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Lipid nanoparticles (LNPs) and ribonucleic acid (RNA) technology are highly versatile tools that can be deployed for diagnostic, prophylactic, and therapeutic applications. In this report, supramolecular chemistry concepts are incorporated into the rational design of a new ionizable lipid, C3-K2-E14, for systemic administration. This lipid incorporates a cone-shaped structure intended to facilitate cell bilayer disruption, and three tertiary amines to improve RNA binding. Additionally, hydroxyl and amide motifs are incorporated to further enhance RNA binding and improve LNP stability. Optimization of messenger RNA (mRNA) and small interfering RNA (siRNA) formulation conditions and lipid ratios produce LNPs with favorable diameter (<150 nm), polydispersity index (<0.15), and RNA encapsulation efficiency (>90%), all of which are preserved after 2 months at 4 or 37 °C storage in ready-to-use liquid form. The lipid and formulated LNPs are well-tolerated in animals and show no deleterious material-induced effects. Furthermore, 1 week after intravenous LNP administration, fluorescent signal from tagged RNA payloads are not detected. To demonstrate the long-term treatment potential for chronic diseases, repeated dosing of C3-K2-E14 LNPs containing siRNA that silences the colony stimulating factor-1 (CSF-1) gene can modulate leukocyte populations in vivo, further highlighting utility., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)

Immunoglobulin (IgG) Fc glycosylation has been shown to be important for the biological activity of antibodies. Fc sialylation is important for the anti-inflammatory activity of IgGs. However, evaluating the structure-activity relationship (SAR) of antibody Fc glycosylation has been hindered using simplified in vitro models in which antibodies are often displayed in monomeric forms. Presenting antibodies in monomeric forms may not accurately replicate the natural environment of the antibodies when binding their antigen in vivo. To address these limitations, we used different Fc-containing molecules, displaying their Fc domains in monovalent and multivalent fashion. Given the inhibitory role of Fc gamma receptor IIb (FcγRIIb) in autoimmune and inflammatory diseases, we focused on evaluating the impact of Fc sialylation on the activation of FcγRIIb. We report for the first time that in human cellular systems, sialic acid mediates the induction of FcγRIIb phosphorylation by IgG-Fc when the IgG-Fc is displayed in a multivalent fashion. This effect was observed with different types of therapeutic agents such as sialylated anti-TNFα antibodies, sialylated IVIg and sialylated recombinant multivalent Fc products. These studies represent the first report of the specific effects of Fc sialylation on FcγRIIb signaling on human immune cells and may help in the characterization of the anti-inflammatory activity of Fc-containing therapeutic candidates.

Lipid nanoparticles (LNPs) are the most clinically advanced delivery vehicles for RNA and have enabled the development of RNA-based drugs such as the mRNA COVID-19 vaccines. Functional delivery of mRNA by an LNP greatly depends on the inclusion of an ionizable lipid, and small changes to these lipid structures can significantly improve delivery. However, the structure-function relationships between ionizable lipids and mRNA delivery are poorly understood, especially for LNPs administered intramuscularly. Here, we show that the iterative design of a novel series of ionizable lipids generates key structure-activity relationships and enables the optimization of chemically distinct lipids with efficacy that is on-par with the current state of the art. We find that the combination of ionizable lipids comprising an ethanolamine core and LNPs with an apparent pK a between 6.6 and 6.9 maximizes intramuscular mRNA delivery. Furthermore, we report a nonlinear relationship between the lipid-to-mRNA mass ratio and protein expression, suggesting that a critical mass ratio exists for LNPs and may depend on ionizable lipid structure. Our findings add to the mechanistic understanding of ionizable lipids and demonstrate that hydrogen bonding, ionization behavior, and lipid-to-mRNA mass ratio are key design parameters affecting intramuscular mRNA delivery. We validate these insights by applying them to the rational design of new ionizable lipids. Overall, our iterative design strategy efficiently generates potent ionizable lipids. This hypothesis-driven method reveals structure-activity relationships that lay the foundation for the optimization of ionizable lipids in future LNP-RNA drugs. We foresee that this design strategy can be extended to other optimization parameters beyond intramuscular expression.

Objective: While airway epithelial biorepositories have established roles in the study of bronchial progenitor stem (basal) cells, the utility of a bank of tracheal basal cells from pediatric patients, who have or are suspected of having an airway disease, has not been established. In vitro study of these cells can enhance options for tracheal restoration, graft design, and disease modeling. Development of a functional epithelium in these settings is a key measure. The aim of this study was the creation a tracheal basal cell biorepository and assessment of recovered cells., Methods: Pediatric patients undergoing bronchoscopy were identified and endotracheal brush ( N  = 29) biopsies were collected. Cells were cultured using the modified conditional reprogramming culture (mCRC) method. Samples producing colonies by day 14 were passaged and cryopreserved. To explore differentiation potential, cells were thawed and differentiated using the air-liquid interface (ALI) method., Results: No adverse events were associated with biopsy collection. Of 29 brush biopsies, 16 (55%) were successfully cultured to passage 1/cryopreserved. Samples with higher initial cell yields were more likely to achieve this benchmark. Ten unique donors were then thawed for analysis of differentiation. The average age was 2.2 ± 2.2 years with five donors (50%) having laryngotracheal pathology. Nine donors (90%) demonstrated differentiation capacity at 21 days of culture, as indicated by detection of ciliated cells (ACT+) and mucous cells (MUC5B+)., Conclusion: Pediatric tracheal basal cells can be successfully collected and cryopreserved. Recovered cells retain the ability to differentiate into epithelial cell types in vitro ., Level of Evidence: Level 3., Competing Interests: The authors have no conflicts of interest to disclose for this study., (© 2022 The Authors. Laryngoscope Investigative Otolaryngology published by Wiley Periodicals LLC on behalf of The Triological Society.)

Background: Serum proteins can be readily assessed during routine clinical care. However, it is unclear to what extent serum proteins reflect the molecular dysregulations of peripheral blood cells (PBCs) or affected end-organs in systemic sclerosis (SSc). We conducted a multiomic comparative analysis of SSc serum profile, PBC, and skin gene expression in concurrently collected samples., Methods: Global gene expression profiling was carried out in skin and PBC samples obtained from 49 SSc patients enrolled in the GENISOS observational cohort and 25 unaffected controls. Levels of 911 proteins were determined by Olink Proximity Extension Assay in concurrently collected serum samples., Results: Both SSc PBC and skin transcriptomes showed a prominent type I interferon signature. The examination of SSc serum profile revealed an upregulation of proteins involved in pro-fibrotic homing and extravasation, as well as extracellular matrix components/modulators. Notably, several soluble receptor proteins such as EGFR, ERBB2, ERBB3, VEGFR2, TGFBR3, and PDGF-Rα were downregulated. Thirty-nine proteins correlated with severity of SSc skin disease. The differential expression of serum protein in SSc vs. control comparison significantly correlated with the differential expression of corresponding transcripts in skin but not in PBCs. Moreover, the differentially expressed serum proteins were significantly more connected to the Well-Associated-Proteins in the skin than PBC gene expression dataset. The assessment of the concordance of between-sample similarities revealed that the molecular profile of serum proteins and skin gene expression data were significantly concordant in patients with SSc but not in healthy controls., Conclusions: SSc serum protein profile shows an upregulation of profibrotic cytokines and a downregulation of soluble EGF and other key receptors. Our multilevel comparative analysis indicates that the serum protein profile in SSc correlates more closely with molecular dysregulations of skin than PBCs and might serve as a reflection of disease severity at the end-organ level., (© 2021. The Author(s).)

