetics, and pharmacodynamic biomarkers in healthy volunteers. Most adverse events (AEs) were mild or moderate. The maximum tolerated i.v. dose in healthy volunteers was 90 μg/kg. Predominant AEs were dose-dependent reversible skin effects consistent with IL-22 pharmacology. UTTR1147A exposure increased approximately dose-proportionally, with a half-life of ~1 week. IL-22 biomarkers (regenerating islet protein 3A (REG3A), serum amyloid A (SAA), and C-reactive protein (CRP)) increased dose-dependently. Neither inflammatory symptoms and signs nor cytokines increased with CRP elevations. UTTR1147A demonstrated acceptable safety, pharmacokinetics, and IL-22R engagement, supporting further clinical development.

Interleukin (IL)-22 plays protective roles in infections and in inflammatory diseases that have been linked to its meditation of innate immunity via multiple mechanisms. IL-22 binds specifically to its heterodimeric receptor, which is expressed on a variety of epithelial tissues. UTTR1147A is a recombinant fusion protein that links the human cytokine IL-22 with the Fc portion of human immunoglobulin (Ig) G4. Here, we report extensive in vitro and in vivo nonclinical studies that were conducted to characterize the pharmacological activity of UTTR1147A. The in vitro activity and potency of UTTR1147A were analyzed using primary human hepatocytes and human colonic epithelial cell lines. Assessment of in vivo efficacy was performed in a mouse colitis model and by measuring relevant pharmacodynamic biomarkers, including antimicrobial peptides REG3A/β, serum amyloid protein A (SAA) and lipopolysaccharide binding protein (LBP). The pharmacokinetic and pharmacodynamic characteristics of UTTR1147A were assessed in healthy mice, rats and cynomolgus monkeys. UTTR1147A induced STAT3 activation through binding to IL-22 receptor expressed in primary human hepatocytes and human colon cell lines. In both, activation occurred in a concentration-dependent manner with similar potencies. In the mouse colitis model, murine IL-22Fc- (muIL-22Fc) treated groups at doses of 1.25 μg and above had statistically lower average histologic colitis scores compared to the control treated group. Administration of muIL-22Fc or UTTR1147A was associated with a dose-dependent induction of PD markers REG3β and SAA in rodents as well as REG3A, SAA and LBP in cynomolgus monkeys. The combined data confirm pharmacological activity of IL-22Fc and support potential regenerative and protective mechanisms in epithelial tissues.

GDC-0853 is a small molecule inhibitor of Bruton's tyrosine kinase (BTK) that is highly selective and noncovalent, leading to reversible binding. In double-blind, randomized, and placebo-controlled phase I healthy volunteer studies, GDC-0853 was well tolerated, with no dose-limiting adverse events (AEs) or serious AEs. The maximum tolerated dose was not reached during dose escalation (≤600 mg, single ascending dose (SAD) study; ≤250 mg twice daily (b.i.d.) and ≤500 mg once daily, 14-day multiple ascending dose (MAD) study). Plasma concentrations peaked 1-3 hours after oral administration and declined thereafter, with a steady-state half-life ranging from 4.2-9.9 hours. Independent assays demonstrated dose-dependent BTK target engagement. Based on pharmacokinetic/pharmacodynamic (PK/PD) simulations, a once-daily dosing regimen (e.g., 100 mg, q.d.) is expected to maintain a high level of BTK inhibition over the dosing interval. Taken together, the safety and PK/PD data support GDC-0853 evaluation in rheumatoid arthritis, lupus, and other autoimmune or inflammatory indications.

Background and aims Lupus nephritis (LN) is a clinical manifestation of systemic lupus erythematosus (SLE) associated with significant morbidity and mortality. Although proteinuria is highly correlated with disease progression in LN, the composition of the LN urinary proteome remains poorly characterised. To address this issue, complementary mass spectrometry (MS)-based approaches were used to identify candidate urinary biomarkers and a targeted proteomics panel was developed to further assess levels in LN samples. Methods LN urine samples were profiled using three MS-based methods: 2D SDS-PAGE, chemical labelling using isobaric mass tags, and data-independent acquisition (DIA). A quantitative, multiple reaction monitoring method was developed to further evaluate levels of these candidate peptide biomarkers in a larger cohort. Results Using these discovery proteomic approaches>2600 proteins were identified, 290 of which are up-regulated in LN samples. While chemical labelling enabled identification of more total proteins, DIA outperformed chemical labelling in identification of proteins significantly up-regulated in LN samples. Further evaluation of a selected panel revealed increases in the majority of candidate peptide biomarkers in LN samples compared to healthy controls, including peptides from proteins involved in inflammation and adaptive immunity. Conclusions These results indicate that peptides from proteins involved in inflammation and adaptive immunity can be quantified in urine of LN patients using a multiplexed MS-based method. Results from this study will be used to inform longitudinal and interventional studies focused on understanding the biological implications of these candidate biomarkers and to direct development of novel tools to evaluate disease progression and treatment efficacy of current and future LN therapeutics.

