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Rationale: Allergic asthma is characterized by reversible airway obstruction, lung inflammation, and airway hyperresponsiveness (AHR). Previous studies using leukotriene B(4) (LTB(4)) receptor 1-deficient mice and adoptive transfer experiments have suggested that LTB(4) plays a role in lung inflammation and AHR.Objectives: In this study, we used a leukotriene A(4) hydrolase (LTA(4)H) inhibitor as a pharmacological tool to directly examine the role of LTB(4) in a mast cell-dependent murine model of allergic airway inflammation.Methods: We used the forced oscillation technique to test the effects of an LTA(4)H inhibitor dosed during the challenge phase on AHR. Lung tissue and lavage were collected for analysis.Measurements and Main Results: Treatment with an LTA(4)H inhibitor improved multiple parameters encompassing AHR and lung function. Significant decreases in inflammatory leukocytes, cytokines, and mucin were observed in the lung lumen. Serum levels of antigen-specific IgE and IgG1 were also decreased. Labeled antigen uptake by lung dendritic cells and subsequent trafficking to draining lymph nodes and the lung were decreased on LTA(4)H inhibitor treatment. Provocatively, inhibition of LTA(4)H increased lipoxin A(4) levels in lung lavage fluid.Conclusions: These data suggest that LTB(4) plays a key role in driving lung inflammation and AHR. Mechanistically, we provide evidence that inhibition of LTA(4)H, affects recruitment of both CD4(+) and CD8(+) T cells, as well as trafficking of dendritic cells to draining lymph nodes, and may beneficially modulate other pro- and antiinflammatory eicosanoids in the lung. Inhibition of LTA(4)H is thus a potential therapeutic strategy that could modulate key aspects of asthma. [ABSTRACT FROM AUTHOR]

Objectives: We have previously reported using gene-deficient mice that the interleukin (IL)-23p19 subunit is required for the development of innate immune-driven arthritic pain and disease. We aimed to explore here, using a number of in vivo approaches, how the IL-23p19 subunit can mechanistically control arthritic pain and disease in a T- and B- lymphocyte-independent manner., Methods: We used the zymosan-induced arthritis (ZIA) model in wild-type and Il23p19 -/- mice, by a radiation chimera approach, and by single cell RNAseq and qPCR analyses, to identify the IL23p19-expressing and IL-23-responding cell type(s) in the inflamed joints. This model was also utilized to investigate the efficacy of IL-23p19 subunit blockade with a neutralizing monoclonal antibody (mAb). A novel IL-23-driven arthritis model was established, allowing the identification of putative downstream mediators of IL-23 in the control of pain and disease. Pain and arthritis were assessed by relative static weight distribution and histology, respectively., Results: We present evidence that (i) IL-23p19 + non-bone marrow-derived macrophages are required for the development of ZIA pain and disease, (ii) prophylactic and therapeutic blockade of the IL-23p19 subunit ameliorate ZIA pain and disease and (iii) systemically administered IL-23 can induce arthritic pain and disease in a manner dependent on TNF, GM-CSF, CCL17 and cyclooxygenase activity, but independently of lymphocytes, CGRP, NGF and substance P., Conclusions: The data presented should aid IL-23 targeting both in the choice of inflammatory disease to be treated and the design of clinical trials., (© 2024. The Author(s).)

The interleukin (IL)-23 pathway is a pathogenic driver in psoriasis, psoriatic arthritis, and inflammatory bowel disease. Currently, no oral therapeutics selectively target this pathway. JNJ-77242113 is a peptide targeting the IL-23 receptor with high affinity (K D : 7.1 pM). In human cells, JNJ-77242113 potently and selectively inhibited proximal IL-23 signaling (IC 50 : 5.6 pM) without impacting IL-12 signaling. JNJ-77242113 inhibited IL-23-induced interferon (IFN)γ production in NK cells, and in blood from healthy donors and psoriasis patients (IC 50 : 18.4, 11 and 9 pM, respectively). In a rat trinitrobenzene sulfonic acid-induced colitis model, oral JNJ-77242113 attenuated disease parameters at doses ≥ 0.3 mg/kg/day. Pharmacologic activity beyond the gastrointestinal tract was also demonstrated. In blood from rats receiving oral JNJ-77242113, dose-dependent inhibition of ex vivo IL-23-stimulated IL-17A production was observed. In an IL-23-induced rat skin inflammation model, JNJ-77242113 inhibited IL-23-induced skin thickening and IL-17A, -17F and -22 gene induction. Oral dosing of JNJ-77242113 in healthy human volunteers inhibited ex vivo IL-23-stimulated IFNγ production in whole blood. Thus, JNJ-77242113 provided selective, systemic IL-23 pathway inhibition in preclinical models which translated to pharmacodynamic activity in healthy human volunteers, supporting the potential for JNJ-77242113 as a selective oral therapy for IL-23-driven immune-mediated diseases., (© 2024. The Author(s).)