The inhibition of Fcγ receptors (FcγR) is an attractive strategy for treating diseases driven by IgG immune complexes (IC). Previously, we demonstrated that an engineered tri-valent arrangement of IgG1 Fc domains (SIF1) potently inhibited FcγR activation by IC, whereas a penta-valent Fc molecule (PentX) activated FcγR, potentially mimicking ICs and leading to Syk phosphorylation. Thus, a precise balance exists between the number of engaged FcγRs for inhibition versus activation. Here, we demonstrate that Fc valency differentially controls FcγR activation and inhibition within distinct subcellular compartments. Large Fc multimer clusters consisting of 5-50 Fc domains predominately recruited Syk-mScarlet to patches on the plasma membrane, whereas PentX exclusively recruited Syk-mScarlet to endosomes in human monocytic cell line (THP-1 cells). In contrast, SIF1, similar to monomeric Fc, spent longer periods docked to FcγRs on the plasma membrane and did not accumulate and recruit Syk-mScarlet within large endosomes. Single particle tracking (SPT) of fluorescent engineered Fc molecules and Syk-mScarlet at the plasma membrane imaged by total internal reflection fluorescence microscopy (SPT-TIRF), revealed that Syk-mScarlet sampled the plasma membrane was not recruited to FcγR docked with any of the engineered Fc molecules at the plasma membrane. Furthermore, the motions of FcγRs docked with recombinant Fc (rFc), SIF1 or PentX, displayed similar motions with D ~ 0.15 μm 2 /s, indicating that SIF1 and PentX did not induce reorganization or microclustering of FcγRs beyond the ligating valency. Multicolor SPT-TIRF and brightness analysis of docked rFc, SIF1 and PentX also indicated that FcγRs were not pre-assembled into clusters. Taken together, activation on the plasma membrane requires assembly of more than 5 FcγRs. Unlike rFc or SIF1, PentX accumulated Syk-mScarlet on endosomes indicating that the threshold for FcγR activation on endosomes is lower than on the plasma membrane. We conclude that the inhibitory effects of SIF1 are mediated by stabilizing a ligated and inactive FcγR on the plasma membrane. Thus, FcγR inhibition can be achieved by low valency ligation with SIF1 that behaves similarly to FcγR docked with monomeric IgG., Competing Interests: AC, DO, JS, AM, and CB were employed by Momenta Pharmaceuticals during the project and have equity interest. CB, AM, and DO are inventors on patent application PCT/US2015/028926 (WO/2015/168643) submitted by Momenta Pharmaceuticals that covers “Compositions and methods related to engineered Fc constructs”. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bailey, Choudhury, Vuppula, Ortiz, Schaeck, Manning, Bosques and Hoppe.)

Background: Plasma is a potentially rich source of protein biomarkers for disease progression and drug response. Large multi-center studies are often carried out to increase the number of samples analyzed in a given study. This may increase the chances of variation in blood processing and handling, leading to altered proteomic results. This study evaluates the impact of blood processing variation on LC-MS/MS proteomic analysis of plasma., Methods: Initially two batches of patient plasma samples (120 and 204 samples, respectively) were analyzed using LC-MS/MS shotgun proteomics. Follow-up experiments were designed and carried out on healthy donor blood in order to examine the effects of different centrifugation conditions, length of delay until first centrifugation, storage temperature and anticoagulant type on results from shotgun proteomics., Results: Variable levels of intracellular proteins were observed in subsets of patient plasma samples from the initial batches analyzed. This observation correlated strongly with the site of collection, implicating variability in blood processing procedures. Results from the healthy donor blood analysis did not demonstrate a significant impact of centrifugation conditions to plasma proteome variation. The time delay until first centrifugation had a major impact on variability, while storage temperature and anticoagulant showed less pronounced but still significant effects. The intracellular proteins associated with study site effect in patient plasma samples were significantly altered by delayed processing also., Conclusions: Variable blood processing procedures contribute significantly to plasma proteomic variation and may give rise to increased intracellular proteins in plasma. Accounting for these effects can be important both at study design and data analysis stages. This understanding will be valuable to incorporate in the planning of protein-based biomarker discovery efforts in the future.

Identification of differentially expressed genes (DEGs) is well recognized to be variable across independent replications of genome-wide transcriptional studies. These are often employed to characterize disease state early in the process of discovery and prioritize novel targets aimed at addressing unmet medical need. Increasing reproducibility of biological findings from these studies could potentially positively impact the success rate of new clinical interventions. This work demonstrates that statistically sound combination of gene expression data with prior knowledge about biology in the form of large protein interaction networks can yield quantitatively more reproducible observations from studies characterizing human disease. The novel concept of Well-Associated Proteins (WAPs) introduced herein-gene products significantly associated on protein interaction networks with the differences in transcript levels between control and disease-does not require choosing a differential expression threshold and can be computed efficiently enough to enable false discovery rate estimation via permutation. Reproducibility of WAPs is shown to be on average superior to that of DEGs under easily-quantifiable conditions suggesting that they can yield a significantly more robust description of disease. Enhanced reproducibility of WAPs versus DEGs is first demonstrated with four independent data sets focused on systemic sclerosis. This finding is then validated over thousands of pairs of data sets obtained by random partitions of large studies in several other diseases. Conditions that individual data sets must satisfy to yield robust WAP scores are examined. Reproducible identification of WAPs can potentially benefit drug target selection and precision medicine studies., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: All authors are currently (or were at the time the study was conducted) employees of Momenta Pharmaceuticals and may own stock and/or stock options in Momenta Pharmaceuticals.

Background: The goal of this study is to use comprehensive molecular profiling to characterize clinical response to anti-TNF therapy in a real-world setting and identify reproducible markers differentiating good responders and non-responders in rheumatoid arthritis (RA)., Methods: Whole-blood mRNA, plasma proteins, and glycopeptides were measured in two cohorts of biologic-naïve RA patients (n = 40 and n = 36) from the Corrona CERTAIN (Comparative Effectiveness Registry to study Therapies for Arthritis and Inflammatory coNditions) registry at baseline and after 3 months of anti-TNF treatment. Response to treatment was categorized by EULAR criteria. A cell type-specific data analysis was conducted to evaluate the involvement of the most common immune cell sub-populations. Findings concordant between the two cohorts were further assessed for reproducibility using selected NCBI-GEO datasets and clinical laboratory measurements available in the CERTAIN database., Results: A treatment-related signature suggesting a reduction in neutrophils, independent of the status of response, was indicated by a high level of correlation (ρ = 0.62; p < 0.01) between the two cohorts. A baseline, response signature of increased innate cell types in responders compared to increased adaptive cell types in non-responders was identified in both cohorts. This result was further assessed by applying the cell type-specific analysis to five other publicly available RA datasets. Evaluation of the neutrophil-to-lymphocyte ratio at baseline in the remaining patients (n = 1962) from the CERTAIN database confirmed the observation (odds ratio of good/moderate response = 1.20 [95% CI = 1.03-1.41, p = 0.02])., Conclusion: Differences in innate/adaptive immune cell type composition at baseline may be a major contributor to response to anti-TNF treatment within the first 3 months of therapy.