Tumor necrosis factor (TNF) and, possibly, lymphotoxin alpha (LTα) signaling contribute to inflammation and rheumatoid arthritis (RA) pathogenesis. Pateclizumab (anti-lymphotoxin- alpha; MLTA3698A) is a humanized monoclonal antibody that blocks and depletes anti-LTα. This phase 2, randomized, head-to-head, active- and placebo-controlled trial examined the safety and efficacy of pateclizumab compared to adalimumab in RA patients with an inadequate response to disease-modifying antirheumatic drugs (DMARD-IR). Patients (n = 214) with active RA (-6 swollen and tender joints, C-reactive protein -10 mg/L) on oral DMARDs were randomized (2:2:1) to receive pateclizumab 360 mg, adalimumab 40 mg, or placebo subcutaneously every 2 weeks. The primary endpoint, 4-variable, 28-joint disease activity score erythrocyte sedimentation rate (DAS28(4)-ESR) response, was evaluated at 12 weeks using an analysis of covariance (ANCOVA) model with adjustments for concomitant DMARD use and geographic region. Secondary efficacy endpoints included American College of Rheumatology (ACR) 20, ACR50, and ACR70 responses at Day 85. Pharmacokinetics, pharmacodynamics, and immunogenicity of pateclizumab were assessed. Pateclizumab reduced the DAS28(4)-ESR response (-1.89) at 12 weeks, however, this did not reach statistical significance compared to placebo (-1.54), while adalimumab (-2.52) differed significantly from both placebo and pateclizumab. Pateclizumab 12-week ACR20, ACR50 and ACR70 response rates (64%, 33%, and 14%) suggested clinical activity but were not statistically significant compared to placebo rates (46%, 24%, and 8%, respectively). CXCL13 serum levels decreased significantly following pateclizumab and adalimumab administration, demonstrating pharmacological target engagement by both drugs. Overall, adverse events (AEs) were comparable among all cohorts. Infections were the most common AE, occurring with comparable frequency in all groups. Serious AEs occurred in 0% of pateclizumab, 5.9% of adalimumab, and 2.3% of placebo patients, with serious infection in 2.3% of adalimumab patients and none in pateclizumab and placebo patients. Pateclizumab had a good safety profile in patients inadequately responsive to DMARDs, but no statistically significant improvement in RA signs and symptoms after 12 weeks of treatment. Adalimumab demonstrated efficacy and safety comparable to published results in this head-to-head comparison in DMARD-IR RA patients. ClinicalTrials.gov NCT01225393 NCT01225393 , Registered 18 October 2010.

The central nucleus of the amygdala (CeA) is an important site for the reinforcing effects of ethanol and has been implicated in the development of alcohol dependence. The CeA GABAA receptor system is particularly vulnerable to the effects of acute and chronic ethanol exposure. Previous work in the CeA focused on ethanol and phasic GABAA receptor signaling, but tonic GABAA receptor signaling in the rat CeA remains understudied. In the present study, we found that the CeA contains two types of tonic conductance that are expressed in a cell-type-specific manner. Low threshold bursting (LTB) and some regular spiking (RS) neurons have an ongoing tonic conductance that is mediated by the α1-GABAA receptor subunit and is insensitive to acute ethanol exposure. Late spiking (LS) and a separate population of RS neurons do not display a persistent tonic conductance but have the potential for tonic signaling that is mediated by the δ-GABAA receptor subunit and can be activated by increasing the ambient GABA concentration or by acute ethanol exposure. Acute ethanol exposure differentially alters the firing discharge of different CeA cell types. Chronic ethanol exposure produces a switch in tonic signaling such that the tonic conductance in LTB and some RS neurons is lost and an ongoing tonic conductance is present in LS and a separate population of RS neurons. Collectively, these data demonstrate cell-type-specific tonic signaling in the CeA and provide new insight into how acute and chronic ethanol exposure differentially alter specific aspects of inhibitory circuitry in the CeA.