Interleukin (IL)-23 is implicated in the pathogenesis of several inflammatory diseases and is usually linked with helper T cell (Th17) biology. However, there is some data linking IL-23 with innate immune biology in such diseases. We therefore examined the effects of IL-23p19 genetic deletion and/or neutralization on in vitro macrophage activation and in an innate immune-driven peritonitis model. We report that endogenous IL-23 was required for maximal macrophage activation by zymosan as determined by pro-inflammatory cytokine production, including a dramatic upregulation of granulocyte-colony stimulating factor (G-CSF). Furthermore, both IL-23p19 genetic deletion and neutralization in zymosan-induced peritonitis (ZIP) led to a specific reduction in the neutrophil numbers, as well as a reduction in the G-CSF levels in exudate fluids. We conclude that endogenous IL-23 can contribute significantly to macrophage activation during an inflammatory response, mostly likely via an autocrine/paracrine mechanism; of note, endogenous IL-23 can directly up-regulate macrophage G-CSF expression, which in turn is likely to contribute to the regulation of IL-23-dependent neutrophil number and function during an inflammatory response, with potential significance for IL-23 targeting particularly in neutrophil-associated inflammatory diseases., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Kevin M.-C. Lee, Tanya Lupancu, John A. Hamilton reports financial support was provided by Janssen Pharmaceuticals Inc. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper]., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Psoriasis is an inflammatory, IL-17-driven skin disease in which autoantigen-induced CD8 + T cells have been identified as pathogenic drivers., Objective: Our study focused on comprehensively characterizing the phenotypic variation of CD8 + T cells in psoriatic lesions., Methods: We used single-cell RNA sequencing to compare CD8 + T-cell transcriptomic heterogeneity between psoriatic and healthy skin., Results: We identified 11 transcriptionally diverse CD8 + T-cell subsets in psoriatic and healthy skin. Among several inflammatory subsets enriched in psoriatic skin, we observed 2 Tc17 cell subsets that were metabolically divergent, were developmentally related, and expressed CXCL13, which we found to be a biomarker of psoriasis severity and which achieved comparable or greater accuracy than IL17A in a support vector machine classifier of psoriasis and healthy transcriptomes. Despite high coinhibitory receptor expression in the Tc17 cell clusters, a comparison of these cells with melanoma-infiltrating CD8 + T cells revealed upregulated cytokine, cytolytic, and metabolic transcriptional activity in the psoriatic cells that differed from an exhaustion program., Conclusion: Using high-resolution single-cell profiling in tissue, we have uncovered the diverse landscape of CD8 + T cells in psoriatic and healthy skin, including 2 nonexhausted Tc17 cell subsets associated with disease severity., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The nuclear receptor retinoid-related orphan receptor gamma t (RORγt) plays a critical role in driving Th17 cell differentiation and expansion, as well as IL-17 production in innate and adaptive immune cells. The IL-23/IL-17 axis is implicated in several autoimmune and inflammatory diseases, and biologics targeting IL-23 and IL-17 have shown significant clinical efficacy in treating psoriasis and psoriatic arthritis. JNJ-61803534 is a potent RORγt inverse agonist, selectively inhibiting RORγt-driven transcription versus closely-related family members, RORα and RORβ. JNJ-61803534 inhibited IL-17A production in human CD4 + T cells under Th17 differentiation conditions, but did not inhibit IFNγ production under Th1 differentiation conditions, and had no impact on in vitro differentiation of regulatory T cells (Treg), nor on the suppressive activity of natural Tregs. In the mouse collagen-induced arthritis model, JNJ-61803534 dose-dependently attenuated inflammation, achieving ~ 90% maximum inhibition of clinical score. JNJ-61803534 significantly inhibited disease score in the imiquimod-induced mouse skin inflammation model, and dose-dependently inhibited the expression of RORγt-regulated genes, including IL-17A, IL-17F, IL-22 and IL-23R. Preclinical 1-month toxicity studies in rats and dogs identified doses that were well tolerated supporting progression into first-in-human studies. An oral formulation of JNJ-61803534 was studied in a phase 1 randomized double-blind study in healthy human volunteers to assess safety, pharmacokinetics, and pharmacodynamics. The compound was well tolerated in single ascending doses (SAD) up to 200 mg, and exhibited dose-dependent increases in exposure upon oral dosing, with a plasma half-life of 164 to 170 h. In addition, dose-dependent inhibition of ex vivo stimulated IL-17A production in whole blood was observed, demonstrating in vivo target engagement. In conclusion, JNJ-61803534 is a potent and selective RORγt inhibitor that exhibited acceptable preclinical safety and efficacy, as well as an acceptable safety profile in a healthy volunteer SAD study, with clear evidence of a pharmacodynamic effect in humans.