Background: The sequelae of Kawasaki disease (KD) vary widely with the greatest risk for future cardiovascular events among those who develop giant coronary artery aneurysms (CAA). We sought to define the molecular signature associated with different outcomes in pediatric and adult KD patients., Methods: Molecular profiling was conducted using mass spectrometry-based shotgun proteomics, transcriptomics, and glycomics methods on 8 pediatric KD patients at the acute, subacute, and convalescent time points. Shotgun proteomics was performed on 9 KD adults with giant CAA and matched healthy controls. Plasma calprotectin was measured by ELISA in 28 pediatric KD patients 1 year post-KD, 70 adult KD patients, and 86 healthy adult volunteers., Results: A characteristic molecular profile was seen in pediatric patients during the acute disease, which resolved at the subacute and convalescent periods in patients with no coronary artery sequelae but persisted in 2 patients who developed giant CAA. We, therefore, investigated persistence of inflammation in KD adults with giant CAA by shotgun proteomics that revealed a signature of active inflammation, immune regulation, and cell trafficking. Correlating results obtained using shotgun proteomics in the pediatric and adult KD cohorts identified elevated calprotectin levels in the plasma of patients with CAA. Investigation of expanded pediatric and adult KD cohorts revealed elevated levels of calprotectin in pediatric patients with giant CAA 1 year post-KD and in adult KD patients who developed giant CAA in childhood., Conclusions: Complex patterns of biomarkers of inflammation and cell trafficking can persist long after the acute phase of KD in patients with giant CAA. Elevated levels of plasma calprotectin months to decades after acute KD and infiltration of cells expressing S100A8 and A9 in vascular tissues suggest ongoing, subclinical inflammation. Calprotectin may serve as a biomarker to inform the management of KD patients following the acute illness.

M281 is a fully human, anti-neonatal Fc receptor (FcRn) antibody that inhibits FcRn-mediated immunoglobulin G (IgG) recycling to decrease pathogenic IgG while preserving IgG production. A randomized, double-blind, placebo-controlled, first-in-human study with 50 normal healthy volunteers was designed to probe safety and the physiological maximum for reduction of IgG. Intravenous infusion of single ascending doses up to 60 mg/kg induced dose-dependent serum IgG reductions, which were similar across all IgG subclasses. Multiple weekly doses of 15 or 30 mg/kg achieved mean IgG reductions of ≈85% from baseline and maintained IgG reductions ≥75% from baseline for up to 24 days. M281 was well tolerated, with no serious or severe adverse events (AEs), few moderate AEs, and a low incidence of infection-related AEs similar to placebo treatment. The tolerability and consistency of M281 pharmacokinetics and pharmacodynamics support further evaluation of M281 in diseases mediated by pathogenic IgG., (© 2018 The Authors Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.)

The importance of IgG glycosylation, Fc-gamma receptor (FcγR) single nucleotide polymorphisms and FcγR copy number variations in fine tuning the immune response has been well established. There is a growing appreciation of the importance of glycosylation of FcγRs in modulating the FcγR-IgG interaction based on the association between the glycosylation of recombinant FcγRs and the kinetics and affinity of the FcγR-IgG interaction. Although glycosylation of recombinant FcγRs has been recently characterized, limited knowledge exists on the glycosylation of endogenous human FcγRs. In order to improve the structural understanding of FcγRs expressed on human cells we characterized the site specific glycosylation of native human FcγRIII from neutrophils of 50 healthy donors and from matched plasma for 43 of these individuals. Through this analysis we have confirmed site specific glycosylation patterns previously reported for soluble FcγRIII from a single donor, identified FcγRIIIb specific Asn45 glycosylation and an allelic effect on glycosylation at Asn162 of FcγRIIIb. Identification of FcγRIIIb specific glycosylation allows for assignment of FcγRIIIb alleles and relative copy number of the two alleles where DNA/RNA is not available. Intriguingly the types of structures found to be elevated at Asn162 in the NA2 allele have been shown to destabilize the Fc:FcγRIII interaction resulting in a faster dissociation rate. These differences in glycosylation may in part explain the differential activity reported for the two alleles which have similar in vitro affinity for IgG., (© 2019 Washburn et al.)

Objective Unplanned readmission within 30 days is a contributor to health care costs in the United States. The use of predictive modeling during hospitalization to identify patients at risk for readmission offers a novel approach to quality improvement and cost reduction. Study Design Two-phase study including retrospective analysis of prospectively collected data followed by prospective longitudinal study. Setting Tertiary academic medical center. Subjects and Methods Prospectively collected data for patients undergoing surgical treatment for head and neck cancer from January 2013 to January 2015 were used to build predictive models for readmission within 30 days of discharge using logistic regression, classification and regression tree (CART) analysis, and random forests. One model (logistic regression) was then placed prospectively into the discharge workflow from March 2016 to May 2016 to determine the model's ability to predict which patients would be readmitted within 30 days. Results In total, 174 admissions had descriptive data. Thirty-two were excluded due to incomplete data. Logistic regression, CART, and random forest predictive models were constructed using the remaining 142 admissions. When applied to 106 consecutive prospective head and neck oncology patients at the time of discharge, the logistic regression model predicted readmissions with a specificity of 94%, a sensitivity of 47%, a negative predictive value of 90%, and a positive predictive value of 62% (odds ratio, 14.9; 95% confidence interval, 4.02-55.45). Conclusion Prospectively collected head and neck cancer databases can be used to develop predictive models that can accurately predict which patients will be readmitted. This offers valuable support for quality improvement initiatives and readmission-related cost reduction in head and neck cancer care.

Study Objectives: The goals of this study were to (1) evaluate the degree of decisional conflict (DC) experienced by caregivers of children with obstructive sleep apnea (OSA) without tonsillar hypertrophy; and (2) describe the association between DC, quality of life (QOL), and OSA severity., Methods: This study comprised children evaluated in the multidisciplinary upper airway center at the Cincinnati Children's Hospital Medical Center from December 2014 to May 2016. Caregivers were asked to complete surveys (Pediatric Quality of Life Inventory 4.0 [PedsQL], OSA-18, Epworth Sleepiness Scale, Family Impact Questionnaire, Decisional Conflict Scale, CollaboRATE scale, and SURE questionnaire) during a clinic visit. Polysomnography data were collected. Analysis included Kruskal-Wallis, Wilcoxon rank-sum, and regression testing., Results: Caregivers of 76 children participated; 16 (21.1%) had high DC. There were no significant differences in demographics between those with low and high DC; the low DC group had a higher obstructive apnea-hypopnea index (13.2 versus 12.3 events/h; P = .013). Overall and disease-specific QOL, sleepiness, family impact scores, and DC did not differ by OSA severity except for the PedsQL physical subcategory ( P = .02). DC was associated with the total PedsQL ( P = .043) on univariate regression; however, this did not persist ( P = .61) after controlling for demographic variables. DC scores correlated well with CollaboRATE and SURE throughout the analysis ( P < .001)., Conclusions: The proportion of caregivers of children with OSA without tonsillar hypertrophy who experienced a high level of DC regarding their child's treatment was 21.1%. Neither DC nor OSA severity was related to QOL in children with OSA. The briefer SURE or CollaboRATE scales were adequate tools to measure DC in these children., (© 2018 American Academy of Sleep Medicine.)

Objectives To evaluate changes in distribution of reported thyroid nodule fine-needle aspiration (FNA) cytopathology results since implementation of the Bethesda classification and revised 2015 American Thyroid Association (ATA) guidelines for selecting nodules for biopsy. Study Design Retrospective review. Setting Tertiary academic medical center. Subjects and Methods Evaluation of ultrasound (US)-guided thyroid FNA by a single surgeon using 2015 ATA nodule selection criteria and Bethesda reporting on 211 thyroid nodules in a 1-year period (2015). Comparison is made to an earlier sample wherein any nodule >1 cm underwent US FNA with cytology reported prior to Bethesda consensus (2006). Results The current cohort involved mostly women (79%); nodules ranged from 1 to 7 cm (mean ± SEM, 2.4 ± 0.07 cm). Mean ± SEM age was 53.5 ± 1.1 years. Bethesda reporting yielded 6% nondiagnostic, 57% benign, 3% malignant, and 34% indeterminate (27% atypia of undetermined significance [AUS]/follicular lesion of undetermined significance [FLUS], 4% follicular neoplasm [FN]/Hürthle neoplasm [HN], and 2% suspicious for malignancy [SFM]). The malignancy rate in indeterminate nodules was 26% (18% AUS/FLUS, 33% FN/HN, and 80% SFM). Age, sex, or nodule size did not correlate with indeterminate cytology. The comparator sample of 447 nodules had significantly different distribution, with 7% nondiagnostic, 80% benign, 5% malignant, and 8% indeterminate ( P < .00001). Conclusion We observed a significantly increased proportion of indeterminate cytology and corresponding decrease in benign nodules compared with an earlier sample, predominately from an increase in AUS/FLUS. Multiple factors are likely involved, including selection of sonographically suspicious nodules for biopsy based upon 2015 ATA guidelines coupled with cytopathological interpretation by a new generation of cytopathologists trained in the era of Bethesda reporting; further study is required to make a definitive conclusion.