Premature birth, the leading cause of perinatal morbidity and mortality in the United States affects between 8% and 12% of all live births. Approximately 80% of premature births are proceeded by premature labor. Significant progress has been made in the physical treatment of premature labor; however, the psychological cost to women being treated needs further study. My purpose in conducting this study was to assess the stress experienced by women treated at home for premature labor and to examine methods used by the women to cope. The theoretical framework was provided by Lazarus's model of Stress, Coping, and Emotion. A total of 50 women participated. Twenty responded to a questionnaire that included the Perceived Stress Scale (PSS) and the Ways of Coping Questionnaire only, 10 were interviewed only, and 20 completed the questionnaire and the interview.The women reported experiencing a moderate amount of stress. Their mean stress score was 26.75, just below the mean for the instrument (M = 28) and slightly less than the stress score of 27.52 reported by women experiencing a "normal" pregnancy. Appraisal was assessed by analyzing the data from the interviews. The women appraised their situation as both threatening and challenging. They described their emotional response most frequently as frustration because of fear concerning the pregnancy outcome, loss of control over their life, and inability to perform their usual roles of mother, wife, and worker. The women reported on the Ways of Coping Questionnaire that they used strategies from three subscales to cope with the premature labor. Those subscales were Seeking Social Support, Planful Problem-Solving, and Positive Reappraisal.

Background Lupus nephritis (LN) is a severe clinical manifestation of systemic lupus erythematosus (SLE) associated with significant morbidity and mortality. Assessment of severity and activity of renal involvement in SLE requires a kidney biopsy, an invasive procedure with limited prognostic value. Despite years of research, a need remains for proximal, non-invasive biomarkers to help inform treatment decisions and to monitor disease activity and progression. Recent retrospective studies have revealed that baseline urine protein-creatinine ratio (UPCR) and reduction in UPCR during induction therapy are significantly associated with renal outcome 1 . Although proteinuria is highly correlated with disease progression in LN, the composition of the urinary proteome of LN patients remains poorly characterized. Objectives To better characterize the urinary LN proteome, we performed a preliminary proteomics study comprised of three complimentary discovery proteomic methods to identify urinary biomarkers of LN. Methods Urine samples from three female patients with biopsy confirmed LN, high proteinuria (>3 mg/mmol, and low serum complement C3 ( Results Using these combined approaches >2600 proteins were identified, 290 of which are up-regulated >2-fold in LN samples compared to healthy controls. While the chemical labeling approach enabled identification of more total proteins (2,598 with chemical labeling vs. 919 with DIA, 1% False Discovery Rate), the DIA approach outperformed the chemical labeling approach in identification of proteins significantly up-regulated in LN samples (52 with chemical labeling vs. 173 with DIA). Conclusions These results suggest that DIA-based approaches are less biased towards high abundance analytes and therefore potentially more suitable for proteomic profiling of biological matrices with a broad dynamic range like urine. Furthermore, candidate biomarkers identified using the DIA method are easily adapted into a targeted, multiplexed mass spectrometry assay suitable for absolute quantitation of candidate biomarkers in a clinical trial. Results from this study will be used to inform longitudinal and interventional studies focused on understanding the biological implications of these candidate biomarkers and to direct development of novel tools to evaluate disease progression and treatment efficacy of current and future LN therapeutics. References Dall9Era et al. Lupus Sci Med. 2015. Disclosure of Interest None declared

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine family of cytokines that mediate leukocyte chemotaxis. The potent and specific activation of monocytes by MCP-1 may mediate the monocytic infiltration of tissues in atherosclerosis and other inflammatory diseases. We have isolated cDNAs that encode two MCP-1-specific receptors with alternatively spliced carboxyl tails. Expression of the receptors in Xenopus oocytes conferred robust mobilization of intracellular calcium in response to nanomolar concentrations of MCP-1 but not to related chemokines. The MCP-1 receptors are most closely related to the receptor for the chemokines macrophage inflammatory protein 1 alpha and RANTES (regulated on activation, normal T expressed and secreted). The identification of the MCP-1 receptor and cloning of two distinct isoforms provide powerful tools for understanding the specificity and signaling mechanisms of this important chemokine.