The nuclear receptor retinoic acid receptor-related orphan receptor gamma t (RORγt) is a transcription factor that drives Th17 cell differentiation and IL-17 production in both innate and adaptive immune cells. The IL-23/IL-17 pathway is implicated in major autoimmune and inflammatory diseases. RORγt lies at the core of this pathway and represents an attractive opportunity for intervention with small molecule therapeutics. Despite diverse chemical series having been reported, combining high potency and nuclear receptor selectivity with good physicochemical properties remains a challenging endeavor in the field of RORγt drug discovery. We recently described the discovery and evaluation of a new class of potent and selective RORγt inverse agonists based on a thiazole scaffold. Herein we describe the successful optimization of this class by incorporation of an additional amide moiety at the 4-position of the thiazole core. In several optimization cycles, we have reduced human PXR activation, improved solubility, and increased potency while maintaining nuclear receptor selectivity. X-ray crystallographic analysis of compound 1g bound in the sterol binding site of the ligand binding domain of RORγt was largely consistent with an earlier structure, guiding further insight into the molecular mechanism for RORγt inhibition with this series. Compound 1g is orally bioavailable, potent in a human whole blood assay and proved to be efficacious in an ex-vivo IL-17A assay, and was selected for preclinical evaluation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Starting from previously identified thiazole-2-carboxamides exemplified by compound 1/6, two new series of RORγt inverse agonists with significantly improved aqueous solubility, ADME parameters and oral PK properties were discovered. These scaffolds were identified from a bioisosteric amide replacement approach. Amongst the variety of heterocycles explored, a 1,3,4-oxadiazole led to compounds with the best overall profile for SAR development and in vivo exploration. In an ex vivo mouse PD model, concentration dependent efficacy was demonstrated and compounds 3/5 and 6/3 were profiled in a 5-day rat tolerability study., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

We have previously reported the syntheses of a series of 3,6-disubstituted quinolines as modulators of the retinoic acid receptor-related orphan receptor gamma t (RORγt). These molecules are potent binders but are high molecular weight and they exhibited poor solubility at pH 2 and pH 7. This manuscript details our efforts at improving physical chemical properties for this series of compounds by increasing the diversity at the 3-position (i.e. introducing heteroatoms and lowering the molecular weight). These efforts have led to molecules which are potent binders with improved solubility., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

We identified 6-substituted quinolines as modulators of the retinoic acid receptor-related orphan receptor gamma t (RORγt). The synthesis of this class of RORγt modulators is reported, and optimization of the substituents at the quinoline 6-position that produced compounds with high affinity for the receptor is detailed. This effort identified molecules that act as potent, full inverse agonists in a RORγt-driven cell-based reporter assay. The X-ray crystal structures of two full inverse agonists from this chemical series bound to the RORγt ligand binding domain are disclosed, and we highlight the interaction of a hydrogen-bond acceptor on the 6-position substituent of the inverse agonist with Glu379:NH as a conserved binding contact., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A high-throughput screen of the ligand binding domain of the nuclear receptor retinoic acid-related orphan receptor gamma t (RORγt) employing a thermal shift assay yielded a quinoline tertiary alcohol hit. Optimization of the 2-, 3- and 4-positions of the quinoline core using structure-activity relationships and structure-based drug design methods led to the discovery of a series of modulators with improved RORγt inhibitory potency and inverse agonism properties., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The IL-23/IL-17 pathway is implicated in autoimmune diseases, particularly psoriasis, where biologics targeting IL-23 and IL-17 have shown significant clinical efficacy. Retinoid-related orphan nuclear receptor gamma t (RORγt) is required for Th17 differentiation and IL-17 production in adaptive and innate immune cells. We identified JNJ-54271074, a potent and highly-selective RORγt inverse agonist, which dose-dependently inhibited RORγt-driven transcription, decreased co-activator binding and promoted interaction with co-repressor protein. This compound selectively blocked Th17 differentiation, significantly reduced IL-17A production from memory T cells, and decreased IL-17A- and IL-22-producing human and murine γδ and NKT cells. In a murine collagen-induced arthritis model, JNJ-54271074 dose-dependently suppressed joint inflammation. Furthermore, JNJ-54271074 suppressed IL-17A production in human PBMC from rheumatoid arthritis patients. RORγt-deficient mice showed decreased IL-23-induced psoriasis-like skin inflammation and cytokine gene expression, consistent with dose-dependent inhibition in wild-type mice through oral dosing of JNJ-54271074. In a translational model of human psoriatic epidermal cells and skin-homing T cells, JNJ-54271074 selectively inhibited streptococcus extract-induced IL-17A and IL-17F. JNJ-54271074 is thus a potent, selective RORγt modulator with therapeutic potential in IL-23/IL-17 mediated autoimmune diseases.