Autoantibody immune complex (IC) activation of Fcγ receptors (FcγRs) is a common pathogenic hallmark of multiple autoimmune diseases. Given that the IC structural features that elicit FcγR activation are poorly understood and the FcγR system is highly complex, few therapeutics can directly block these processes without inadvertently activating the FcγR system. To address these issues, the structure activity relationships of an engineered panel of multivalent Fc constructs were evaluated using sensitive FcγR binding and signaling cellular assays. These studies identified an Fc valency with avid binding to FcγRs but without activation of immune cell effector functions. These observations directed the design of a potent trivalent immunoglobulin G-Fc molecule that broadly inhibited IC-driven processes in a variety of immune cells expressing FcγRs. The Fc trimer, Fc3Y, was highly efficacious in three different animal models of autoimmune diseases. This recombinant molecule may represent an effective therapeutic candidate for FcγR-mediated autoimmune diseases., (Copyright © 2016, American Association for the Advancement of Science.)

Autoantibody immune complexes (ICs) mediate pathogenesis in multiple autoimmune diseases via direct interference with target function, complement fixation, and interaction with Fc-gamma receptors (FcγRs). Through high avidity interactions, ICs are able to crosslink low affinity FcγRs expressed on a wide variety of effector cells, leading to secretion of pro-inflammatory mediators and inducing cytotoxicity, ultimately resulting in tissue injury. Given their relevance in numerous autoimmune diseases, FcγRs have been considered as attractive therapeutic targets for the last three decades. However, a limited number of investigational drug candidates have been developed targeting FcγRs and only a few approved therapeutics have been associated with impacting FcγRs. This review provides a historical overview of the different therapeutic approaches used to target FcγRs for the treatment of autoimmune and inflammatory diseases., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Most protective components in human milk are stable during prolonged storage at 4ºC; however, pasteurization reduces some microbicidal activities responsible for suppressing microbial growth and protecting against infection. Donor milk used by neonatal intensive care units (NICUs) is frozen pasteurized donor human milk (PDHM) defrosted and stored at 4ºC. Current Human Milk Banking Association of North America (HMBANA) Best Practice guidelines recommend that milk be discarded 24 hours after being thawed, but experimental data on the duration of microbiological purity in thawed PDHM are sparse., Objective: This study evaluates microbiological purity of thawed PDHM during prolonged storage at 4ºC., Methods: A total of 42 independent, randomly selected PDHM samples were thawed at 4ºC. As is typical in NICUs, each bottle was opened at 3-hour intervals and 3 mL was withdrawn with a sterile syringe and transferred into a sterile tube. The 3 mL samples removed at 0, 24, 48, 72, 96 hours, and 9 days were tested for the presence of any microorganisms by a clinical laboratory that routinely screens PDHM for microbes., Results: No evidence of microbial growth was observed in cultured samples taken at 0 to 9 days after thawing of the milk samples., Conclusion: There was no evidence of microbes in PDHM as dispensed by HMBANA milk banks when defrosted and stored at 4ºC for up to 9 days. Extended storage of PDHM in the NICU could reduce waste of donor milk, thereby increasing availability of human milk to vulnerable neonatal patients., (© The Author(s) 2015.)

Fidelity of glycan structures is a key requirement for biotherapeutics, with carbohydrates playing an important role for therapeutic efficacy. Comprehensive glycan profiling techniques such as liquid chromatography (LC) and mass spectrometry (MS), while providing detailed description of glycan structures, require glycan cleavage, labeling, and paradigms to deconvolute the considerable data sets they generate. On the other hand, lectins as probes on microarrays have recently been used in orthogonal approaches for in situ glycoprofiling but require analyte labeling to take advantage of the capabilities of automated microarray readers and data analysis they afford. Herein, we describe a lectin-based microtiter assay (lectin-enzyme-linked immunosorbent assay [ELISA]) to quantify terminal glycan moieties, applicable to in vitro and in-cell glycan-engineered Fc proteins as well as intact IgGs from intravenous immunoglobulin (IVIG), a blood product containing pooled polyvalent IgG antibodies extracted from plasma from healthy human donors. We corroborate our findings with industry-standard LC-MS profiling. This "customizable" ELISA juxtaposes readouts from multiple lectins, focusing on a subset of glycoforms, and provides the ability to discern single- versus dual-arm glycosylation while defining levels of epitopes at sensitivities comparable to MS. Extendable to other biologics, this ELISA can be used stand-alone or complementary to MS for quantitative glycan analysis., (© 2015 Society for Laboratory Automation and Screening.)

Despite the beneficial therapeutic effects of intravenous immunoglobulin (IVIg) in inflammatory diseases, consistent therapeutic efficacy and potency remain major limitations for patients and physicians using IVIg. These limitations have stimulated a desire to generate therapeutic alternatives that could leverage the broad mechanisms of action of IVIg while improving therapeutic consistency and potency. The identification of the important anti-inflammatory role of fragment crystallizable domain (Fc) sialylation has presented an opportunity to develop more potent Ig therapies. However, translating this concept to potent anti-inflammatory therapeutics has been hampered by the difficulty of generating suitable sialylated products for clinical use. Therefore, we set out to develop the first, to our knowledge, robust and scalable process for generating a well-qualified sialylated IVIg drug candidate with maximum Fc sialylation devoid of unwanted alterations to the IVIg mixture. Here, we describe a controlled enzymatic, scalable process to produce a tetra-Fc-sialylated (s4-IVIg) IVIg drug candidate and its qualification across a wide panel of analytic assays, including physicochemical, pharmacokinetic, biodistribution, and in vivo animal models of inflammation. Our in vivo characterization of this drug candidate revealed consistent, enhanced anti-inflammatory activity up to 10-fold higher than IVIg across different animal models. To our knowledge, this candidate represents the first s4-IVIg suitable for clinical use; it is also a valuable therapeutic alternative with more consistent and potent anti-inflammatory activity.

Stapled α-helical peptides have emerged as a promising new modality for a wide range of therapeutic targets. Here, we report a potent and selective dual inhibitor of MDM2 and MDMX, ATSP-7041, which effectively activates the p53 pathway in tumors in vitro and in vivo. Specifically, ATSP-7041 binds both MDM2 and MDMX with nanomolar affinities, shows submicromolar cellular activities in cancer cell lines in the presence of serum, and demonstrates highly specific, on-target mechanism of action. A high resolution (1.7-Å) X-ray crystal structure reveals its molecular interactions with the target protein MDMX, including multiple contacts with key amino acids as well as a role for the hydrocarbon staple itself in target engagement. Most importantly, ATSP-7041 demonstrates robust p53-dependent tumor growth suppression in MDM2/MDMX-overexpressing xenograft cancer models, with a high correlation to on-target pharmacodynamic activity, and possesses favorable pharmacokinetic and tissue distribution properties. Overall, ATSP-7041 demonstrates in vitro and in vivo proof-of-concept that stapled peptides can be developed as therapeutically relevant inhibitors of protein-protein interaction and may offer a viable modality for cancer therapy.