A method to facilitate exploratory or discovery-oriented psychotherapy process research is presented. The personal and professional contexts out of which the need for this new approach to psychotherapy process research arose are described. Several changes in scientific values and beliefs that have occurred as a consequence of the development and use of this methodology are noted.

s an evening supervisor, I was recently paged by an emergency department nurse. "I have a teenage boy with a broken toe," she told me. "But that's not the problem. What concerns me is the Ilizarov device on his right femur. Can you tell me how to handle it?" Another evening, the recovery room nurse paged me with a similar request: The Ilizarov device on the right tibia of her 12-year-old appendectomy patient was the first one she'd ever seen, and she had several questions about its purpose and care. The Ilizarov external fixator is a circular, stainless steel device of wires, rings, and bolts designed to correct bony or soft tissue defects and deformities. Although the Ilizarov can immobilize fractures, it's more commonly used to lengthen bone shortened by a congenital defect, trauma, or nonunion of a fracture. The apparatus weighs about 3.6 kg (8 lb) when assembled and can be worn by patients of all ages, though the majority are seven to 17 years old. Developed in 1951 by Dr. G. Ilizarov of Siberia, the Ilizarov technique was slow to move west. By 1991, however, several hundred American surgeons had been trained to perform the procedure. Today, there are approximately 400 centers in the United States where an Ilizarov device may be applied and at least 1,500 surgeons trained to do the procedure.

Background Pateclizumab (formerly MLTA3698A), a novel humanized IgG1 antibody against soluble and membrane isoforms of lymphotoxin-alpha, is being investigated as a targeted therapy for rheumatoid arthritis (RA). Objectives To compare the efficacy and safety of subcutaneous Pateclizumab (PTZ) vs placebo (PBO) and adalimumab (ADA) in patients with active RA and an inadequate response to DMARDs. Methods RA patients on oral DMARDs were randomized (2:2:1) to receive PTZ 360 mg, ADA 40 mg, or PBO every 2 weeks. The primary endpoint, DAS28-ESR, was evaluated at 12 weeks. Pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of PTZ were assessed. Results 217 patients were enrolled, 85 received PTZ, 85 received ADA, and 44 received PBO. Baseline demographics [mean (±SD)], age 50 years (±13.3), disease duration 8.7 yrs (±8.1), and clinical characteristics, were balanced across treatment arms. Mean baseline characteristics included a SJC of 18.6/17.4/15.8, TJC of 28.6/28.1/23.2, HAQ scores of 1.8/1.7/1.8, CRP values of 2.2/2.2/2.4 mg/dL, and DAS28-ESR scores of 6.95/6.82/6.79, for PTZ/ADA/PBO, respectively. RF/anti-CCP status and use of methotrexate or leflunomide as DMARD agent were comparable across groups at baseline. 86% of PTZ, 87% of ADA, and 77% of PBO subjects completed 10 weeks of dosing for the efficacy analyses. PTZ 12wk DAS28-ESR response (-1.96) was not significantly different from PBO (-1.66), while ADA (-2.63) differed significantly from both PBO (P