The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4(+) Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7β, 27-dihydroxycholesterol (7β, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ- or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7β, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an RORγt-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7β, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17-producing cells, including CD4(+) and γδ(+) T cells, similar to the deficiency observed in RORγt knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these RORγt agonist ligands, which we propose as RORγt endogenous ligands, driving both innate and adaptive IL-17-dependent immune responses.

Leukotrienes (LT's) are known to play a physiological role in inflammatory immune response. Leukotriene A(4) hydrolase (LTA(4)H) is a cystolic enzyme that stereospecifically catalyzes the transformation of LTA(4) to LTB(4). LTB(4) is a known pro-inflammatory mediator. This paper describes the identification and synthesis of substituted benzofurans as LTH(4)H inhibitors. The benzofuran series demonstrated reduced mouse and human whole blood LTB(4) levels in vitro and led to the identification one analog for advanced profiling. Benzofuran 28 showed dose responsive target engagement and provides a useful tool to explore a LTA(4)H inhibitor for the treatment of inflammatory diseases, such as asthma and inflammatory bowel disease (IBD)., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Previously, benzthiazole containing LTA(4)H inhibitors were discovered that were potent (1-3), but were associated with the potential for a hERG liability. Utilizing medicinal chemistry first principles (e.g., introducing rigidity, lowering cLogD) a new benzthiazole series was designed, congeners of 1-3, which led to compounds 7a, 7c, 12a-d which exhibited LTA(4)H IC(50)=3-6 nM and hERG Dofetilide Binding IC(50)=8.9-> >10 μM., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In recent years, the classic paradigm of Th1/Th2 CD4(+) T cell-mediated immunity has evolved to include the IL-17A-producing Th17 subset, a distinct proinflammatory CD4(+) T cell lineage. Accumulating evidence suggests that IL-17A and the Th17 pathway may play an important role in the pathology of psoriasis and in other immune-mediated inflammatory diseases. This review summarizes the preclinical and clinical evidence implicating Th17 cells in psoriasis and the therapeutic approaches, approved or under investigation, to target this pathway in psoriasis.

Leukotriene A4 hydrolase (LTA4H) is a ubiquitously expressed enzyme that catalyzes the final step in the synthesis of leukotriene B4 (LTB4), a potent proinflammatory lipid mediator derived from arachidonic acid. Although LTB4 was identified 30 years ago, several recent findings have refocused attention on this mediator as a target for inflammatory and autoimmune diseases. While LTB4 was once thought to be a chemoattractant and activator only of leukocytes mediating acute, innate inflammatory responses, LTB4 receptors have since been discovered on multiple cell types, including T-lymphocytes and antigen-presenting dendritic cells. Thus, the inhibition of LTB4 synthesis demonstrates potential for targeting chronic, autoimmune-driven inflammation. In addition to genetic data in animals and humans linking the LTB4 pathway to cardiovascular disease, variants in the LTA4H gene have been linked with susceptibility to asthma. Several companies have initiated drug discovery efforts to identify potent, selective LTA4H inhibitors. Selected molecules have demonstrated oral efficacy in preclinical models of asthma, inflammatory bowel disease and arthritis, suggesting therapeutic potential for multiple indications. This review focuses on developments with therapeutic relevance for inhibitors of LTA4H as anti-inflammatory drugs, and particularly in the treatment of respiratory disease.

LTA 4H is a ubiquitously distributed 69 kDa zinc-containing cytosolic enzyme with both hydrolase and aminopeptidase activity. As a hydrolase, LTA 4H stereospecifically catalyzes the transformation of the unstable epoxide LTA 4 to the diol LTB 4, a potent chemoattractant and activator of neutrophils and a chemoattractant of eosinophils, macrophages, mast cells, and T cells. Inhibiting the formation of LTB 4 is expected to be beneficial in the treatment of inflammatory diseases such as inflammatory bowel disease (IBD), asthma, and atherosclerosis. We developed a pharmacophore model using a known inhibitor manually docked into the active site of LTA 4H to identify a subset of compounds for screening. From this work we identified a series of benzoxazole, benzthiazole, and benzimidazole inhibitors. SAR studies resulted in the identification of several potent inhibitors with an appropriate cross-reactivity profile and excellent PK/PD properties. Our efforts focused on further profiling JNJ 27265732, which showed encouraging efficacy in a disease model relevant to IBD.

Little is known about inhalable dust concentrations in soybean processing plants in southern Africa. This project measured inhalable dust in soybean plants in the region and correlated dust measurements with total protein and soy trypsin inhibitor. Sixty-four personal inhalable dust measurements were taken in three processing plants. Levels of total protein and soy trypsin inhibitor were determined in only two of the three plants. Correlations between inhalable dust, total protein and trypsin inhibitor were determined for 44 of 64 samples. In plants' production areas, inhalable dust levels were 0.24-35.02 mg/m3 (median 2.58 mg/m3). Total protein and soy trypsin inhibitor levels were 29.41-448.82 microg/m3 (median 90.09 microg/m3) and 0.05-2.58 microg/m3 (median 0.07 microg/m3), respectively. No statistically significant correlations between presence of inhalable dust and soy trypsin inhibitor were found. Total protein and soy trypsin inhibitor were better correlated. This study indicates that total protein might be a good proxy for soybean specific protein concentrations in soybean processing plants.