Purpose: The objective of this study was to compare the differences in lipid loss from 24 samples of banked donor human milk (DHM) among 3 feeding methods: DHM given by syringe pump over 1 hour, 2 hours, and by bolus/gravity gavage., Design: Comparative, descriptive. There were no human subjects., Methods: Twenty-four samples of 8 oz of DHM were divided into four 60-mL aliquots. Timed feedings were given by Medfusion 2001 syringe pumps with syringes connected to narrow-lumened extension sets designed for enteral feedings and connected to standard silastic enteral feeding tubes. Gravity feedings were given using the identical syringes connected to the same silastic feeding tubes. All aliquots were analyzed with the York Dairy Analyzer. Univariate repeated-measures analyses of variance were used for the omnibus testing for overall differences between the feeding methods. Lipid content expressed as grams per deciliter at the end of each feeding method was compared with the prefed control samples using the Dunnett's test. The Tukey correction was used for other pairwise multiple comparisons., Results: The univariate repeated-measures analysis of variance conducted to test for overall differences between feeding methods showed a significant difference between the methods (F = 58.57, df = 3, 69, P < .0001). Post hoc analysis using the Dunnett's approach revealed that there was a significant difference in fat content between the control sample and the 1-hour and 2-hours feeding methods (P < .0001), but we did not find any significant difference in fat content between the control and the gravity feeding methods (P = .3296). Pairwise comparison using the Tukey correction revealed a significant difference between both gravity and 1-hour feeding methods (P < .0001), and gravity and 2-hour feeding method (P < .0001). There was no significant difference in lipid content between the 1-hour and 2-hour feeding methods (P = .2729)., Conclusions: Unlike gravity feedings, the timed feedings resulted in a statistically significant loss of fat as compared with their controls. These findings should raise questions about how those infants in the neonatal intensive care unit are routinely gavage fed.

IRAK-1 and IRAK-4 are protein kinases that mediate signaling by Toll/IL1/Plant R (TIR) domain-containing receptors including the IL-1, IL-18, and Toll-like receptors (TLRs). Although well studied in mouse systems, the mechanism by which they function in human systems is less clear. To extend our knowledge of how these proteins regulate inflammatory signaling in human cells, we genetically and pharmacologically manipulated IRAK-1 and IRAK-4 kinase activities in vitro. Ablation of IRAK-4 expression in human umbilical vein endothelial cells (HUVEC) with siRNA suppressed IL-1beta induced IL-6 and IL-8 production whereas IRAK-1 siRNA suppressed TNFalpha induced but not IL-1beta induced cytokine production. Complementation of IRAK-4-depleted cells with a kinase-inactive allele restored IL-1beta induced cytokine gene expression suggesting that the IRAK-4 kinase activity is dispensable relative to its scaffolding function. Consistent with this finding, an IRAK-4 selective kinase inhibitor (RO6245) that inhibited IRAK-1 degradation failed to block IL-1beta induced cytokine production. In contrast, an inhibitor of both IRAK-1 and IRAK-4 (RO0884) reduced IL-1beta induced p38 MAP kinase, c-Jun N-terminal kinase activation, and IL-6 production in HUVEC. RO0884 also antagonized IL-1beta, TNFalpha, and TLR-mediated cytokine production in human fibroblast-like synoviocytes and peripheral blood mononuclear cells. Therefore in human cells the non-kinase functions of IRAK-4 are essential, whereas the kinase activity of IRAK-4 appears redundant with that of IRAK-1. Pharmacologic inhibition of both kinases appears necessary to block pro-inflammatory cytokine production.

P38alpha is a protein kinase that regulates the expression of inflammatory cytokines, suggesting a role in the pathogenesis of diseases such as rheumatoid arthritis (RA) or systemic lupus erythematosus. Here, we describe the preclinical pharmacology of pamapimod, a novel p38 mitogen-activated protein kinase inhibitor. Pamapimod inhibited p38alpha and p38beta enzymatic activity, with IC(50) values of 0.014 +/- 0.002 and 0.48 +/- 0.04 microM, respectively. There was no activity against p38delta or p38gamma isoforms. When profiled across 350 kinases, pamapimod bound only to four kinases in addition to p38. Cellular potency was assessed using phosphorylation of heat shock protein-27 and c-Jun as selective readouts for p38 and c-Jun NH(2)-terminal kinase (JNK), respectively. Pamapimod inhibited p38 (IC(50), 0.06 microM), but inhibition of JNK was not detected. Pamapimod also inhibited lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) alpha production by monocytes, interleukin (IL)-1beta production in human whole blood, and spontaneous TNFalpha production by synovial explants from RA patients. LPS- and TNFalpha-stimulated production of TNFalpha and IL-6 in rodents also was inhibited by pamapimod. In murine collagen-induced arthritis, pamapimod reduced clinical signs of inflammation and bone loss at 50 mg/kg or greater. In a rat model of hyperalgesia, pamapimod increased tolerance to pressure in a dose-dependent manner, suggesting an important role of p38 in pain associated with inflammation. Finally, an analog of pamapimod that has equivalent potency and selectivity inhibited renal disease in lupus-prone MRL/lpr mice. Our study demonstrates that pamapimod is a potent, selective inhibitor of p38alpha with the ability to inhibit the signs and symptoms of RA and other autoimmune diseases.

Objective: To assess the frequency of heart murmurs in overtly healthy cats., Design: Prospective study., Sample Population: 103 healthy domestic cats., Procedure: Background information and physical characteristics were assessed in cats that were candidates for blood donation during an 8-month period. For cats with heart murmurs, additional information collected included murmur timing, grade, point of maximal intensity, and presence of additional heart sounds., Results: Heart murmurs were detected in 22 of the 103 (21%) cats. Echocardiography was performed in 7 of those 22 cats. The echocardiogram was considered normal in 1 cat; in the other 6 cats, diagnoses included hypertrophic cardiomyopathy (interventricular septal hypertrophic form [IVSH]; n = 4), left ventricular concentric hypertrophy with valvular disease (1), and equivocal IVSH (1). Thirteen cats had more than 1 examination during the study; 3 of them developed heart murmurs. There were no significant differences in age, sex, breed, coat color, eye color, or heart rate between cats with and without murmurs. Among the 103 cats, there were 6 pairs of siblings from 6 multiple-cat households and 16 cats from 7 multiple-cat households in which the cats were not related; the proportion of cats with murmurs was higher in the related cats (5/12) than in the unrelated cats (3/16), but the difference was not significant., Conclusions and Clinical Relevance: Results indicated that heart murmurs are detectable in a large proportion of overtly healthy cats and that many murmurs appear to be caused by structural heart disease that is in a clinically latent state.

The c-Jun NH(2)-terminal kinases (JNKs) phosphorylate and activate members of the activator protein-1 (AP-1) transcription factor family and other cellular factors implicated in regulating altered gene expression, cellular survival and proliferation in response to cytokines and growth factors, noxious stimuli and oncogenic transformation. Because these events are commonly associated with the pathogenesis of a number of human diseases, the potential of JNK inhibitors as therapeutics has attracted considerable interest. Here we discuss the evidence supporting the application of JNK inhibitors in inflammatory, vascular, neurodegenerative, metabolic and oncological diseases in humans, and describe the present status of drug discovery targeting JNK.

Oxygen (O2) supplementation increases the O2 content of blood, increases the partial pressure of oxygen (PO2) in the capillary blood, and improves tissue delivery of O2. In addition to improving tissue oxygenation, the administration of O2 may improve the function of O2-dependent cellular systems, such as the cytochrome P450 system, which is important to drug and toxin metabolism; nitric oxide synthase, which regulates vasodilation; and host defense systems. Improved tissue oxygenation is also beneficial for wound healing. Given the important contributions that supplemental O2 can make, it is no wonder that O2 is one of the most common drugs administered in the emergency and intensive care settings.