Background RA pts treated with TCZ showed superior clinical benefit vs pts treated with ADA in the ADACTA study 1 ; however, it is unclear whether distinct RA subpopulations responded preferentially to anti-IL6R vs anti-TNFα therapy. Objectives To evaluate biomarker-defined pt subpopulations and their response to TCZ and ADA monotherapy. Methods ADACTA was a randomised, double-blind, 24-wk study in pts with RA of ≥6-mo duration and DAS28 >5.1 who were MTX intolerant or for whom continued treatment with MTX was considered ineffective or inappropriate. Pts received TCZ 8 mg/kg IV every 4 wks (+ placebo [PBO] ADA) or ADA 40 mg SC every 2 wks (+ PBO TCZ) for 24 wks as monotherapy. Serum biomarkers previously identified in TCZ or ADA studies—including CXCL13, CXCL10, CCL2, CCL13, IL8, IL6, IL2, IL10, TNFα, IFNγ, GM-CSF, IL12p70, IL1β, sICAM1, sCD25, MMP1, MMP3 and MMP9—were measured using quantitative immunoassay methods. Baseline (BL) levels (wk 0, n=203) were assessed against ACR20/50/70 response and ΔDAS28-ESR at wk 24 using local regression. Biomarkers were determined to enrich for clinical response if both efficacy endpoints showed improvement. Post-dose changes were assessed at wks 4 and 24 for biomarkers demonstrating enrichment for response. Statistical assessment for post-dose changes was performed using the Wilcoxon rank sum test comparing absolute biomarker levels at wk 0 vs 24, without correction for multiple comparisons. Post-dose changes vs efficacy compared wk 24 levels in ACR50 responders [R] vs non-responders [NR], except sICAM1 wk 4 as indicated. Results Analysis of BL biomarker levels vs efficacy at wk 24 revealed that pts with BL levels above the median for MMP3, CXCL13 or CXCL10 had increased ACR50 response rates and decreased DAS28-ESR scores after TCZ treatment vs pts with BL levels below the median; higher BL levels of these biomarkers did not enrich for ADA response (Table). Conversely, BL levels above the median for sICAM1 were associated with enhanced ADA but not TCZ clinical response. TCZ reduced post-dose levels of MMP3 in all comers (wk 0 vs 24, p Image/graph Conclusions Distinct patterns of biomarkers enriching for response suggest that TCZ and ADA may preferentially demonstrate efficacy in different subpopulations of RA pts. Higher BL levels of distinct biomarkers (MMP3, CXCL13 or CXCL10 for TCZ; sICAM1 for ADA) may enrich for clinical response. Furthermore, post-dose data suggest BL biomarker levels may enrich for efficacy independently of modulation by treatment. References Gabay C et al. Lancet . In press Disclosure of Interest A. Herman Employee of: Genentech, a member of the Roche group, D. Musselman Employee of: Genentech, a member of the Roche group, S. Fischer Employee of: Genentech, a member of the Roche group, A. Setiadi Employee of: Genentech, a member of the Roche group, C. Gabay Grant/research support from: Roche, Consultant for: Roche, Abbvie, Pfizer, UCB, BMS, MSD, A. Kavanaugh Grant/research support from: Roche, Abbott, Amgen, UCB, BMS, Pfizer, Janssen, M. Klearman Employee of: Genentech, a member of the Roche group, A. Song Shareholder of: Roche, Employee of: Genentech, a member of the Roche group, M. Townsend Shareholder of: Genentech, a member of the Roche group, Employee of: Genentech, a member of the Roche group

Background Lymphotoxin (LT)-α, a member of the TNF superfamily, is required for lymph node development and recruitment of B and T cells into lymph nodes via induction of CXCL13 and CCL19 ([1][1]). LTα has been implicated in the development of ectopic lymphoid tissue found in rheumatoid arthritis (RA) synovium ([2][2]). In pre-clinical xenograft models, the anti-LTα antibody pateclizumab (PTZ) inhibited the binding of soluble LTα3 to TNF receptors and membrane-bound LTα1β2 to LTβ receptor, and depleted LT-expressing T and B cells ([3][3]). In a Phase 1 study, patients treated with PTZ had decreased circulating CXCL13 levels ([4][4]). Objectives PTZ and the anti-TNFα antibody adalimumab (ADA) target distinct and overlapping pathways; thus, pharmacodynamic (PD) biomarkers representative of each pathway were assessed to understand contributions of each to disease and therapeutic response. Methods A Phase 2 study was conducted in DMARD-IR RA patients. Patients received PTZ (360 mg), ADA (40 mg), or placebo, SC every 2 weeks for 10 weeks. All patients remained on background DMARDs. Serum was collected to assess PD biomarkers on Day 1 (pre-dose), and at multiple timepoints thereafter. Chemokines, cytokines, adhesion molecules, matrix metalloproteases (MMPs), and inflammatory markers were measured using quantitative immunoassay methods. Statistical assessments were performed using the Wilcoxon rank sum test to compare the change in biomarker levels to placebo at Week 12. Results ADA had superior efficacy to PTZ in DMARD-IR RA patients ([5][5]). Circulating levels of CXCL13 and CCL19 were reduced in PTZ treated patients, demonstrating target engagement in the LTα pathway. ADA also blocked CXCL13, but not CCL19, suggesting a unique role for LTα (likely as LTα1β2) in CCL19 production. Blockade of the TNFα pathway via ADA down-regulated several additional biomarkers involved in TNF biology. These included MMP-1, MMP-3, and IL-6; and the inflammatory markers CRP, ESR, and S100A8. In contrast, PTZ did not induce changes in inflammatory markers or MMPs. Although PTZ was expected to inhibit the interaction of soluble LTα3 with TNFRs, TNFR-driven biological activity remained apparent in PTZ-treated patients. Conclusions Blockade of TNFα vs. LTα in DMARD-IR RA patients demonstrated distinct and overlapping patterns in downstream biomarkers of disease activity. TNFα blockade led to decreases in multiple biomarkers related to TNFR signaling. LTα blockade demonstrated significant reduction in levels of CCL19, as well as CXCL13, suggesting an impact on T and B cell migration and/or lymphogenesis. However, the decrease in chemokines observed with LTα blockade was associated with only modest reduction in clinical activity ([5][5]). References 1. Ngo VN et al. 1999 JEM 189:403. 2. Grogan JL & Ouyang W. 2012 EJI 42:2255. 3. Chiang EY et al. 2012 PLoS One 7:e33106. 4. Emu et al. 2012 Arthritis Res Ther 14:R6. 5. Kennedy WP et al. EULAR 2013 (submitted). Disclosure of Interest A. Herman Employee of: Genentech, Inc., W. Kennedy Employee of: Genentech, Inc., M. Townsend Employee of: Genentech, Inc., M. Williams Employee of: Genentech, Inc., J. Grogan Employee of: Genentech, Inc., S. Fischer Employee of: Genentech, Inc., S. Iyer Employee of: Genentech, Inc., A. Song Employee of: Genentech, Inc., J. Davis Jr Employee of: Genentech, Inc. [1]: #p-6 [2]: #p-7 [3]: #p-8 [4]: #p-9 [5]: #p-10