Background and Purpose: Leukotriene B(4) (LTB(4)), formed by the sequential actions of the 5-lipoxygenase (5-LO) and leukotriene A(4) hydrolase (LTA(4)H), is a pro-inflammatory mediator implicated in the pathogenesis of inflammatory bowel disease. However, inhibitors of 5-LO have not proved to be consistent in their therapeutic efficacy in colitis. Another approach to inhibiting LTB(4) synthesis is through the use of inhibitors of LTA(4)H, such as the novel, potent and selective compound, JNJ 26993135., Experimental Approach: The effect of oral administration of JNJ 26993135 has been evaluated in a rat model of colitis provoked by colonic instillation of trinitrobenzenesulphonic acid (TNBS). The extent and severity of the macroscopic inflammatory response, the colonic levels of myeloperoxidase (MPO) and LTB(4) and of the pro-inflammatory cytokines, tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were measured., Key Results: Oral administration of JNJ 26993135 (5, 15 and 30 mg kg(-1), twice a day) dose-dependently reduced both the extent and intensity of the colonic inflammatory damage observed 3 days after TNBS challenge. JNJ 26993135 also dose-dependently reduced the elevated colonic levels of LTB(4), as well as the inflammatory biomarkers, MPO, IL-6 and TNF-alpha. This dosing regimen was supported by the pharmacokinetic profile of JNJ 26993135, along with the demonstration of the inhibition of ex vivo production of LTB(4) in whole blood following oral administration., Conclusions and Implications: These results with JNJ 26993135 in the rat TNBS model support the role of LTB(4) in colitis and the potential value of targeting LTA(4)H for the treatment of inflammatory bowel diseases.

Leukotriene A(4) hydrolase (LTA(4)H) catalyzes production of the proinflammatory lipid mediator, leukotriene (LT) B(4), which is implicated in a number of inflammatory diseases. We have identified a potent and selective inhibitor of both the epoxide hydrolase and aminopeptidase activities of recombinant human LTA(4)H (IC(50), approximately 10 nM). In a murine model of arachidonic acid-induced ear inflammation, the LTA(4)H inhibitor, JNJ-26993135 (1-[4-(benzothiazol-2-yloxy)-benzyl]-piperidine-4-carboxylic acid), dose-dependently inhibited ex vivo LTB(4) production in blood, in parallel with dose-dependent inhibition of neutrophil influx (ED(50), 1-3 mg/kg) and ear edema. In murine whole blood and in zymosan-induced peritonitis, JNJ-26993135 selectively inhibited LTB(4) production, without affecting cysteinyl leukotriene production, while maintaining or increasing production of the anti-inflammatory mediator, lipoxin (LX) A(4). The 5-lipoxygenase (5-LO) inhibitor zileuton showed inhibition of LTB(4), LTC(4), and LXA(4) production. Although zileuton inhibited LTB(4) production in the peritonitis model more effectively than the LTA(4)H inhibitor, the influx of neutrophils into the peritoneum after 1 and 2 h was significantly higher in zileuton- versus JNJ-26993135-treated animals. This difference may have been mediated by the increased LXA(4) levels in the presence of the LTA(4)H inhibitor. The selective inhibition of LTB(4) production by JNJ-26993135, while increasing levels of the anti-inflammatory mediator, LXA(4), may translate to superior therapeutic efficacy versus 5-LO or 5-LO-activating protein inhibitors in LTB(4)-mediated inflammatory diseases.

Leukotriene B4 (LTB4) is a potent activator and chemoattractant for leukocytes and is implicated in several inflammatory diseases. The actions of LTB4 are mediated by two cell surface receptors, BLT1, which is predominantly expressed in peripheral blood leukocytes, and BLT2, which is expressed more ubiquitously. Recently, BLT1 expression and LTB4-dependent chemotaxis have been reported in immature mast cells (MCs). We now show the first evidence for BLT2 mRNA expression, in addition to BLT1, in murine bone marrow-derived MCs (mBMMCs) and in a human MC line (HMC-1). Protein expression of BLT1 was confirmed by mAb staining in HMC-1 cells and shown to be predominantly intracellular. Both HMC-1 cells and mBMMCs migrated to LTB4 in a dose-dependent manner in chemotaxis assays. Migration to LTB4 could be inhibited by either a BLT1- or BLT2-selective antagonist. Significant dose-dependent migration of mBMMCs also was observed to 12-(S)-hydroxyeicosotetraenoic acid, a BLT2-selective agonist, demonstrating functional BLT2 activity in these cells. Stimulation of mBMMCs with LTB4 induced transient, dose-dependent, ERK phosphorylation and changes in Akt phosphorylation. Dose-dependent ERK phosphorylation also was observed in response to 12-(S)-hydroxyeicosotetraenoic acid, indicating signaling downstream of BLT2. Pretreatment of mBMMCs with stem cell factor significantly down-regulated expression of BLT1 and BLT2 mRNA and inhibited their migration to LTB4. This study demonstrates expression of functional LTB4 receptors, both BLT1 and BLT2, in murine and human MCs and a regulatory role for stem cell factor in their expression. These receptors may mediate recruitment and accumulation of MCs in response to LTB4 production in areas of inflammation.