In this study, we examined the role of the nuclear factor-kappaB (NF-kappaB)-inducing kinase (NIK) in distinct signaling pathways leading to NF-kappaB activation. We show that a dominant-negative form of NIK (dnNIK) delivered by adenoviral (Ad5dnNIK) vector inhibits Fas-induced IkappaBalpha phosphorylation and NF-kappaB-dependent gene expression in HT-29 and HeLa cells. Interleukin (IL)-1beta- and tumor necrosis factor-alpha (TNF-alpha)-induced NF-kappaB activation and kappaB-dependent gene expression are inhibited in HeLa cells but not in Ad5dnNIK-infected HT-29 cells. Moreover, Ad5dnNIK failed to sensitize HT-29 cells to TNF-alpha-induced apoptosis at an early time point. However, cytokine- and Fas-induced signals to NF-kappaB are finally integrated by the IkappaB kinase (IKK) complex, since IkappaBalpha phosphorylation, NF-kappaB DNA binding activity, and IL-8 gene expression were strongly inhibited in HT-29 and HeLa cells overexpressing dominant-negative IKKbeta (Ad5dnIKKbeta). Our findings support the concept that cytokine signaling to NF-kappaB is redundant at the level of NIK. In addition, this study demonstrates for the first time the critical role of NIK and IKKbeta in Fas-induced NF-kappaB signaling cascade.

Jun N-terminal kinase (JNK) is a stress-activated protein kinase that can be induced by inflammatory cytokines, bacterial endotoxin, osmotic shock, UV radiation, and hypoxia. We report the identification of an anthrapyrazolone series with significant inhibition of JNK1, -2, and -3 (K(i) = 0.19 microM). SP600125 is a reversible ATP-competitive inhibitor with >20-fold selectivity vs. a range of kinases and enzymes tested. In cells, SP600125 dose dependently inhibited the phosphorylation of c-Jun, the expression of inflammatory genes COX-2, IL-2, IFN-gamma, TNF-alpha, and prevented the activation and differentiation of primary human CD4 cell cultures. In animal studies, SP600125 blocked (bacterial) lipopolysaccharide-induced expression of tumor necrosis factor-alpha and inhibited anti-CD3-induced apoptosis of CD4(+) CD8(+) thymocytes. Our study supports targeting JNK as an important strategy in inflammatory disease, apoptotic cell death, and cancer.

Abnormal electrolyte concentrations occur commonly in hospitalized patients and may produce a variety of clinical symptoms, cause lack of response to therapeutics for primary clinical conditions, and affect clinical outcome. Recognition of electrolyte disturbances requires a high index of suspicion by the clinician for such a disturbance and prompt therapy to ensure a positive and timely outcome for the patient. This article discusses electrolyte abnormalities that occur in critically ill patients, with a review of diseases commonly associated with each electrolyte disturbance, and their recommended management.

After liver injury, hepatic stellate cells (HSCs) undergo a process of activation with expression of smooth muscle alpha-actin (alpha-SMA), an increased proliferation rate, and a dramatic increase in synthesis of type I collagen. The intracellular signaling mechanisms of activation and perpetuation of the activated phenotype in HSCs are largely unknown. In this study the role of the stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38, were evaluated in primary cultures of rat HSCs. The effect of JNK was assessed by using an adenovirus expressing a dominant negative form of transforming growth factor beta (TGF-beta)-activated kinase 1 (TAK1) (Ad5dnTAK1) and a new selective pharmacologic inhibitor SP600125. The effect of p38 was assessed with the selective pharmacologic inhibitor SB203580. These kinases were inhibited starting either in quiescent HSCs (culture day 1) or in activated HSCs (culture day 5). Although blocking TAK1/JNK and p38 decreased the expression of alpha-SMA protein in early stages of HSC activation, no effect was observed when TAK1/JNK or p38 were inhibited in activated HSCs. JNK inhibition increased and p38 inhibition decreased collagen alpha1(I) mRNA level as measured by RNase protection assays, with maximal effects observed in early stages of HSC activation. Furthermore, TAK1/JNK inhibition decreased HSC proliferation, whereas p38 inhibition led to an increased proliferation rate of HSCs, independently of its activation status. These results show novel roles for the TAK1/JNK pathway and p38 during HSC activation in culture. Despite similar activators of TAK1/JNK and p38, their functions in HSCs are distinct and opposed.

Objective: Inhibitor of nuclear factor kappaB kinase beta (IkappaB kinase beta, or IKKbeta) has emerged as a key regulator of the transcription factor nuclear factor kappaB (NF-kappaB). Since IKKbeta could have both pro- and antiinflammatory activity, we examined whether its constitutive activation was sufficient to cause a chronic inflammatory disease such as rheumatoid arthritis., Methods: Normal Lewis rats were evaluated for paw swelling by plethysmometry and histologic assessment after intraarticular injection of an adenoviral construct encoding the IKKbeta wild-type gene (Ad.IKKbeta-wt); controls received an adenoviral construct encoding green fluorescent protein (Ad.GFP). The rats were killed after 7 days. Additionally, rats were killed 48 hours after intraarticular injection of Ad.IKKbeta-wt or Ad.GFP for studies of IKK activity and NF-kappaB binding. For studies of the effects of inhibition of IKKbeta activity, Lewis rats were immunized with Mycobacterium tuberculosis in mineral oil. The ankle joints were injected on day 12 with an adenoviral construct encoding IKKbeta K-->M (dominant negative, IKKbeta-dn) or Ad.GFP. We evaluated paw swelling and NF-kappaB expression on day 25., Results: Intraarticular gene transfer of IKKbeta-wt into the joints of normal rats resulted in significant paw swelling and histologic evidence of synovial inflammation. Increased IKK activity was detectable in the IKKbeta-wt-injected ankle joints, coincident with enhanced NF-kappaB DNA binding activity. Intraarticular gene transfer of IKKbeta-dn significantly ameliorated the severity of adjuvant arthritis, accompanied by a significant decrease in NF-kappaB DNA expression in the joints of Ad.IKKbeta-dn-treated animals., Conclusion: IKKbeta plays a key role in rodent synovial inflammation. Intraarticular gene therapy to inhibit IKKbeta activity represents an attractive strategy for the treatment of chronic arthritis.

Mitogen-activated protein kinase (MAPK) cascades are involved in inflammation and tissue destruction in rheumatoid arthritis (RA). In particular, c-Jun N-terminal kinase (JNK) is highly activated in RA fibroblast-like synoviocytes and synovium. However, defining the precise function of this kinase has been difficult because a selective JNK inhibitor has not been available. We now report the use of a novel selective JNK inhibitor and JNK knockout mice to determine the function of JNK in synoviocyte biology and inflammatory arthritis. The novel JNK inhibitor SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one) completely blocked IL-1--induced accumulation of phospho-Jun and induction of c-Jun transcription in synoviocytes. Furthermore, AP-1 binding and collagenase mRNA accumulation were completely suppressed by SP600125. In contrast, complete inhibition of p38 had no effect, and ERK inhibition had only a modest effect. The essential role of JNK was confirmed in cultured synoviocytes from JNK1 knockout mice and JNK2 knockout mice, each of which had a partial defect in IL-1--induced AP-1 activation and collagenase-3 expression. Administration of SP600125 modestly decreased the rat paw swelling in rat adjuvant-induced arthritis. More striking was the near-complete inhibition of radiographic damage that was associated with decreased AP-1 activity and collagenase-3 gene expression. Therefore, JNK is a critical MAPK pathway for IL-1--induced collagenase gene expression in synoviocytes and in joint arthritis, indicating that JNK is an important therapeutic target for RA.