In today's environment, the transportation and maintenance of military forces is nearly as important as combat operations. Rapid deployment to regions of low-intensity conflict will become a very common training scenario for the U.S. military. Thus it is desirable to apply distributed simulation technology to train logistics personnel in their combat support roles. Currently, distributed interactive simulation (DIS) only contains rudimentary logistics protocols. This paper introduces new protocols designed to handle the logistics problem. The Newtonian protocol takes a physics-based approach to modeling interactions on the simulation network. This protocol consists of a family of protocol data units (PDUs) which are used to communicate forces in different circumstances. The protocol implements a small set of physical relations. This represents a flexible and general mechanism to describe battlefield interactions between network entities. The migratory object protocol (MOP) family addresses the transfer of control. General mechanisms provide the means to simulate resupply, repair, and maintenance of entities at any level of abstraction (individual soldier to division). It can also increase the fidelity of mine laying, enable handover of weapons for terminal guidance, allow for the distribution of aggregate-level simulation entities, provide capabilities for the simulation of personnel, etc.© (1995) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The glycine-rich G-loop controls ATP binding and phosphate transfer in protein kinases. Here we show that the functions of Src family and Abl protein tyrosine kinases require an electrostatic interaction between oppositely charged amino acids within their G loops that is conserved in multiple other phylogenetically distinct protein kinases from plants to humans. By limiting G-loop flexibility, it controls ATP binding, catalysis and inhibition by ATP-competitive compounds such as Imatinib. In WeeB mice, mutational disruption of the interaction results in expression of a Lyn protein with reduced catalytic activity, and in perturbed B cell receptor signaling. Like Lyn-/- mice, WeeB mice show profound defects in B cell development and function and succumb to autoimmune glomerulonephritis. This demonstrates the physiological importance of the conserved G-loop salt bridge and at the same time distinguishes the in vivo requirement for the Lyn kinase activity from other potential functions of the protein.

Elevated plasma MB creatine kinase (CK) is considered the most sensitive and specific diagnostic indicator of myocardial infarction. However, attempts to purify human MB CK have been unsuccessful. The need for purified human MB CK was further enhanced with the development of a radioimmunoassay for CK isoenzymes which would provide more prompt and specific detection of myocardial infarction. The major protein contaminant of MB CK is albumin which has been difficult to separate due to their similar electrophoretic mobility. Human hearts were obtained within 2 h postmortem and the tissue homogenized in 50 m m Tris-HCl (pH 7.4), 2 m m mercaptoethanol. The CK was recovered from the supernatant (31,000 g ) by ethanol extraction (50–70%). The resuspended pellet was fractionated on DEAE Sephadex A-50 with a salt gradient (50–500 m m , pH 8.0). The MB fraction contained about 90% albumin. The preparation was bound to an Affigel blue column and contaminating proteins other than albumin were eluted with 50 m m Tris-HCl (pH 8.0), 2 m m mercaptoethanol. MB CK was eluted with 250 m m NaCl, but the albumin remained bound. The MB fraction with a specific activity of 453 IU/mg represented an 80-fold increase in purity and exhibited a single protein band on polyacrylamide gels. Purified MB CK labeled with 125 I exhibited no binding to human albumin antiserum, but bound to MB CK antiserum, and unlabeled MB CK competitively inhibited binding of 125 I-MB CK in the radioimmunoassay system exhibiting a sensitivity for detection of plasma MB CK at the nanogram level.