Aggrecan is the major proteoglycan in cartilage, endowing this tissue with the unique capacity to bear load and resist compression. In arthritic cartilage, aggrecan is degraded by one or more 'aggrecanases' from the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteinases. ADAMTS1, 8 and 9 have weak aggrecan-degrading activity. However, they are not thought to be the primary aggrecanases because ADAMTS1 null mice are not protected from experimental arthritis, and cleavage by ADAMTS8 and 9 is highly inefficient. Although ADAMTS4 and 5 are expressed in joint tissues, and are known to be efficient aggrecanases in vitro, the exact contribution of these two enzymes to cartilage pathology is unknown. Here we show that ADAMTS5 is the major aggrecanase in mouse cartilage, both in vitro and in a mouse model of inflammatory arthritis. Our data suggest that ADAMTS5 may be a suitable target for the development of new drugs designed to inhibit cartilage destruction in arthritis, although further work will be required to determine whether ADAMTS5 is also the major aggrecanase in human arthritis.

The ADAM family of disintegrin metalloproteases plays important roles in "ectodomain shedding," the process by which biologically active, soluble forms of cytokines, growth factors, and their receptors are released from membrane-bound precursors. Whereas ADAM8, ADAM15, and MDC-L (ADAM28) are expressed in specific cell types and tissues, their in vivo functions and substrates are not known. By screening a library of synthetic peptides as potential substrates, we show that soluble recombinant forms of these enzymes have similar proteolytic substrate specificity, clearly distinct from that of ADAM17 (TNFalpha-converting enzyme). A number of tumor necrosis factor (TNF) family proteins and CD23 were screened as potential substrates for ectodomain cleavage. We found that ADAM8, ADAM15, and MDC-L, but not ADAM17, catalyzed ectodomain shedding of CD23, the low affinity IgE receptor. ADAM8-dependent, soluble CD23 release required proteolytically active ADAM8, and a physical association of ADAM8 was observed with the membrane-bound form of CD23. The ADAM8-dependent release of sCD23 and the endogenous release from B cell lines could be similarly inhibited by a hydroxamic acid, metalloprotease inhibitor compound. We conclude that ADAM8 could contribute to ectodomain shedding of CD23 and may thus be a potential target for therapeutic intervention in allergy and inflammation.

While the exact aetiology of myeloma is unknown, genetic factors feature among the potential risk factors. The HLA phenotypes in African blacks with myeloma (the commonest haematopoietic malignancy in this group) have not been characterized. The purpose of this study was to determine the HLA class I and class II phenotypes of patients with multiple myeloma and to compare the findings to an ethnically matched control group of 100 individuals. Analysis of the HLA class I and class II phenotypes in 62 myeloma patients revealed: (i) a corresponding statistically significant association with HLA B18 [odds ratio (OR) 6.3; 95% confidence interval (CI) 1.013-39.727; P < 0.005]; (ii) no statistically significant association with HLA B13, Cw2, Cw6 or the DR and DQ antigens; and (iii) a statistically significant negative (protective) association with HLA Cw7 (OR 0.4; 95% CI 0.21-0.87; P < 0.005). This study suggests that although genetic factors may play a role in the multifactorial aetiology of multiple myeloma, with the exception of HLA B18, there is no specific association between HLA types and multiple myeloma in South African blacks.

Vertebrate cells contain at least 12 different genes for Hsp70 proteins, 3 of which are encoded in the major histocompatibility complex (MHC) class III region. In the human MHC, these are named Hsp70-1, -2, and -Hom. To characterize these proteins, we have determined their substrate binding specificity, their cellular and tissue distribution, and the regulation of their expression. We show for the first time (1) peptide binding specificity of Hsp70-Hom; (2) endogenous expression of Hsp70-Hom in human cell lines; (3) cytoplasmic location of Hsp70-Hom protein under basal conditions and concentration in the nucleus after heat shock; (4) unique RNA expression profiles in human tissues for each of the MHC-encoded Hsp70s, significantly different from that for the constitutive Hsc70; (5) a relative increase in levels of Hsp70-Hom protein, compared with other Hsp70s, in response to interferon gamma; and (6) a specific increase on lipopolysaccharide (LPS) treatment of in vivo messenger RNA levels for the MHC-encoded Hsp70s and the DnaJ homologue, hdj2, relative to other chaperones. The unique tissue distributions and specific up-regulation by LPS of the MHC-encoded Hsp70s suggest some specialization of functions for these members of the Hsp70 family, possibly in the inflammatory response.