Objective: To prospectively evaluate a survival prediction index (SPI) in dogs admitted to intensive care units (ICU) and to generate and test an improved SPI (ie, SPI2)., Sample Population: Medical records of 624 critically ill dogs admitted to an ICU., Procedure: Data were collected from dogs within 24 hours after admission to an ICU. Variables recorded reflected function of vital organ systems, severity of underlying physiologic derangement, and extent of physiologic reserve; outcome was defined as dogs that survived or did not survive until 30 days after admission to the ICU. Probabilities of survival were calculated, using an established model (SPI). We then performed another logistic regression analysis, thereby reestimating the variables to create the new SPI2. Cross-validation of the models obtained was performed by randomly assigning the total sample of 624 dogs into an estimation group of 499 dogs and validation group of 125 dogs., Results: Testing of SPI resulted in an area under the curve (AUC) of 0.723. Testing of SPI2 revealed an AUC of 0.773. A backwards-elimination procedure was used to create a model containing fewer variables, and variables were sequentially eliminated. The AUC for the reduced model of SPI2 was 0.76, indicating little loss in predictive accuracy., Conclusions and Clinical Relevance: The new SPI2 objectively stratified clinical patients into groups according to severity of disease. This index could provide an important tool for clinical research.

Background and Aims: Hepatocyte apoptosis is induced by tumor necrosis factor alpha (TNF-alpha) and Fas ligand. Although nuclear factor-kappaB (NF-kappaB) activation protects hepatocytes from TNF-alpha-mediated apoptosis, the NF-kappaB responsive genes that protect hepatocytes are unknown. Our aim was to study the role of NF-kappaB activation and inducible nitric oxide synthases (iNOSs) in TNF-alpha- and Fas-mediated apoptosis in hepatocytes., Methods: Primary cultures of hepatocytes from wild-type and iNOS knockout mice were treated with TNF-alpha, the Fas agonistic antibody Jo2, a nitric oxide (NO) donor (S-nitroso-N-acetylpenicillamine), an NO inhibitor (N(G)-methyl-L-arginine acetate), and/or adenovirus-expressing NF-kappaB inhibitors., Results: The IkappaB superrepressor and a dominant-negative form of IkappaB kinase beta (IKKbeta) inhibited NF-kappaB binding activity by TNF-alpha or Jo2 and sensitized hepatocytes to TNF-alpha- and Jo2-mediated apoptosis. TNF-alpha and Jo2 induced iNOS messenger RNA and protein levels through the induction of NF-kappaB. S-nitroso-N-acetylpenicillamine inhibited Bid cleavage, the mitochondrial permeability transition, cytochrome c release, and caspase-8 and -3 activity, and reduced TNF-alpha- and Fas-mediated death in hepatocytes expressing IkappaB superrepressor. N(G)-methyl-L-arginine acetate partially sensitized hepatocytes to TNF-alpha- and Fas-mediated cell killing. TNF-alpha alone or Jo2 alone induced moderate cell death in hepatocytes from iNOS(-)/(-) mice., Conclusions: NO protects hepatocytes from TNF-alpha- and Fas-mediated apoptosis. Endogenous iNOS, which is activated by NF-kappaB via IKKbeta, provides partial protection from apoptosis.

Nuclear factor-kappa B (NF-kappa B) protects hepatocytes from undergoing apoptosis during embryonic development and during liver regeneration. Activation of NF-kappa B is mediated through phosphorylation of its inhibitor, I kappa B, by a kinase complex that contains 2 I kappa B kinases. We analyzed the differential role of I kappa B kinase 1 (IKK1) and I kappa B kinase 2 (IKK2) in tumor necrosis factor alpha (TNF-alpha)- and interleukin-1 beta (IL-1 beta)-mediated NF-kappa B activation in primary rat hepatocytes. Maximal induction of IKK activity was observed 5 minutes after TNF-alpha and 15 minutes after IL-1 beta treatment, and activated IKK was able to phosphorylate GST-I kappa B (1-54) and GST-p65 (354-551), but not a GST-p65 (354-551) substrate with a serine-to-alanine substitution at position 536. Infection with an adenovirus containing catalytically inactive IKK2K44M (Ad5IKK2dn) completely blocked both TNF-alpha- and IL-1 beta-induced GST-I kappa B and GST-p65 phosphorylation, I kappa B degradation, and NF-kappa B DNA binding. Adenovirally transduced, catalytically inactive IKK1K44M (Ad5IKK1dn) reduced IKK activity and NF-kappa B DNA binding only slightly. Accordingly, Ad5IKK2dn induced apoptosis in 75% (+/-6%) of hepatocytes after 12 hours of TNF-alpha, which was accompanied by activation of caspases 3 and 8, nuclear fragmentation, and DNA laddering. In contrast, Ad5IKK1dn led to 21% (+/-2%) apoptosis in TNF-alpha-treated hepatocytes after 12 hours and comparatively low activity of caspases 3 and 8. Furthermore, Ad5IKK2dn completely blocked the induction of inducible nitric oxide synthase (iNOS), whereas Ad5IKK1dn had no influence on the expression of iNOS. Thus, IKK2 is the main mediator for cytokine-induced NF-kappa B activation in primary hepatocytes and protects against TNF-alpha-induced apoptosis, whereas IKK1 kinase activity is not required for NF-kappa B activation.

We investigated the structure-activity relationship studies of N-[3, 5-bis(trifluoromethyl)phenyl][2-chloro-4-(trifluoromethyl)pyrimidin-5 -yl]carboxamide (1), an inhibitor of transcription mediated by both NF-kappaB and AP-1 transcription factors, with the goal of improving its potential oral bioavailability. Compounds were examined for cell-based activity, were fit to Lipinski's rule of 5, and were examined for potential gastrointestinal permeability using the intestinal epithelial cell line, Caco-2. Selected groups were substituted at the 2-, 4-, and 5-positions of the pyrimidine ring using solution-phase combinatorial methodology. The introduction of a fluorine in the place of 2-chlorine of 1 resulted in a compound with comparable activity. However, other substitutions at the 2-position resulted in a loss of activity. The trifluoromethyl group at the 4-position could be replaced with a methyl, ethyl, chlorine, or phenyl without a substantial loss of activity. The carboxamide group at the 5-position is critical for activity. If it was moved to the 6-position, the activity was lost. The 2-methyl analogue of 1 (81) showed comparable in vitro activity and improved Caco-2 permeability compared to 1.

In an effort to identify novel inhibitors of AP-1 and NF-kappaB mediated transcriptional activation, several analogues of ethyl 4-[(3-methyl-2,5-dioxo(3-pyrrolinyl))amino]-2-(trifluoromethyl)pyr imidine-5-carboxylate (1) were synthesized and tested in two in vitro assays. The 2-(2'-thienyl) substituted compound (11) was identified as the most potent in this series.