In vitro PA28 binds and activates proteasomes. It is shown here that mice with a disrupted PA28b gene lack PA28a and PA28b polypeptides, demonstrating that PA28 functions as a hetero-oligomer in vivo. Processing of antigenic epitopes derived from exogenous or endogenous antigens is altered in PA28-/- mice. Cytotoxic T lymphocyte responses are impaired, and assembly of immunoproteasomes is greatly inhibited in mice lacking PA28. These results show that PA28 is necessary for immunoproteasome assembly and is required for efficient antigen processing, thus demonstrating the importance of PA28-mediated proteasome function in immune responses.

Mutations within conserved regions of the tumor suppressor protein, p53, result in oncogenic forms of the protein with altered tertiary structures. In most cases, the mutant p53 proteins are selectively recognized and bound by members of the HSP70 family of molecular chaperones, but the binding site(s) in p53 for these chaperones have not been clearly defined. We have screened a library of overlapping biotinylated peptides, spanning the entire human p53 sequence, for binding to the HSP70 proteins, Hsc70 and DnaK. We show that most of the high affinity binding sites for these proteins map to secondary structure elements, particularly beta-strands, in the hydrophobic core of the central DNA binding domain, where the majority of oncogenic p53 mutations are found. Although peptides corresponding to the C-terminal region of p53 also contain potential binding sites, p53 proteins with C-terminal deletions are capable of binding to Hsc70, indicating that this region is not required for complex formation. We propose that mutations in the p53 protein alter the tertiary structure of the central DNA binding domain, thus exposing high affinity HSP70 binding sites that are cryptic in the wild-type molecule.

We have studied the binding of synthetic peptides to three hsp70 molecular chaperones, DnaK, BiP, and hsc70, as a model for the interaction of hsp70 proteins with unfolded regions of target polypeptides. We measured the ability of 53 peptides to inhibit the formation of complexes between the hsp70 proteins and denatured lactalbumin. Peptides that bound with highest affinity to all three hsp70 proteins contained stretches of at least 7 residues that included large hydrophobic and basic amino acids, but few or no acidic residues. Amino acid substitutions within one heptameric peptide showed that an important feature for its binding to all three chaperones was a large hydrophobic residue in position 4, while specificity differences between the chaperones were revealed by substitutions at positions 2 and 6. Such specificity differences were frequently observed with other peptides, the most extreme example being a peptide rich in basic residues that bound with high affinity to DnaK, intermediate affinity to hsc70, and negligible affinity to BiP. Substitution of a lysine residue at position 2 in this peptide by tyrosine abolished the specificity difference by increasing the affinities of the DnaK and hsc70 proteins 5- and 20-fold, respectively, and that of BiP by greater than 2 orders of magnitude. Thus, hsp70 proteins can exhibit common or exclusive binding specificities, depending on the peptide sequence.

The molecular chaperone BiP purified from bovine liver (bBiP) exhibits a low basal level of ATPase activity that can be stimulated 3-6-fold by synthetic peptides (Flynn, G. C., Chappell, T. G., and Rothman, J. E. (1989) Science 245, 385-390). By contrast, recombinant murine BiP (rBiP) purified to homogeneity following expression in Escherichia coli exhibits a higher basal level of ATPase activity and is much less stimulated by synthetic peptides. Nondenaturing gel electrophoresis showed that rBiP is predominantly monomeric, while bBiP exists in multiple forms probably corresponding to differentially modified monomeric, dimeric, and higher oligomeric species. Some, but not all, synthetic peptides cause conversion of the oligomeric and modified species of bBiP to a monomeric form. We propose that the peptide-dependent ATPase stimulation observed for BiP reflects the conversion of inactive oligomeric and/or modified species into active monomers.

LDL receptors, expressed in cultured fibroblasts from patients homozygous for the FH Afrikaner-1 (FH1) mutation (Asp206 to Glu), are transported from the endoplasmic reticulum (ER) to the Golgi apparatus more slowly than in normal cells. In the present study, binding characteristics of FH1 cells for lipoprotein ligands (LDL and beta VLDL) and for receptor-specific monoclonal antibodies pointed to the existence of two surface forms of the same mutant receptor. One of these forms bound lipoproteins with normal high affinity whereas another did not. Binding studies of transfected hamster cells expressing only the mutant human gene confirmed the single-gene origin of the different forms. The existence of functionally distinct forms of the receptor protein was supported by the observation that only lipoprotein-binding receptor molecules were trapped intracellularly and degraded following ammonium chloride treatment of cells in the presence of ligand. The lipoprotein-binding receptor population was indistinguishable from normal receptors with respect to its affinity for LDL and beta VLDL, uptake and degradation of lipoprotein, and receptor recycling. Ligand blotting versus immunoblotting of receptors revealed normal-sized mutant receptors that were not recognized by lipoprotein ligand. Despite these differences, both mutant forms of the receptor were degraded at rates similar to those of normal receptors. We propose that the single amino acid substitution in this receptor interferes with the folding and/or posttranslational processing of precursor molecules in such a way that receptors adopt alternative stable structures.

The region of the low-density-lipoprotein (LDL) receptor showing sequence similarity to the epidermal-growth-factor (EGF) precursor is required for LDL binding and the acid-induced dissociation of ligand and receptor. We describe here a naturally occurring mutant LDL receptor, found in a patient with homozygous familial hypercholesterolaemia, which lacks the first two growth-factor-like repeats of the EGF-precursor-like ('homology') domain. The mutation in the receptor gene is a 2.5 kb deletion including exons 7 and 8. The molecular mass of the mutant receptor (145 kDa) was approx. 15 kDa smaller than the normal LDL receptor. The mutant receptors were derived from precursors (105 kDa) that apparently underwent normal processing. Fibroblasts from the patient had high-affinity binding sites for the the apolipoprotein E-containing ligand, beta VLDL, but did not bind LDL. In the presence of beta VLDL, receptors were rapidly degraded. The mutant receptors also displayed an abnormally rapid turnover, about four times faster than that of normal receptors, in the absence of ligand; this accelerated degradation accounted for the low level of expression of mutant receptors in up-regulated cells. These data support a role for the growth-factor-like repeats in the binding of LDL (but not beta VLDL) and in receptor recycling, and indicate that a normal rate of turnover of unoccupied receptors is dependent on the integrity of these segments of the protein.

The effects of cardiotoxin on the ATPase activity and Ca2+-transport of guinea pig erythrocyte and rabbit muscle sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase (E.C.3.6.1.3) were investigated. Erythrocyte (Ca2+ + Mg2+)-ATPase was inhibited by cardiotoxin in a time- and dose-dependent fashion and inhibition appears to be irreversible. Micromolar calcium prevented this inhibitory effect. Specificity for (Ca2+ + Mg2+)-ATPase inhibition by cardiotoxin was indicated since a homologous neurotoxin had no effect. Cardiotoxin did not affect (Ca2+ + Mg2+)-ATPase activity from sarcoplasmic reticulum, but Ca2+-transport was 50% inhibited. This inhibition was not due to an increased Ca2+-efflux and could be the result of an intramolecular uncoupling of ATPase activity from Ca2+-transport. Inhibition of Ca2+-transport by cardiotoxin could not be prevented by millimolar concentrations of Ca2+. It is suggested that the biological effects of cardiotoxin could be a consequence of inhibition of plasma membrane (Ca2+ + Mg2+)-ATPases.

The ligand-binding domain of the low density lipoprotein (LDL) receptor is composed of seven cysteine-rich repeats, each approximately 40 amino acids long. Previous studies by van Driel et al. [van Driel, I. R., Goldstein, J. L., Sudhof, T. C. & Brown, M. S. (1987) J. Biol. Chem. 262, 17443-17449] showed that if the first repeat of the ligand-binding domain (encoded by exon 2) is deleted, the receptor fails to bind an anti-LDL receptor monoclonal antibody (IgG-C7) but continues to bind LDL with high affinity. Cultured fibroblasts from a Black South African Xhosa patient (TT) with the clinical syndrome of homozygous familial hypercholesterolemia demonstrated high-affinity cell-surface binding of 125I-labeled LDL but not 125I-labeled IgG-C7. Previous haplotype analysis, using 10 restriction fragment length polymorphic sites, suggested that the patient inherited two identical LDL receptor alleles. The polymerase chain reaction technique was used to selectively amplify exon 2 of the LDL receptor gene from this patient. Sequence analysis of the amplified fragment disclosed a deletion of six base pairs that removes two amino acids, aspartic acid and glycine, from the first cysteine-rich ligand binding repeat. The mutation creates a new PstI restriction site that can be used to detect the deletion. The existence of this mutant allele confirms that the epitope of IgG-C7 is located in the first cysteine-rich repeat and that this repeat is not necessary for LDL binding. The mutant gene produced a normally sized 120-kilodalton LDL receptor precursor protein that matured to the 160-kilodalton form at less than one-fourth the normal rate. Thus, deletion of two amino acids within the first cysteine-rich repeat retards receptor transport from the endoplasmic reticulum to the cell surface, in contrast to deletion of the entire first repeat, which has no effect on receptor maturation.

Two distinct mutant low-density-lipoprotein receptors in South African Afrikaners exhibit retarded posttranslational processing to mature forms. One mutation gives rise to cell-surface receptors that are subject to abnormally rapid degradation, whereas the other is associated with a functionally heterogeneous surface population degraded at a normal rate.