NF-kappa B plays a key role in the production of cytokines in inflammatory diseases. The effects of a novel T cell-specific NF-kappa B inhibitor, SP100030, were evaluated in cultured Jurkat cells and in murine collagen-induced arthritis (CIA). Chemical libraries were screened for NF-kappa B-inhibitory activity. SP100030, a compound identified in this process, inhibited NF-kappa B activation in PMA/PHA-activated Jurkat cells by EMSA at a concentration of 1 microM. Jurkat cells and the monocytic cell line THP-1 were transfected with an NF-kappa B promotor/luciferase construct and activated. SP100030 inhibited luciferase production in the Jurkat cells (IC50 = 30 nM). ELISA and RT-PCR confirmed that IL-2, IL-8, and TNF-alpha production by activated Jurkat and other T cell lines were inhibited by SP100030. However, cytokine expression was not blocked by the compound in THP-1 cells, fibroblasts, endothelial cells, or epithelial cells. Subsequently, DBA/1J mice were immunized with type II collagen. Treatment with SP100030 (10 mg/kg/day i.p. beginning on day 21) significantly decreased arthritis severity from onset of clinical signs to the end of the study on day 34 (arthritis score, 5.6 +/- 1.7 for SP100030 and 9.8 +/- 1.5 for control; p < 0.001). Histologic evaluation demonstrated a trend toward improvement in SP100030-treated animals. EMSA of arthritic mouse ankles in CIA showed that synovial NF-kappa B binding was suppressed in the SP100030-treated mice. SP100030 inhibits NF-kappa B activation in T cells, resulting in reduced NF-kappa B-regulated gene expression and decreased CIA. Its selectivity for T cells could provide potent immunosuppression with less toxicity than other NF-kappa B inhibitors.

NF-kappa B transcription factors play an important role in the activation of the IL-2 gene in response to TCR ligation. The release of NF-kappa B factors to the nucleus requires phosphorylation and degradation of the inhibitory kappa-B proteins (I kappa Bs). I kappa B alpha and I kappa B beta phosphorylation is dependent on dual signaling by the TCR and the CD28 accessory receptor. This pathway involves a multisubunit I kappa B kinase (IKK) complex, which includes the IKK alpha (IKK-1) and IKK beta (IKK-2) kinases. We demonstrate that stimulation of primary human CD4+ T cells by CD3/CD28 activates two distinct endogenous IKK complexes, a heterodimeric IKK alpha/beta and a homodimeric IKK beta complex. IKK beta overexpression in a Jurkat cell line resulted in the formation of a constitutively active IKK complex, which was CD3/CD28 inducible. In contrast, ectopic expression of IKK alpha assembled into a complex with negligible I kappa B kinase activity. Moreover, IKK beta, but not IKK alpha, overexpression enhanced transcriptional activation of the CD28 response element in the IL-2 promoter. Conversely, only kinase-inactive IKK beta interfered in the activation of the IL-2 promoter. Sodium salicylate, an inhibitor of IKK beta, but not IKK alpha, activity, inhibited IL-2 promoter activation as well as IL-2 secretion and interfered in activation of both the heterodimeric as well as the homodimeric IKK complexes in primary CD4+ T cells. Taken together, these data demonstrate the presence of an IKK beta-mediated signaling pathway that is activated by TCR and CD28 coligation and regulates IL-2 promoter activity.

A myriad of unrelated exogenous or endogenous agents that represent a threat to the organism are capable of inducing NF-kappaB activity, including viral infection, bacterial lipids, DNA damage, oxidative stress and chemotherapuetic agents. Likewise, NF-kappaB regulates the expression of an equally diverse array of cellular genes. These findings are indicative of the widespread significance of NF-kappaB as a mediator of cellular stress. Remarkably, the NF-kappaB pathway displays the capacity to activate, in a cell- and stimulus-specific manner, only a subset of the total repertoire of NF-kappaB-responsive genes. The seemingly promiscuous nature of NF-kappaB activation poses a regulatory quagmire as to how specificity is achieved at the level of gene expression. The review will summarize recent findings and explore how they further our understanding of the mechanism by which stimulus-specific activation of NF-kappaB is achieved in response to cellular stress.

Potential mechanisms of joint destruction in rheumatoid arthritis (RA) were examined by studying the regulation of mitogen-activated protein kinases and collagenase gene expression in fibroblast-like synoviocytes (FLS). The three main mitogen-activated protein kinase families [p38, Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases (ERKs)] were constitutively expressed in RA and osteoarthritis (OA) FLS. p38 and ERK1/2 were readily phosphorylated in both RA and OA FLS after interleukin-1 (IL-1) stimulation. JNK was phosphorylated in RA FLS but not OA FLS after IL-1 stimulation. Reverse transcription-polymerase chain reaction studies suggested that JNK2 is the major isoform of the JNK family expressed by FLS. Northern blot analysis of collagenase gene expression demonstrated that RA FLS contained significantly more collagenase mRNA than OA FLS after IL-1 stimulation. The roles of JNK and p38 kinase were evaluated with the p38/JNK inhibitor SB 203580. Low concentrations of SB 203580 (1 microM, a concentration that only inhibits p38) had no significant effect on IL-1-induced collagenase expression in RA FLS whereas 25 microM (which inhibits p38, JNK2, and c-raf) blocked collagenase mRNA accumulation. IL-1-stimulated AP-1 binding was also inhibited by 25 microM SB 203580 in RA FLS. These studies suggest that OA and RA FLS have a different pattern of JNK phosphorylation, which might lead to enhanced collagenase gene expression in RA.

Protein phosphorylation is a key cellular regulatory mechanism. Protein kinases and phosphatases regulate cell-cycle progression, transcription, translation, protein sorting and cell adhesion events that are critical to the inflammatory process. Two of the best- characterized immunosuppressants, cyclosporin and rapamycin, are also effective anti-inflammatory drugs. They act directly on protein phosphorylation and, as such, validate the concept that small-molecule modulators of phosphorylation cascades possess anti- inflammatory properties. The authors describe studies that outline progress in defining specific protein kinase signal-transduction cascades, the key drug discovery targets in these cascades and progress towards developing selective agents that have potential in treating numerous inflammatory diseases.

Inflammatory and autoimmune diseases, including rheumatoid arthritis, inflammatory bowel diseases, multiple sclerosis, psoriasis and asthma, provide drug discoverers with a tremendous challenge. The precise causes of these diseases are not known, but our understanding of the molecular and cellular mechanisms associated with inflammatory diseases has increased dramatically. As a consequence, a wide array of gene targets have emerged that control cell influx and activation, inflammatory mediator release and activity, and tissue proliferation and degradation. Since multiple gene products have been identified at the sites of inflammation, there has been a surge of interest in identifying intracellular signaling targets, including transcription factors that control inflammatory gene expression and which are amenable to drug discovery.

NF-kappa B is a key regulator of inflammatory gene transcription and is activated in the rheumatoid arthritis (RA) synovium. In resting cells, NF-kappa B is retained as an inactive cytoplasmic complex by its inhibitor, I kappa B. Phosphorylation of I kappa B targets it for proteolytic degradation, thereby releasing NF-kappa B for nuclear translocation. Recently, two related I kappa B kinases (IKK-1 and IKK-2) were identified in immortalized cell lines that regulate NF-kappa B activation by initiating I kappa B degradation. To determine whether IKK regulates NF-kappa B in primary cells isolated from a site of human disease, we characterized IKK in cultured fibroblast-like synoviocytes (FLS) isolated from synovium of patients with RA or osteoarthritis. Immunoreactive IKK protein was found to be abundant in both RA and osteoarthritis FLS by Western blot analysis. Northern blot analysis showed that IKK-1 and IKK-2 genes were constitutively expressed in all FLS lines. IKK function in FLS extracts was determined by measuring phosphorylation of recombinant I kappa B in vitro. IKK activity in both RA and osteoarthritis FLS was strongly induced by TNF-alpha and IL-1 in a concentration-dependent manner. Activity was significantly increased within 10 min of stimulation and declined to near basal levels within 80 min. Activation of IKK in FLS was accompanied by phosphorylation and degradation of endogenous I kappa B alpha as determined by Western blot analysis. Concomitant activation and nuclear translocation of NF-kappa B was documented by EMSA and immunohistochemistry. Transfection with a dominant negative IKK-2 mutant prevented TNF-alpha-mediated NF-kappa B nuclear translocation, whereas a dominant negative IKK-1 mutant had no effect. This is the first demonstration that IKK-2 is a pivotal regulator of NF-kappa B in primary human cells.